Other Formats: [Citation Format] Links: [101 medline neighbors] [JBC Online] UI - 98389750 AU - Mattingly JR Jr AU - Torella C AU - Iriarte A AU - Martinez-Carrion M TI - Conformation of aspartate aminotransferase isozymes folding under different conditions probed by limited proteolysis [In Process Citation] LA - Eng DA - 19980910 DP - 1998 Sep 4 IS - 0021-9258 TA - J Biol Chem PG - 23191-202 SB - M SB - X CY - UNITED STATES IP - 36 VI - 273 JC - HIV AA - AUTHOR AB - The partially homologous mitochondrial (mAAT) and cytosolic (cAAT) aspartate aminotransferase have nearly identical three-dimensional structures but differ in their folding rates in cell-free extracts and in their affinity for binding to molecular chaperones. In its native state, each isozyme is protease-resistant. Using limited proteolysis as an index of their conformational states, we have characterized these proteins (a) during the early stages of spontaneous refolding; (b) as species trapped in stable complexes with the chaperonin GroEL; or (c) as newly translated polypeptides in cell-free extracts. Treatment of the refolding proteins with trypsin generates reproducible patterns of large proteolytic fragments that are consistent with the formation of defined folding domains soon after initiating refolding. Binding to GroEL affords considerable protection to both isozymes against proteolysis. The tryptic fragments are similar in size for both isozymes, suggesting a common distribution of compact and flexible regions in their folding intermediates. cAAT synthesized in cell-free extracts becomes protease-resistant almost instantaneously, whereas trypsin digestion of the mAAT translation product produces a pattern of fragments qualitatively akin to that observed with the protein refolding in buffer. Analysis of the large tryptic peptides obtained with the GroEL-bound proteins reveals that the cleavage sites are located in analogous regions of the N-terminal portion of each isozyme. These results suggest that (a) binding to GroEL does not cause unfolding of AAT, at least to an extent detectable by proteolysis; (b) the compact folding domains identified in AAT bound to GroEL (or in mAAT fresh translation product) are already present at the early stages of refolding of the proteins in buffer alone; and (c) the two isozymes seem to bind in a similar fashion to GroEL, with the more compact C- terminal portion completely protected and the more flexible N-terminal first 100 residues still partially accessible to proteolysis. AD - Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499, USA. RO - O:099 PMID- 0009722549 SO - J Biol Chem 1998 Sep 4;273(36):23191-202 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [114 medline neighbors] [JBC Online] UI - 98136136 AU - Lain B AU - Yanez A AU - Iriarte A AU - Martinez-Carrion M TI - Aminotransferase variants as probes for the role of the N-terminal region of a mature protein in mitochondrial precursor import and processing. LA - Eng MH - Amino Acid Sequence MH - Animal MH - Aspartate Transaminase/chemistry/genetics/*metabolism MH - Biological Transport MH - Cell Compartmentation MH - Enzyme Precursors/chemistry/genetics/*metabolism MH - Mitochondria, Liver/enzymology/*metabolism MH - Molecular Probes MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed MH - *Protein Processing, Post-Translational MH - Rats MH - Support, U.S. Gov't, P.H.S. RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Enzyme Precursors) RN - 0 (Molecular Probes) PT - JOURNAL ARTICLE ID - HL-38412/HL/NHLBI ID - GM-38341/GM/NIGMS DA - 19980319 DP - 1998 Feb 20 IS - 0021-9258 TA - J Biol Chem PG - 4406-15 SB - M SB - X CY - UNITED STATES IP - 8 VI - 273 JC - HIV AA - Author EM - 199805 AB - Of the two homologous isozymes of aspartate aminotransferase that are also nearly identical in their folded structures, only the mitochondrial form (mAAT) is synthesized as a precursor (pmAAT). After its in vitro synthesis in rabbit reticulocyte lysate, it can also be efficiently imported into isolated rat liver mitochondria, where it is processed to its native form by removal of the N-terminal presequence. The homologous cytosolic isoenzyme (cAAT) is not imported into mitochondria, even after fusion of the mitochondrial presequence from pmAAT to its N-terminal end. Substitution of the 30-residue N-terminal peptide of the mature portion of pmAAT with the corresponding sequence from the homologous, import-incompetent cytosolic isozyme (pcmAAT) does not prevent import but reduces substantially its processing in the matrix. A detectable amount of the pcmAAT chimera is found associated with the inner mitochondrial membrane. Single and double substitution mutants of Trp-5 and Trp-6 at the N-terminal end of the mature protein are imported into mitochondria with efficiency similar to that of wild type. However, replacement of Trp-5 with proline, or of both tryptophans with either alanine (W5A/W6A mutant) or valine and alanine (W5V/W6A mutant), allows import but interferes with the correct processing of the imported protein despite the presence of an intact cleavage site for the processing peptidase. Similar cleavage results were obtained using newly synthesized proteins and mitochondrial matrix extracts. These results indicate that translocation and processing for a precursor are independent events and that sequences C-terminal to the cleavage site are indeed important for the correct maturation of pmAAT in the matrix, probably because of their contribution to the conformation and flexibility of the peptide region surrounding the cleavage site required for efficient processing. The same region from the mature component of the protein may play a role in the commitment of the passenger protein to complete its translocation into the matrix. AD - Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499, USA. PMID- 0009468492 SO - J Biol Chem 1998 Feb 20;273(8):4406-15 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [113 medline neighbors] [JBC Online] UI - 98129795 AU - Torella C AU - Mattingly JR Jr AU - Artigues A AU - Iriarte A AU - Martinez-Carrion M TI - Insight into the conformation of protein folding intermediate(s) trapped by GroEL. LA - Eng MH - Aspartate Transaminase/chemistry/metabolism MH - Cysteine/metabolism MH - Endopeptidase K/metabolism MH - GroEL Protein/*metabolism MH - GroES Protein/metabolism MH - Guanidine/pharmacology MH - Mitochondria/enzymology MH - Models, Molecular MH - Molecular Chaperones/metabolism MH - Peptide Fragments/analysis MH - Peptide Peptidohydrolases/metabolism MH - *Protein Conformation/drug effects MH - Protein Denaturation MH - *Protein Folding MH - Sulfhydryl Reagents/metabolism MH - Support, U.S. Gov't, P.H.S. MH - Trypsin/metabolism RN - EC 2.6.1.1 (Aspartate Transaminase) RN - EC 3.4.- (Peptide Peptidohydrolases) RN - EC 3.4.21.4 (Trypsin) RN - EC 3.4.21.64 (Endopeptidase K) RN - 0 (GroEL Protein) RN - 0 (GroES Protein) RN - 0 (Molecular Chaperones) RN - 0 (Peptide Fragments) RN - 0 (Sulfhydryl Reagents) RN - 113-00-8 (Guanidine) RN - 4371-52-2 (Cysteine) PT - JOURNAL ARTICLE ID - HL-38412/HL/NHLBI ID - GM-38341/GM/NIGMS DA - 19980323 DP - 1998 Feb 13 IS - 0021-9258 TA - J Biol Chem PG - 3915-25 SB - M SB - X CY - UNITED STATES IP - 7 VI - 273 JC - HIV AA - Author EM - 199805 AB - Many aspects of the mechanism by which the GroEL/ES chaperonins mediate protein folding are still unclear, including the amount of structure present in the substrate bound to GroEL. To address this issue we have analyzed the susceptibility to limited proteolysis and to alkylation of cysteine residues of mitochondrial aspartate aminotransferase (mAAT) bound to GroEL. Several regions of the N-terminal portion of GroEL- bound mAAT are highly susceptible to proteolysis, whereas a large core of about 200 residues containing the C-terminal half of the polypeptide chain is protected in the complex. This protection does not extend to the mAAT sulfhydryl groups which in the GroEL-mAAT complex have similar reactivity as in fully unfolded mAAT. These results suggest that the mAAT species bound to GroEL represent folding intermediates with a conformation that is substantially more disorganized than that of the native state. The N-terminal half of the molecule is more flexible and lies exposed at the mouth of the central cavity of GroEL. The more compact C-terminal section of mAAT, which contains residues located at the subunit interface in the native dimer, appears to be hidden in the central cavity of GroEL. Thus, the bulk of the interactions in the GroEL.mAAT complex seems to involve residues from the more compact C- terminal section of the substrate. AD - Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499, USA. PMID- 0009461576 SO - J Biol Chem 1998 Feb 13;273(7):3915-25 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [98 medline neighbors] [Archives of Biochemistry and Biophysics] UI - 97108728 AU - Berezov A AU - Iriarte A AU - Martinez-Carrion M TI - Interaction of a dimeric mitochondrial precursor with phospholipid vesicles: direct association of each subunit with the membrane is required for loss of functionality. LA - Eng MH - Animal MH - Aspartate Transaminase/*metabolism MH - Cysteine/chemistry MH - Enzyme Precursors/metabolism MH - Fluorescent Dyes MH - Liposomes/chemistry MH - Microscopy, Electron MH - Mitochondria, Liver/enzymology MH - Phosphatidylglycerols MH - Phospholipids/chemistry MH - Rats MH - Spectrometry, Fluorescence MH - Structure-Activity Relationship MH - Support, U.S. Gov't, P.H.S. MH - 1,2-Dipalmitoylphosphatidylcholine RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Enzyme Precursors) RN - 0 (Fluorescent Dyes) RN - 0 (Liposomes) RN - 0 (Phosphatidylglycerols) RN - 0 (Phospholipids) RN - 2644-64-6 (1,2-Dipalmitoylphosphatidylcholine) RN - 4371-52-2 (Cysteine) RN - 4537-77-3 (1,2-dipalmitoylphosphatidylglycerol) PT - JOURNAL ARTICLE ID - GM-38341/GM/NIGMS DA - 19970116 DP - 1996 Dec 1 IS - 0003-9861 TA - Arch Biochem Biophys PG - 173-83 SB - M SB - X CY - UNITED STATES IP - 1 VI - 336 JC - 6SK AA - Author EM - 199703 AB - Binding of the precursor to mitochondrial aspartate aminotransferase to anionic phospholipid vesicles results in the loss of catalytic activity, apparently due to the inability of the bound protein to undergo the conformational transitions required for catalysis [A. Berezov, A. Iriarte, and M. Martinez-Carrion (1994) J. Biol. Chem. 269, 22222-22229]. Light scattering and electron microscopy analysis indicate that presequence-dependent binding of the precursor leads to extensive vesicle aggregation brought about by their cross-linking through interaction of each of the two presequences of this dimeric protein with separate vesicles. To evaluate the possible contribution of this aggregation to the properties of the bound protein, we analyzed the membrane interaction of a hybrid dimer containing a single presequence peptide. This dimer still binds to vesicles but does not cause aggregation. The properties of the bound hybrid are intermediate between those of the free and bound homo-precursor dimer with only the presequence-carrying subunit showing alterations in its structural and functional properties. These results indicate that the conformational perturbation of the mature moiety of lipid-bound precursor is caused by the direct interaction of each subunit with the membrane through its own N-terminal presequence peptide. AD - School of Biological Sciences, University of Missouri at Kansas City, 64110-2499, USA. PMID- 0008951049 CU - 1997 SO - Arch Biochem Biophys 1996 Dec 1;336(1):173-83 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [163 medline neighbors] [JBC Online] UI - 96027561 AU - Lain B AU - Iriarte A AU - Mattingly JR Jr AU - Moreno JI AU - Martinez-Carrion M TI - Structural features of the precursor to mitochondrial aspartate aminotransferase responsible for binding to hsp70. LA - Eng MH - Amino Acid Sequence MH - Animal MH - Aspartate Transaminase/*chemistry/metabolism MH - Base Sequence MH - Enzyme Precursors/*chemistry/metabolism MH - Heat-Shock Proteins 70/chemistry/*metabolism MH - Mitochondria/*enzymology MH - Molecular Sequence Data MH - Protein Folding MH - Rabbits MH - Support, U.S. Gov't, P.H.S. RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Enzyme Precursors) RN - 0 (Heat-Shock Proteins 70) PT - JOURNAL ARTICLE ID - HL-38412/HL/NHLBI ID - GM-38341/GM/NIGMS DA - 19951121 DP - 1995 Oct 20 IS - 0021-9258 TA - J Biol Chem PG - 24732-9 SB - M SB - X CY - UNITED STATES IP - 42 VI - 270 JC - HIV AA - Author EM - 199601 AB - The precursor (pmAspAT) and mature (mAspAT) forms of mitochondrial aspartate aminotransferase interact with hsp70 very early during translation when synthesized in either rabbit reticulocyte lysate or wheat germ extract (Lain, B., Iriarte, A., and Martinez-Carrion. (1994) J. Biol. Chem. 269, 15588-15596). The nature of the structural elements responsible for recognition and binding of this protein to hsp70 has been studied by examining the folding and potential association with the chaperone of several engineered forms of this enzyme. Whereas pmAspAT and mAspAT bind hsp70 very early during translation, the cytosolic form of this enzyme (cAspAT) does not interact with hsp70. A fusion protein consisting of the mitochondrial presequence peptide attached to the amino terminus of cAspAT associates with hsp70 only after the protein has acquired its native-like conformation, apparently through binding to the presequence exposed on the surface of the folded protein. Deletion of the amino-terminal segment of mAspAT or its replacement with the corresponding domain from the cytosolic isozyme eliminates the cotranslational binding of hsp70 to the mitochondrial protein. We conclude that both the presequence and NH2 We conclude that both the presequence and NH2-terminal region of pmAspAT represent recognition signals for binding of hsp70 to the newly synthesized mitochondrial precursor. Results from competition studies with synthetic peptides support this conclusion. The ability of hsp70 to discriminate between these two highly homologous proteins probably involves the recognition of specific sequence elements in the NH2- terminal portion of the mitochondrial protein and may relate to their separate localization in the cell. A slower folding rate and higher affinity for cytosolic chaperones may represent evolutionary adaptations of translocated mitochondrial proteins to ensure their efficient importation into the organelle. AD - Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City 64110-2499, USA. PMID- 0007559589 CU - 1997 SO - J Biol Chem 1995 Oct 20;270(42):24732-9 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [202 medline neighbors] [JBC Online] UI - 95138101 AU - Mattingly JR Jr AU - Iriarte A AU - Martinez-Carrion M TI - Homologous proteins with different affinities for groEL. The refolding of the aspartate aminotransferase isozymes at varying temperatures. LA - Eng MH - Animal MH - Aspartate Transaminase/*metabolism MH - Cytosol/enzymology MH - GroEL Protein/*metabolism MH - Isoenzymes/*metabolism MH - Mitochondria/enzymology MH - Protein Binding MH - *Protein Folding MH - Rabbits MH - Rats MH - Support, U.S. Gov't, P.H.S. MH - Temperature RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (GroEL Protein) RN - 0 (Isoenzymes) PT - JOURNAL ARTICLE ID - HL-38412/HL/NHLBI ID - GM-38341/GM/NIGMS DA - 19950224 DP - 1995 Jan 20 IS - 0021-9258 TA - J Biol Chem PG - 1138-48 SB - M SB - X CY - UNITED STATES IP - 3 VI - 270 JC - HIV AA - Author EM - 199505 AB - The homologous cytosolic and mitochondrial isozymes of aspartate aminotransferase (c- and mAspAT, respectively) seem to follow very different folding pathways after synthesis in rabbit reticulocyte lysate, suggesting that the nascent proteins interact differently with molecular chaperones (Mattingly, J. R., Jr., Iriarte, A., and Martinez- Carrion, M. (1993) J. Biol. Chem. 268, 26320-26327). In an attempt to discern the structural basis for this phenomenon, we have begun to study the effect of temperature on the refolding of the guanidine hydrochloride-denatured, purified proteins and their interaction with the groEL/groES molecular chaperone system from Escherichia coli. In the absence of chaperones, temperature has a critical effect on the refolding of the two isozymes, with mAspAT being more susceptible than cAspAT to diminishing refolding yields at increasing temperatures. No refolding is observed for mAspAT at physiological temperatures. The molecular chaperones groEL and groES can extend the temperature range over which the AspAT isozymes successfully refold; however, cAspAT can still refold at higher temperatures than mAspAT. In the absence of groES and MgATP, the two isozymes interact differently with groEL, groEL arrests the refolding of mAspAT throughout the temperature range of 0-45 degrees C. Adding only MgATP releases very little mAspAT from groEL; both groES and MgATP are required for significant refolding of mAspAT in the presence of groEL. On the other hand, the extent to which groEL inhibits the refolding of cAspAT depends upon the temperature of the refolding reaction, only slowing the reaction at 0 degrees C but arresting it completely at 30 degrees C. MgATP alone is sufficient to effect the release of cAspAT from groEL at any temperature examined; inclusion of groES along with MgATP has no effect on the refolding yield but does increase the refolding rate at temperatures greater than 15 degrees C. These results demonstrate that groEL can have significantly different affinities for proteins with highly homologous final tertiary and quarternary structures and suggest that dissimilarities in the primary sequence of the protein substrates may control the structure of the folding intermediates captured by groEL and/or the composition of the surfaces through which the folding proteins interact with groEL. AD - Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City 64110-2499. PMID- 0007836372 CU - 1997 SO - J Biol Chem 1995 Jan 20;270(3):1138-48 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [114 medline neighbors] UI - 96108466 AU - Martinez-Carrion M AU - Artigues A AU - Berezov A AU - Bianconi ML AU - Reyes AM AU - Iriarte A TI - Probes for analysis of stability of different variants of aspartate aminotransferase. LA - Eng MH - Acrylamides/pharmacology MH - Amino Acid Sequence MH - Aspartate Transaminase/*chemistry/genetics/metabolism MH - Calorimetry, Differential Scanning MH - Cytoplasm/enzymology MH - Enzyme Stability MH - Fluorescence MH - Guanidines/pharmacology MH - Isoenzymes/chemistry/genetics MH - Kinetics MH - Liposomes/metabolism MH - Mitochondria/enzymology MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed MH - Protein Conformation MH - Protein Denaturation MH - Protein Folding MH - Recombinant Proteins/chemistry MH - Support, U.S. Gov't, P.H.S. MH - Temperature RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Acrylamides) RN - 0 (Guanidines) RN - 0 (Isoenzymes) RN - 0 (Liposomes) RN - 0 (Recombinant Proteins) RN - 113-00-8 (Guanidine) RN - 79-06-1 (acrylamide) PT - JOURNAL ARTICLE ID - GM-38341/GM/NIGMS DA - 19960205 DP - 1995 IS - 0076-6879 TA - Methods Enzymol PG - 590-608 SB - M CY - UNITED STATES VI - 259 JC - MVA EM - 199604 AD - School of Biological Sciences, University of Missouri-Kansas City 64110, USA. PMID- 0008538474 CU - 1997 SO - Methods Enzymol 1995;259:590-608 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [122 medline neighbors] UI - 94350976 AU - Berezov A AU - Iriarte A AU - Martinez-Carrion M TI - Binding to phospholipid vesicles impairs substrate-mediated conformational changes of the precursor to mitochondrial aspartate aminotransferase. LA - Eng MH - Animal MH - Aspartate Transaminase/chemistry/*metabolism MH - Binding Sites MH - Dithionitrobenzoic Acid MH - Enzyme Precursors/chemistry/*metabolism MH - Fluorescent Dyes MH - Liposomes MH - Mitochondria, Liver/*enzymology MH - Phospholipids/*metabolism MH - Protein Conformation MH - Rats MH - Spectrometry, Fluorescence MH - Support, U.S. Gov't, P.H.S. RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Enzyme Precursors) RN - 0 (Fluorescent Dyes) RN - 0 (Liposomes) RN - 0 (Phospholipids) RN - 69-78-3 (Dithionitrobenzoic Acid) PT - JOURNAL ARTICLE ID - GM-38341/GM/NIGMS DA - 19940929 DP - 1994 Sep 2 IS - 0021-9258 TA - J Biol Chem PG - 22222-9 SB - M SB - X CY - UNITED STATES IP - 35 VI - 269 JC - HIV AA - Author EM - 199412 AB - Specific labeling of both the mature (mAspAT) and precursor (pmAspAT) forms of rat liver mitochondrial aspartate aminotransferase with three different spectroscopic probes (monobromotrimethylammoniobimane, N- (iodoacetylaminoethyl)-5-naphthalene-1-sulfonic acid, and N-(1- pyrenyl)maleimide) was used to assess the possible conformational consequences of the interaction of a mitochondrial precursor protein with lipid membranes by means of fluorescence spectroscopy. The three probes react with the same cysteine residue causing a partial loss of catalytic activity whose extent depends on the nature of the probe introduced. The fluorescence intensity of the attached probes decreases upon addition of substrates or substrate analogues, indicating that the modified enzymes can undergo the open-closed conformational transitions that accompany catalysis. Both unmodified and labeled precursor proteins bind to negatively charged phospholipid vesicles, whereas the mature enzyme is unable to bind. Binding to liposomes does not affect the fluorescent properties of the attached probes. However, addition of the pseudosubstrate alpha-methylaspartate to liposome-bound precursor fails to induce the characteristic conformational changes observed with the protein free in solution. Furthermore, upon binding to liposomes the precursor protein loses enzymatic activity, and the reactive cysteine residue becomes inaccessible to reaction with thiol reagents. In contrast, the presence of liposomes has no effect on the activity, cysteine reactivity, or syncatalytic conformational transitions of the mature enzyme. It appears that interaction of pmAspAT with negatively charged phospholipids prevents the protein from undergoing the conformational transitions required for catalysis, "freezing" the enzyme in a sterically hindered but open-like conformation. AD - Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City 64110-2499. PMID- 0008071348 CU - 1997 SO - J Biol Chem 1994 Sep 2;269(35):22222-9 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [258 medline neighbors] UI - 94350941 AU - Artigues A AU - Iriarte A AU - Martinez-Carrion M TI - Acid-induced reversible unfolding of mitochondrial aspartate aminotransferase. LA - Eng MH - Animal MH - Aspartate Transaminase/*metabolism MH - Binding Sites MH - Circular Dichroism MH - Coenzymes/metabolism MH - Hydrogen-Ion Concentration MH - Mitochondria/*enzymology MH - Potassium Chloride MH - Protein Denaturation MH - *Protein Folding MH - Protein Structure, Secondary MH - Spectroscopy, Fourier Transform Infrared MH - Support, U.S. Gov't, P.H.S. MH - Ultracentrifugation RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Coenzymes) RN - 7447-40-7 (Potassium Chloride) PT - JOURNAL ARTICLE ID - GM-38341/GM/NIGMS ID - GM-38184/GM/NIGMS DA - 19940929 DP - 1994 Sep 2 IS - 0021-9258 TA - J Biol Chem PG - 21990-9 SB - M SB - X CY - UNITED STATES IP - 35 VI - 269 JC - HIV AA - Author EM - 199412 AB - The acid-induced reversible unfolding of several forms of the mitochondrial isoenzyme of mammalian aspartate aminotransferase, including its precursor form, has been characterized under equilibrium conditions. A minimum of two transitions can be detected for the holoenzyme (pyridoxal form). One transition takes place at pH 3.6 and corresponds to the monomerization of the dimeric protein. The second transition is centered at pH 3.3 and represents the disappearance of much of the tertiary and secondary structures. The presequence peptide in the precursor protein does not affect the equilibria nor the rate of unfolding in the pH range from 7.5 to 2.0. The presence of the cofactor, pyridoxal 5'-phosphate, stabilizes the protein against acid denaturation. At pH 2.0, the protein retains significant amounts of secondary structure (26% alpha-helix, 20% beta-structure). Increasing the ionic strength at pH 2.0 results in significant changes in the secondary structure of the unfolded protein that acquires some of the characteristics ascribed to a compact molten globule. According to the circular dichroism spectra these changes are characterized by an increase in beta-structure, although Fourier transform infrared spectroscopy analysis indicates that this increase in beta-structure is due mostly to the formation of intermolecular beta-sheet as a consequence of protein aggregation. The formation of high molecular weight aggregates has been confirmed by analytical ultracentrifugation. Following neutralization of the acid-unfolded state at low ionic strength both mature and precursor proteins refold to their native active state (> 80% yield). By contrast the compact state present at pH 2.0 and high ionic strength is unable to recover its activity following neutralization. Thus, this compact state does not appear to represent an intermediate in the folding pathway of the protein, but rather a dead end product of aggregation, which may reflect the intrinsic tendencies of the unfolded protein to oligomerize at intracellular salt concentrations unless controlled by factors such as chaperones present in the cellular environment. AD - Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City 64110-2499. PMID- 0008071319 CU - 1997 SO - J Biol Chem 1994 Sep 2;269(35):21990-9 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [185 medline neighbors] UI - 94267208 AU - Mihovilovic M AU - Donnelly-Roberts D AU - Richman DP AU - Martinez-Carrion M TI - Pathogenesis of hyperacute experimental autoimmune myasthenia gravis. Acetylcholine receptor/cholinergic site/receptor function/autoimmunity. LA - Eng MH - Animal MH - Antibodies, Monoclonal/metabolism/pharmacology MH - Autoimmunity MH - Bungarotoxins/metabolism/pharmacology MH - Carbachol/metabolism/pharmacology MH - Disease Models, Animal MH - Human MH - In Vitro MH - Myasthenia Gravis/*etiology/immunology/metabolism MH - Rats MH - Receptors, Cholinergic/antagonists & inhibitors/*immunology/metabolism MH - Support, Non-U.S. Gov't MH - Support, U.S. Gov't, Non-P.H.S. MH - Support, U.S. Gov't, P.H.S. RN - 0 (Antibodies, Monoclonal) RN - 0 (Bungarotoxins) RN - 0 (Receptors, Cholinergic) RN - 51-83-2 (Carbachol) PT - JOURNAL ARTICLE ID - NS15462/NS/NINDS ID - NS19779/NS/NINDS ID - NS07113/NS/NINDS ID - + DA - 19940713 DP - 1994 Jun 15 IS - 0022-1767 TA - J Immunol PG - 5997-6002 SB - A SB - M SB - X CY - UNITED STATES IP - 12 VI - 152 JC - IFB AA - Author EM - 199409 AB - Three mAbs, mAbs 249E, 370, and 383C, directed against the alpha- bungarotoxin (alpha BgTx) binding site of the acetylcholine receptor (AChR) induce a hyperacute form of experimental autoimmune myasthenia gravis (EAMG), characterized by death within hours of mAb injection. To analyze the mechanisms of this effect, purified AChR-mAb complexes were investigated for their ability to bind the cholinergic agonist carbamoylcholine and to undergo agonist-induced activation of the cholinergic ionophore. The three mAbs inhibited carbamylcholine binding, and, conversely, their binding to AChR was inhibited by carbamylcholine. All three completely inhibited carbamylcholine-induced T1+ influxes to AChR-rich vesicles. These data indicate that the severe hyperacute EAMG induced by these mAbs results from blockage of AChR function and that the role of such potent Abs (even if present in small amounts) in the pathogenesis of human myasthenia gravis deserves further investigation. AD - Department of Medicine, Duke University, Durham, NC 27710. PMID- 0008207224 SO - J Immunol 1994 Jun 15;152(12):5997-6002 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [111 medline neighbors] UI - 94253142 AU - Lain B AU - Iriarte A AU - Martinez-Carrion M TI - Dependence of the folding and import of the precursor to mitochondrial aspartate aminotransferase on the nature of the cell-free translation system. LA - Eng MH - Animal MH - Aspartate Transaminase/*biosynthesis MH - Cell-Free System MH - Comparative Study MH - Cytosol/metabolism MH - Enzyme Precursors/*chemistry/*metabolism MH - Ethylmaleimide/pharmacology MH - Heat-Shock Proteins/metabolism MH - Kinetics MH - Mitochondria/*enzymology MH - Peptide Mapping MH - *Protein Folding MH - Rabbits MH - Recombinant Proteins/biosynthesis/chemistry/metabolism MH - Reticulocytes/*metabolism MH - Seeds/metabolism MH - Support, Non-U.S. Gov't MH - Support, U.S. Gov't, P.H.S. MH - *Translation, Genetic MH - Trypsin MH - Wheat/metabolism RN - EC 2.6.1.1 (Aspartate Transaminase) RN - EC 3.4.21.4 (Trypsin) RN - 0 (Enzyme Precursors) RN - 0 (Heat-Shock Proteins) RN - 0 (Recombinant Proteins) RN - 128-53-0 (Ethylmaleimide) PT - JOURNAL ARTICLE ID - GM-38341/GM/NIGMS ID - GM-38184/GM/NIGMS DA - 19940630 DP - 1994 Jun 3 IS - 0021-9258 TA - J Biol Chem PG - 15588-96 SB - M SB - X CY - UNITED STATES IP - 22 VI - 269 JC - HIV AA - Author EM - 199409 AB - The precursor to mitochondrial aspartate amino-transferase (pmAspAT), when newly synthesized in vitro using either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE), is highly susceptible to proteolysis. Treatment of these translation products with trypsin generates a characteristic pattern of proteolytic fragments which differs between WGE and RRL. pmAspAT synthesized in RRL acquires over time the trypsin resistance characteristic of purified recombinant pmAspAT in a process that reflects folding of the nascent protein in a cytosolic-like environment and results in the loss of its ability to be imported into mitochondria (Mattingly, J.R., Jr., Youssef, J., Iriarte, A., and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 3925-3937). Folding in RRL is temperature-dependent. By contrast, pmAspAT synthesized in WGE does not acquire trypsin resistance at any temperature and is unable to be imported into isolated mitochondria. Yet, addition of RRL to the protein synthesized in WGE restores both the folding and import processes. This effect is temperature and N- ethylmaleimide-sensitive and requires ATP. We have also detected that newly synthesized pmAspAT, in both the mammalian (RRL) and plant (WGE) environments, associates at an early stage, possibly cotranslationally, with cytosolic hsp70. This complex is transient and short-lived in RRL but is stable for protein synthesized in WGE. The mature form of the protein is also found associated with hsp70 early on during its synthesis. Yet, in RRL, it dissociates from the complex more easily than its precursor form. This suggests that the interaction between the precursor and hsp70 is at least partially dependent on the signal peptide. The dissociation of the precursor/hsp70 complex seems to precede ATP-requiring subsequent folding steps. Furthermore, release of precursor from this complex, either to be imported into mitochondria or to resume folding when in the absence of mitochondria, appears to be influenced by other factor(s) present in RRL but absent or inhibited in WGE. AD - Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City 64110-2499. PMID- 0008195205 CU - 1997 SO - J Biol Chem 1994 Jun 3;269(22):15588-96 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [116 medline neighbors] UI - 94280523 AU - Perez-Ramirez B AU - Iriarte A AU - Martinez-Carrion M TI - Residues 377-389 from the delta subunit of Torpedo californica acetylcholine receptor are located in the cytoplasmic surface. LA - Eng MH - Amino Acid Sequence MH - Animal MH - Binding Sites MH - Cell Membrane/*metabolism/ultrastructure MH - Chromatography, High Pressure Liquid MH - Kinetics MH - Macromolecular Systems MH - Models, Structural MH - Molecular Sequence Data MH - Peptide Fragments/*analysis MH - *Protein Structure, Secondary MH - Pyridoxal Phosphate/metabolism MH - Receptors, Nicotinic/*chemistry/metabolism MH - Spectrometry, Fluorescence MH - Support, U.S. Gov't, P.H.S. MH - Thermodynamics MH - Torpedo MH - Trypsin RN - EC 3.4.21.4 (Trypsin) RN - 0 (Macromolecular Systems) RN - 0 (Peptide Fragments) RN - 0 (Receptors, Nicotinic) RN - 54-47-7 (Pyridoxal Phosphate) PT - JOURNAL ARTICLE ID - GM-38341/GM/NIGMS DA - 19940726 DP - 1994 Jan IS - 0277-8033 TA - J Protein Chem PG - 67-76 SB - M CY - UNITED STATES IP - 1 VI - 13 JC - AEJ AA - Author EM - 199409 AB - Torpedo californica acetylcholine receptor (AcChR) enriched, sealed vesicles have been specifically labeled on the cytoplasmic surface with pyridoxal 5'-phosphate (Perez-Ramirez, B., and Martinez-Carrion, M., 1989, Biochemistry 28, 5034-5040). After chromatography of the peptide fragments produced by trypin digestion of labeled AcChR, several fractions containing the phosphopyridoxyl label were obtained. Edman degradation identified one of the fractions, with sequence SRSELMFEKQSER, as corresponding to residues 377-389 in the delta subunit (primary structure). The latter must be a cytoplasmic region of this transmembranous protein, and residue delta K385 must reside in a water-soluble exposed domain of the cytosolic side of the membrane. Introduction of phosphopyridoxyl residues allows for their potential use as probes of conformational changes in the cytosolic surface of the receptor molecule. AD - Division of Cell Biology and Biphysics, School of Biological Sciences, University of Missouri-Kansas City, Missouri 64110. PMID- 0008011073 SO - J Protein Chem 1994 Jan;13(1):67-76 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [104 medline neighbors] UI - 94075315 AU - Mattingly JR Jr AU - Iriarte A AU - Martinez-Carrion M TI - Structural features which control folding of homologous proteins in cell-free translation systems. The effect of a mitochondrial-targeting presequence on aspartate aminotransferase. LA - Eng MH - Amino Acid Sequence MH - Animal MH - Aspartate Transaminase/genetics/*metabolism MH - Base Sequence MH - Cell-Free System MH - Enzyme Precursors/chemistry/genetics MH - Isoenzymes/genetics/*metabolism MH - Mitochondria/*enzymology MH - Molecular Sequence Data MH - *Protein Folding MH - Rabbits MH - Reticulocytes/enzymology MH - Support, U.S. Gov't, P.H.S. MH - *Translation, Genetic MH - Trypsin/metabolism MH - Wheat/metabolism RN - EC 2.6.1.1 (Aspartate Transaminase) RN - EC 3.4.21.4 (Trypsin) RN - 0 (Enzyme Precursors) RN - 0 (Isoenzymes) PT - JOURNAL ARTICLE ID - HL-38412/HL/NHLBI ID - GM-38184/GM/NIGMS DA - 19940113 DP - 1993 Dec 15 IS - 0021-9258 TA - J Biol Chem PG - 26320-7 SB - M SB - X CY - UNITED STATES IP - 35 VI - 268 JC - HIV AA - Author EM - 199403 AB - When the precursor to mitochondrial aspartate aminotransferase (pmAspAT) is synthesized in a rabbit reticulocyte lysate translation system (RRL), its properties are quite unlike those of the purified protein (Mattingly, J.R., Jr., Youssef, J., Iriarte, A., and Martinez- Carrion, M. (1993) J. Biol. Chem. 268, 3925-3937). These results suggest that molecular chaperones present in RRL modulate the folding of pmAspAT. To investigate the structural basis for this, we have used protease resistance to monitor the extent of folding for several related AspATs after synthesis in RRL and in wheat germ extract (WGE). In addition to pmAspAT, the following proteins were examined: the mature form of pmAspAT (delta 2-28 pmAspAT), its cytosolic counterpart (cAspAT), a chimeric protein consisting of the presequence of pmAspAT attached to the amino terminus of cAspAT (pcAspAT), and a pmAspAT variant in which the presequence and the amino-terminal domain of the mature enzyme are deleted (delta 2-57 pmAspAT). In RRL, delta 2-28 pmAspAT folds somewhat faster than intact pmAspAT, whereas the truncated delta 2-57 pmAspAT is unable to fold. In contrast, cAspAT and pcAspAT both fold with extreme rapidity. After synthesis in WGE, pmAspAT and delta 2-28 pmAspAT never acquire a protease-resistant conformation, whereas the folding of cAspAT and pcAspAT still occurs rapidly. We conclude that the presequence has only a minor role in determining the folding rate of the pmAspAT mitochondrial precursor protein in RRL or WGE and has no influence on the folding of the homologous cAspAT. Rather, the primary sequence of the mature part of the protein seems to dictate whether or how molecular chaperones regulate folding events. AD - Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City 64110-2499. PMID- 0008253754 CU - 1997 SO - J Biol Chem 1993 Dec 15;268(35):26320-7 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [128 medline neighbors] UI - 94042973 AU - Reyes AM AU - Iriarte A AU - Martinez-Carrion M TI - Refolding of the precursor and mature forms of mitochondrial aspartate aminotransferase after guanidine hydrochloride denaturation [published erratum appears in J Biol Chem 1994 Feb 18;269(7):5480] LA - Eng MH - Apoenzymes/chemistry/metabolism MH - Aspartate Transaminase/*chemistry/metabolism MH - Comparative Study MH - Enzyme Precursors/*chemistry/metabolism MH - Guanidines/*pharmacology MH - Isoenzymes/chemistry/metabolism MH - Kinetics MH - Macromolecular Systems MH - Mathematics MH - Mitochondria/*enzymology MH - Protein Conformation MH - Protein Denaturation MH - *Protein Folding MH - Pyridoxal Phosphate/pharmacology MH - Spectrometry, Fluorescence MH - Support, U.S. Gov't, P.H.S. RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Apoenzymes) RN - 0 (Enzyme Precursors) RN - 0 (Guanidines) RN - 0 (Isoenzymes) RN - 0 (Macromolecular Systems) RN - 113-00-8 (Guanidine) RN - 54-47-7 (Pyridoxal Phosphate) PT - JOURNAL ARTICLE ID - GM-38184/GM/NIGMS ID - HL-38412/HL/NHLBI DA - 19931201 DP - 1993 Oct 25 IS - 0021-9258 TA - J Biol Chem PG - 22281-91 SB - X SB - M CY - UNITED STATES IP - 30 VI - 268 JC - HIV AA - Author EM - 199402 AB - The mitochondrial isozyme of aspartate aminotransferase (mAspAT), a dimeric pyridoxal phosphate (PLP)-dependent enzyme, is encoded by the nuclear genome and synthesized in the cytoplasm as a precursor protein (pmAspAT) containing a 29-residue amino-terminal signal peptide which is essential for its targeting and import into mitochondria. In the cytosolic-like environment of rabbit reticulocyte lysate, newly synthesized rat liver pmAspAT has been found to slowly fold and bind PLP (Mattingly, J. R., Jr., Youssef, J., Iriarte, A. and Martinez- Carrion, M. (1993) J. Biol. Chem. 268, 3925-3937). On the other hand, isolated mammalian (pig) mAspAT, when denatured with guanidine hydrochloride, seems unable to refold to a catalytically active state (West, S. M., and Price, N. C. (1990) Biochem. J. 265, 45-50). With the availability of rat liver recombinant precursor and mature forms of mAspAT as homogeneous, stable preparations, an assessment of the influence of the signal peptide on the in vitro refolding of this protein can be made. Following unfolding induced by guanidine hydrochloride, we have investigated the refolding process of this complex, dimeric coenzyme-dependent protein system by activity, fluorescence, and circular dichroism. Both mAspAT and pmAspAT can be efficiently renatured after rapid dilution of the denaturing agent at low protein concentrations. The equilibrium unfolding/refolding transitions and the kinetics of folding are protein concentration- independent and identical for both protein forms. Binding of coenzyme into the active site pocket seems to occur at a late step in the folding process of both mAspAT and pmAspAT, suggesting that in these proteins the coenzyme does not direct the folding of the polypeptide chain. These results indicate that the in vitro refolding of mAspAT is not regulated or influenced by the presence of the amino-terminal signal peptide. On the other hand, in vitro refolding in buffer is significantly faster than the folding of newly synthesized precursor protein in reticulocyte lysate examined in our previous report (reference above), pointing at the likely influence of cytosolic factors in modulating folding in the cell. AD - Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City 64110-2499. RO - M:MWS PMID- 0008226737 LR - 19940405 CU - 1997 SO - J Biol Chem 1993 Oct 25;268(30):22281-91 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [107 medline neighbors] UI - 93179389 AU - Mattingly JR Jr AU - Youssef J AU - Iriarte A AU - Martinez-Carrion M TI - Protein folding in a cell-free translation system. The fate of the precursor to mitochondrial aspartate aminotransferase. LA - Eng MH - Adenosine Triphosphate/chemistry MH - Animal MH - Aspartate Transaminase/*chemistry/genetics MH - Biological Transport MH - Cell-Free System MH - Coenzymes/metabolism MH - Detergents MH - Edetic Acid/chemistry MH - Enzyme Precursors/*chemistry/genetics MH - Escherichia coli/genetics MH - Ethylmaleimide/chemistry MH - Mitochondria/*enzymology MH - Polyethylene Glycols MH - *Protein Folding MH - Rabbits MH - Rats MH - RNA, Messenger/metabolism MH - Support, U.S. Gov't, P.H.S. MH - *Translation, Genetic MH - Trypsin/metabolism RN - EC 2.6.1.1 (Aspartate Transaminase) RN - EC 3.4.21.4 (Trypsin) RN - 0 (Coenzymes) RN - 0 (Detergents) RN - 0 (Enzyme Precursors) RN - 0 (Polyethylene Glycols) RN - 0 (RNA, Messenger) RN - 128-53-0 (Ethylmaleimide) RN - 56-65-5 (Adenosine Triphosphate) RN - 60-00-4 (Edetic Acid) RN - 9002-93-1 (Octoxynol) PT - JOURNAL ARTICLE ID - HL-22265/HL/NHLBI ID - GM-38184/GM/NIGMS DA - 19930326 DP - 1993 Feb 25 IS - 0021-9258 TA - J Biol Chem PG - 3925-37 SB - M SB - X CY - UNITED STATES IP - 6 VI - 268 JC - HIV AA - Author EM - 199306 AB - The precursor to rat mitochondrial aspartate aminotransferase (pmAspAT) can be expressed in and purified from Escherichia coli as a fully active enzyme with remarkable trypsin resistance. Only two sites within the presequence are readily hydrolyzed (Martinez-Carrion, M., Altieri, F., Iriarte, A., Mattingly, J. R., Youssef, J., and Wu, T. (1990) Ann. N.Y. Acad. Sci. 585, 346-356). In contrast, pmAspAT freshly synthesized in rabbit reticulocyte lysate is significantly less resistant to proteolysis and is completely digested by trypsin. Extended incubation of the pmAspAT translation product slowly converts it to a species with qualitatively the same trypsin resistance as the purified pmAspAT. In addition, this species binds pyridoxal 5'-phosphate, exhibits catalytic activity, and loses its ability to be imported into mitochondria. This process appears to reflect protein folding. The rate of folding is unaffected by the addition of cofactor or the depletion of endogenous cofactor and is not significantly affected by the concentration of translation product in the reaction. Agents that decrease the availability of ATP partially inhibit the folding, whereas the sulfhydryl alkylating reagent N-ethylmaleimide and the detergent Triton X-100 completely prevent the conversion. Although the folding of pmAspAT in reticulocyte lysate is slow, folding is rapid once the translation product is sequestered within the mitochondria as the mature form of the enzyme. These results are presented as a model for the in vivo folding of pyridoxal-dependent, oligomeric mitochondrial precursors in the presence of cytoplasmic components and for the fate of true mitochondrial precursor proteins when not imported. AD - Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City 64110-2499. PMID- 0008440686 CU - 1997 SO - J Biol Chem 1993 Feb 25;268(6):3925-37 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [204 medline neighbors] UI - 92190152 AU - Martinez-Liarte JH AU - Iriarte A AU - Martinez-Carrion M TI - Inorganic phosphate binding and electrostatic effects in the active center of aspartate aminotransferase apoenzyme. LA - Eng MH - Animal MH - Anions MH - Apoenzymes/*metabolism MH - Aspartate Transaminase/*metabolism MH - Binding Sites MH - Electricity MH - Enzyme Stability MH - Fourier Analysis MH - Heat MH - Hydrogen-Ion Concentration MH - Myocardium/enzymology MH - Phosphates/*metabolism MH - Pyridoxal Phosphate/metabolism MH - Pyridoxamine/metabolism MH - Spectrophotometry, Infrared MH - Support, U.S. Gov't, P.H.S. MH - Swine RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Anions) RN - 0 (Apoenzymes) RN - 0 (Phosphates) RN - 54-47-7 (Pyridoxal Phosphate) RN - 85-87-0 (Pyridoxamine) PT - JOURNAL ARTICLE ID - GM-38341/GM/NIGMS ID - HL-38412/HL/NHLBI DA - 19920422 DP - 1992 Mar 17 IS - 0006-2960 TA - Biochemistry PG - 2712-9 SB - M CY - UNITED STATES IP - 10 VI - 31 JC - A0G AA - Author EM - 199206 AB - The ionization state of the phosphate group bound at the aspartate aminotransferase apoenzyme's active site has been investigated utilizing Fourier-transform infrared spectroscopy following the band corresponding to the symmetric stretching of the dianionic phosphate. Unlike free phosphate, when inorganic phosphate is bound at the enzyme's active site, the integrated intensity value of the dianionic band does not change with pH within the studied range, and this value is similar to that for free dianionic phosphate at pH 8.3. From these results, we propose a dianionic state for the phosphate ion bound to cytosolic aspartate aminotransferase throughout the pH range of 5.7- 8.3. The presence of other anions such as acetate and chloride or the substrate aspartate and its analogues produces a pH-dependent phosphate removal from the active site which is favored at low pH values. Elimination of the charged primary amine at the active-site Lys-258, through formation of a Schiff base with pyridoxal or chemical modification by carbamylation, also produces a pH-independent phosphate release. These results are interpreted as Lys-258 together with the active-site alpha-helix and other residues may be involved in stabilizing phosphate as a dianion in the apoenzyme phosphate pocket which anchors the phosphate ester of pyridoxal phosphate in the holoenzyme. It is proposed that the dianionic phosphate contributes to the apoenzyme's thermal stability through formation of strong hydrogen bond and salt bridges with the amino acid residues forming the phosphate binding pocket with assistance of Lys-258, and other active- site cationic components. AD - School of Basic Life Sciences, University of Missouri-Kansas City 64110- 2499. PMID- 0001547211 CU - 1997 SO - Biochemistry 1992 Mar 17;31(10):2712-9 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [2 protein links] [205 medline neighbors] UI - 92201207 AU - Arnone MI AU - Birolo L AU - Giamberini M AU - Cubellis MV AU - Nitti G AU - Sannia G AU - Marino G TI - Limited proteolysis as a probe of conformational changes in aspartate aminotransferase from Sulfolobus solfataricus. LA - Eng MH - Amino Acid Sequence MH - Animal MH - Aspartate Transaminase/antagonists & inhibitors/*chemistry/metabolism MH - Comparative Study MH - Cytosol/enzymology MH - Electrophoresis, Polyacrylamide Gel MH - Enzyme Inhibitors MH - Molecular Sequence Data MH - Myocardium/enzymology MH - Peptide Hydrolases/*chemistry/metabolism MH - Protein Conformation MH - Structure-Activity Relationship MH - Sulfolobus/*enzymology MH - Support, Non-U.S. Gov't MH - Swine MH - Temperature MH - Thermolysin/pharmacology RN - EC 2.6.1.1 (Aspartate Transaminase) RN - EC 3.4 (Peptide Hydrolases) RN - EC 3.4.24.- (Thermolysin) RN - 0 (Enzyme Inhibitors) PT - JOURNAL ARTICLE DA - 19920424 DP - 1992 Mar 15 IS - 0014-2956 TA - Eur J Biochem PG - 1183-9 SB - M SB - X CY - GERMANY IP - 3 VI - 204 JC - EMZ AA - Author EM - 199207 AB - The analysis of conformational transitions using limited proteolysis was carried out on a hyperthermophilic aspartate aminotransferase isolated from the archaebacterium Sulfolobus solfataricus, in comparison with pig cytosolic aspartate aminotransferase, a thoroughly studied mesophilic aminotransferase which shares about 15% similarity with the archaebacterial protein. Aspartate aminotransferase from S. solfataricus is cleaved at residue 28 by thermolysin and residues 32 and 33 by trypsin; analogously, pig heart cytosolic aspartate aminotransferase is cleaved at residues 19 and 25 [Iriarte, A., Hubert, E., Kraft, K. & Martinez-Carrion, M. (1984) J. Biol. Chem. 259, 723- 728] by trypsin. In the case of aspartate aminotransferase from S. solfataricus, proteolytic cleavages also result in transaminase inactivation thus indicating that both enzymes, although evolutionarily distinct, possess a region involved in catalysis and well exposed to proteases which is similarly positioned in their primary structure. It has been reported that the binding of substrates induces a conformational transition in aspartate aminotransferases and protects the enzymes against proteolysis [Gehring, H. (1985) in Transaminases (Christen, P. & Metzler, D. E., eds) pp. 323-326, John Wiley & Sons, New York]. Aspartate aminotransferase from S. solfataricus is protected against proteolysis by substrates, but only at high temperatures (greater than 60 degrees C). To explain this behaviour, the kinetics of inactivation caused by thermolysin were measured in the temperature range 25-75 degrees C. The Arrhenius plot of the proteolytic kinetic constants measured in the absence of substrates is not rectilinear, while the same plot of the constants measured in the presence of substrates is a straight line. Limited proteolysis experiments suggest that aspartate aminotransferase from S. solfataricus undergoes a conformational transition induced by the binding of substrates. Another conformational transition which depends on temperature and occurs in the absence of substrates could explain the non-linear Arrhenius plot of the proteolytic kinetic constants. The latter conformational transition might also be related to the functioning of the archaebacterial aminotransferase since the Arrhenius plot of kcat is non-linear as well. AD - Dipartimento di Chimica Organica e Biologica, Universita di Napoli, Italy. PMID- 0001551394 CU - 1997 SO - Eur J Biochem 1992 Mar 15;204(3):1183-9 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [205 medline neighbors] UI - 91378308 AU - Sanchez-Ruiz JM AU - Iriarte A AU - Martinez-Carrion M TI - The ionization states of the 5'-phosphate group in the various coenzyme forms bound to mitochondrial aspartate aminotransferase. LA - Eng MH - Animal MH - Aspartate Transaminase/*metabolism MH - Fourier Analysis MH - Kinetics MH - Mitochondria, Heart/*enzymology MH - Oxidation-Reduction MH - Pyridoxal Phosphate/chemistry/*metabolism MH - Pyridoxamine/*analogs & derivatives/chemistry/metabolism MH - Solutions MH - Spectrophotometry, Infrared/methods MH - Support, Non-U.S. Gov't MH - Support, U.S. Gov't, Non-P.H.S. MH - Support, U.S. Gov't, P.H.S. MH - Swine RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Solutions) RN - 529-96-4 (pyridoxamine phosphate) RN - 54-47-7 (Pyridoxal Phosphate) RN - 85-87-0 (Pyridoxamine) PT - JOURNAL ARTICLE ID - HL-38412/HL/NHLBI ID - GM-38184/GM/NIGMS DA - 19911024 DP - 1991 Apr IS - 0003-9861 TA - Arch Biochem Biophys PG - 38-45 SB - M SB - X CY - UNITED STATES IP - 1 VI - 286 JC - 6SK AA - Author EM - 199112 AB - We have carried out a Fourier transform infrared spectroscopic study of mitochondrial aspartate aminotransferase in the spectral region where phosphate monoesters give rise to absorption. Infrared spectra in the above-mentioned region are dominated by protein absorption. Yet, below 1020 cm-1 protein interferences are minor, permitting the detection of the band arising from the symmetric stretching of dianionic phosphate monoesters [T. Shimanouchi, M. Tsuboi, and Y. Kyogoku (1964) Adv. Chem. Phys. 8, 435-498]. The integrated intensity of this band in several enzyme forms (pyridoxal phosphate, pyridoxamine phosphate, and sodium borohydride-reduced, pyridoxyl phosphate form) does not change with pH in the range 5-9. This behavior contrasts that of free pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP) in solution, where the dependence of the same infrared band intensity with pH can be correlated to the known pK values for the 5'-phosphate ester in solution. The integrated intensity value of this infrared band for the PLP enzyme form before and after reduction with sodium borohydride is close to that given by free PLP at pH 8-9. These results are taken as evidence that in the active site of mitochondrial aspartate aminotransferase the 5'-phosphate group of PLP remains mostly dianionic even at a pH near 5. Thus, it is suggested that the chemical shift changes associated with pH titrations of various PLP forms reported in a previous 31P NMR study of this enzyme [M. E. Mattingly, J. R. Mattingly, and M. Martinez-Carrion (1982) J. Biol. Chem. 257, 8872] are due to the fact that the phosphorus chemical shift senses the O-P-O bond distortions induced by the ionization of a nearby residue. Since no chemical shift changes were observed in pH titrations of the PMP forms (lacking an ionizable internal aldimine) of this isozyme, the Schiff base between PLP and Lys-258 at the active site is the most likely candidate for the ionizing group influencing the phosphorus chemical shift in this enzyme. AD - Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri-Kansas City 64110-2499. PMID- 0001897957 CU - 1997 SO - Arch Biochem Biophys 1991 Apr;286(1):38-45 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [201 medline neighbors] UI - 91158805 AU - Garcia-Borron JC AU - Chinchetru MA AU - Martinez-Carrion M TI - Selective labeling of alpha-bungarotoxin with fluorescein isothiocyanate and its use for the study of toxin-acetylcholine receptor interactions. LA - Eng MH - Animal MH - Binding Sites MH - Binding, Competitive MH - Bungarotoxins/isolation & purification/*metabolism/pharmacology MH - Comparative Study MH - Electric Organ MH - *Fluoresceins MH - *Fluorescent Dyes MH - Kinetics MH - Protein Binding MH - Receptors, Cholinergic/analysis/drug effects/*metabolism MH - Spectrometry, Fluorescence MH - Support, U.S. Gov't, P.H.S. MH - *Thiocyanates MH - Torpedo RN - 0 (Bungarotoxins) RN - 0 (Fluoresceins) RN - 0 (Fluorescent Dyes) RN - 0 (Receptors, Cholinergic) RN - 0 (Thiocyanates) RN - 3326-32-7 (Fluorescein-5-isothiocyanate) PT - JOURNAL ARTICLE ID - GM-38341/GM/NIGMS ID - NS24304/NS/NINDS DA - 19910415 DP - 1990 Dec IS - 0277-8033 TA - J Protein Chem PG - 683-93 SB - M CY - UNITED STATES IP - 6 VI - 9 JC - AEJ AA - Author EM - 199106 AB - The main product of the reaction of fluorescein isothiocyanate (FITC) and bungarotoxin (Bgt) under near stoichiometric conditions is a monofluorescein derivative preferentially labeled at Lys 26, a highly conserved residue known to be involved in the binding (McDaniel, C.S., Manshouri, T., and Atassi, M.Z. (1987) J. Prot. Chem. 6, 455-461; Garcia-Borron, J.C., Bieber, A.L., and Martinez-Carrion, M. (1987) Biochemistry 26, 4295-4303) of postsynaptic neurotoxins specific for the nicotinic acetylcholine receptor (AcChR). The fluorescently labeled toxin retains a high affinity for the AcChR, and an unaltered specificity. Binding of FITC-Bgt to AcChR results in a significant decrease in the fluorescence intensity of the probe. This AcChR- mediated quenching of FITC-Bgt fluorescence allows for a continuous monitoring of the binding process. The quenching of free and bound FITC- Bgt by charged and neutral quenchers shows few fluorophore accessibility changes as induced by the toxin-bound state. The results are consistent with a model in which the positively charged concave surface of the toxin interacts with a negatively charged complementary surface in the receptor molecule. AD - Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, Kansas City 64110-2499. PMID- 0002127357 CU - 1992 SO - J Protein Chem 1990 Dec;9(6):683-93 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [199 medline neighbors] UI - 90290050 AU - Martinez-Carrion M AU - Altieri F AU - Iriarte AJ AU - Mattingly J AU - Youssef J AU - Wu TH TI - Precursor forms of mitochondrial aspartate transaminase. LA - Eng MH - Amino Acid Sequence MH - Animal MH - Aspartate Transaminase/*metabolism MH - Biological Transport MH - Chemistry, Physical MH - Comparative Study MH - Electrophoresis, Polyacrylamide Gel MH - Intracellular Membranes/enzymology MH - Mitochondria, Liver/enzymology MH - Molecular Sequence Data MH - Peptide Fragments/metabolism MH - Peptide Hydrolases/metabolism MH - Protein Precursors/*metabolism MH - Rats MH - Support, U.S. Gov't, P.H.S. MH - Translation, Genetic RN - EC 2.6.1.1 (Aspartate Transaminase) RN - EC 3.4 (Peptide Hydrolases) RN - 0 (Peptide Fragments) RN - 0 (Protein Precursors) PT - JOURNAL ARTICLE ID - GM-38341/GM/NIGMS ID - HL-38412/HL/NHLBI DA - 19900723 DP - 1990 IS - 0077-8923 TA - Ann N Y Acad Sci PG - 346-56 SB - M SB - X CY - UNITED STATES VI - 585 JC - 5NM EM - 199009 AD - Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri-Kansas City 64110. PMID- 0002192618 CU - 1997 SO - Ann N Y Acad Sci 1990;585:346-56 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [207 medline neighbors] UI - 90057442 AU - Marquez J AU - Iriarte A AU - Martinez-Carrion M TI - Covalent modification of a critical sulfhydryl group in the acetylcholine receptor: cysteine-222 of the alpha-subunit. LA - Eng MH - Amino Acid Sequence MH - Amino Acids/analysis MH - Animal MH - Chemistry MH - Chromatography, High Pressure Liquid MH - Cysteine/*metabolism MH - Fluorescent Dyes/pharmacology MH - Maleimides/pharmacology MH - Molecular Sequence Data MH - Peptide Mapping MH - Receptors, Cholinergic/*metabolism MH - Sulfhydryl Compounds/*metabolism MH - Support, U.S. Gov't, P.H.S. MH - Torpedo RN - 0 (Amino Acids) RN - 0 (Fluorescent Dyes) RN - 0 (Maleimides) RN - 0 (Receptors, Cholinergic) RN - 0 (Sulfhydryl Compounds) RN - 42189-56-0 (N-(3-pyrene)maleimide) RN - 4371-52-2 (Cysteine) PT - JOURNAL ARTICLE ID - GM-38341/GM/NIGMS DA - 19900111 DP - 1989 Sep 5 IS - 0006-2960 TA - Biochemistry PG - 7433-9 SB - M CY - UNITED STATES IP - 18 VI - 28 JC - A0G AA - Author EM - 199003 AB - Chemical modification of the Torpedo californica acetylcholine receptor (AcChR) by the fluorescent agent N-(1-pyrenyl)maleimide (PM) under nonreducing conditions resulted in the labeling of cysteine residues in all subunits and marked inhibition of the AcChR ion channel opening [Clarke, J. H., & Martinez-Carrion, M. (1986) J. Biol. Chem. 261, 10063- 10072]. The PM alkylation kinetics are not affected by the presence of agonists or a competitive antagonist. The PM-labeled alpha-subunit has been purified and digested with both CNBr and trypsin. The resulting fragments from both cleavages were fractionated by high-performance liquid chromatography. The amino acid analysis and sequencing data of the PM-labeled peptides identified cysteine-222 as the only residue labeled by PM on the alpha-subunit primary structure. Cysteine-222 is located in the middle of a hydrophobic domain designated M1, which contains a homologous class of cysteines (Cys-241 in the aligned sequences) conserved in the four subunits of the AcChR. Because of its reactivity and fluorescent properties of the bound probe, alpha Cys-222 seems to be free sulfhydryl group accessible through a hydrophobic pocket, and these properties should be incorporated into proposed folding models for the alpha-subunit. AD - Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, Kansas City 64110. PMID- 0002819078 CU - 1996 SO - Biochemistry 1989 Sep 5;28(18):7433-9 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [190 medline neighbors] UI - 89352584 AU - Perez-Ramirez B AU - Martinez-Carrion M TI - Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: application to the nicotinic acetylcholine receptor. LA - Eng MH - Affinity Labels MH - Animal MH - Cell Membrane Permeability MH - Cytoplasm/*analysis MH - Electrophoresis, Polyacrylamide Gel MH - Freezing MH - Hydrolysis MH - Membrane Proteins/*analysis MH - Peptide Hydrolases MH - Pyridoxal Phosphate/*analysis/chemical synthesis MH - Pyridoxaminephosphate Oxidase/isolation & purification MH - Receptors, Nicotinic/*analysis/isolation & purification MH - Spectrometry, Fluorescence MH - Support, U.S. Gov't, P.H.S. MH - Torpedo RN - EC 1.4.3.5 (Pyridoxaminephosphate Oxidase) RN - EC 3.4 (Peptide Hydrolases) RN - 0 (Affinity Labels) RN - 0 (Membrane Proteins) RN - 0 (Receptors, Nicotinic) RN - 54-47-7 (Pyridoxal Phosphate) PT - JOURNAL ARTICLE ID - GM-38341/GM/NIGMS DA - 19891006 DP - 1989 Jun 13 IS - 0006-2960 TA - Biochemistry PG - 5034-40 SB - M CY - UNITED STATES IP - 12 VI - 28 JC - A0G AA - Author EM - 198912 AB - A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with [3H]pyridoxine 5-phosphate ([3H]PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of [3H]PNP at 37 degrees C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The resulting Schiff's bases between PLP and AcChR amino groups were reduced with NaCNBH3, and the pyridoxylated proteins were analyzed by fluorography. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Proteinase K treatment of labeled AcChR vesicles generated a peptide of 13 kDa that could be detected with anti-PLP antibodies only when the pyridoxylation was carried out on the internal surface of the vesicles. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the alpha- subunit, internal surface labeling by PLP was greatest for the highest molecular weight (delta) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion [Strader, C. D., & Raftery, M. A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5807-5811] that is proportional to their subunit molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS) AD - Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, Kansas City 64110. PMID- 0002669967 CU - 1996 SO - Biochemistry 1989 Jun 13;28(12):5034-40 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [216 medline neighbors] UI - 89352471 AU - Chinchetru MA AU - Marquez J AU - Garcia-Borron JC AU - Richman DP AU - Martinez-Carrion M TI - Interaction of nicotinic acetylcholine receptor with two monoclonal antibodies recognizing different epitopes. LA - Eng MH - Animal MH - *Antibodies, Monoclonal MH - Binding Sites MH - Binding, Competitive MH - Epitopes MH - Ethidium/metabolism MH - Kinetics MH - Peptides/chemical synthesis/immunology MH - Protein Conformation MH - Receptors, Nicotinic/*immunology/metabolism MH - Support, U.S. Gov't, P.H.S. MH - Torpedo RN - 0 (Antibodies, Monoclonal) RN - 0 (Epitopes) RN - 0 (Peptides) RN - 0 (Receptors, Nicotinic) RN - 3546-21-2 (Ethidium) PT - JOURNAL ARTICLE ID - GM-38341/GM/NIGMS ID - NS-24304/NS/NINDS DA - 19890928 DP - 1989 May 16 IS - 0006-2960 TA - Biochemistry PG - 4222-9 SB - M CY - UNITED STATES IP - 10 VI - 28 JC - A0G AA - Author EM - 198912 AB - The interactions of nicotinic acetylcholine receptor (nAChR) with two monoclonal antibodies (mAb370A and mAb371A) which block the agonist- induced ion flux into nicotinic acetylcholine receptor vesicles [Donnelly, D., Mihovilovic, M., Gonzalez-Ros, J. M., Ferragut, J. A., Richman, D., & Martinez-Carrion, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7999] have been studied by a combination of immunochemical and spectroscopic techniques. Both mAbs are specific for the alpha- subunit of the receptor, but they recognize different epitopes. We have detected specific binding of the mAb370A to a synthetic peptide corresponding to residues alpha 187-205, a sequence known to contain the alpha-bungarotoxin binding site. By contrast, mAb371A seems to recognize an epitope which is largely silent after proteolytic digestion of the subunit. Binding of mAb370A to the receptor is inhibited by cholinergic agonist and alpha-neurotoxins but not by competitive antagonists or local anesthetics. By contrast, none of these ligands interferes with binding of mAb371A. The spectroscopic properties of the fluorescent probe ethidium have been used to investigate the effect of the mAbs on the interaction of the agonist carbamylcholine with nAChR in membranes. mAb370A, but not mAb371A, blocks both the agonist-induced increase in the fluorescence intensity of receptor-bound ethidium and the agonist-induced increase in the polarization value of the probe. In addition, measurements of ethidium binding followed by stopped-flow techniques showed that mAb370A, but not mAb371A, blocked the agonist-induced association of the probe to nAChR membranes.(ABSTRACT TRUNCATED AT 250 WORDS) AD - Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri-Kansas City 64110. PMID- 0002475163 CU - 1996 SO - Biochemistry 1989 May 16;28(10):4222-9 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [196 medline neighbors] UI - 89174748 AU - Altieri F AU - Mattingly JR Jr AU - Rodriguez-Berrocal FJ AU - Youssef J AU - Iriarte A AU - Wu TH AU - Martinez-Carrion M TI - Isolation and properties of a liver mitochondrial precursor protein to aspartate aminotransferase expressed in Escherichia coli. LA - Eng MH - Animal MH - Aspartate Transaminase/genetics/*isolation & purification/metabolism MH - Blotting, Western MH - Catalysis MH - Coenzymes/metabolism MH - Electrophoresis, Polyacrylamide Gel MH - Escherichia coli/enzymology/*genetics MH - Kinetics MH - Mitochondria, Liver/*metabolism MH - Plasmids MH - Protein Precursors/genetics/*isolation & purification/metabolism MH - Rats MH - Support, U.S. Gov't, P.H.S. RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Coenzymes) RN - 0 (Plasmids) RN - 0 (Protein Precursors) PT - JOURNAL ARTICLE ID - GM-38184/GM/NIGMS ID - HL-38412/HL/NHLBI DA - 19890425 DP - 1989 Mar 25 IS - 0021-9258 TA - J Biol Chem PG - 4782-6 SB - M SB - X CY - UNITED STATES IP - 9 VI - 264 JC - HIV AA - Author EM - 198907 AB - The precursor to rat liver mitochondrial aspartate aminotransferase has been expressed in Escherichia coli JM105 using the pKK233-2 expression vector. This mammalian natural precursor has been isolated as a soluble dimeric protein. The amino-terminal sequence and the amino acid composition of the isolated protein correspond to those predicted from the inserted cDNA (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). The isolated precursor contains bound pyridoxal phosphate and shows catalytic activity with a specific activity equal to that of the mature form of the enzyme. This precursor can also be processed by mitochondria into a form with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of mature enzyme. The isolation of this precursor as a stable and catalytically active entity indicates that the presequence peptide does not necessarily interfere with much of the folding and basic structural properties of the mature protein component. AD - Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, Kansas City 64110. PMID- 0002647743 CU - 1997 SO - J Biol Chem 1989 Mar 25;264(9):4782-6 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [3 protein links] [1 nucleotide link] [200 medline neighbors] UI - 89034271 AU - Pave-Preux M AU - Ferry N AU - Bouguet J AU - Hanoune J AU - Barouki R TI - Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase. LA - Eng MH - Adrenalectomy MH - Amino Acid Sequence MH - Animal MH - Aspartate Transaminase/*genetics MH - Base Sequence MH - Blotting, Northern MH - Cloning, Molecular MH - Codon MH - Cytosol/enzymology MH - *Gene Expression Regulation/drug effects MH - *Genes, Structural/drug effects MH - Hydrocortisone/*pharmacology MH - Isoenzymes/*genetics MH - Liver/*enzymology MH - Male MH - Mitochondria/enzymology MH - Molecular Sequence Data MH - Organ Specificity MH - Rats MH - Rats, Inbred Strains MH - Reference Values MH - RNA, Messenger/drug effects/*genetics MH - Support, Non-U.S. Gov't RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Codon) RN - 0 (Isoenzymes) RN - 0 (RNA, Messenger) RN - 50-23-7 (Hydrocortisone) PT - JOURNAL ARTICLE DA - 19881220 DP - 1988 Nov 25 IS - 0021-9258 TA - J Biol Chem PG - 17459-66 SB - M SB - X CY - UNITED STATES IP - 33 VI - 263 JC - HIV AA - Author EM - 198902 AB - Cytosolic and mitochondrial aspartate aminotransferase cDNAs were cloned from a lambda gt11 rat liver cDNA library. The complete coding sequence and the 3' non-coding sequence of the cytosolic isozyme mRNA were obtained from two overlapping cDNA clones. Partial sequences of the mitochondrial enzyme cDNAs were found to be identical to the recently published complete sequence (Mattingly, J. R., Jr., Rodriguez- Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). A single mRNA (2.4 kb (kilobase pair] hybridizing to the mitochondrial cDNA probe was detected by Northern blot analysis, whereas the cytosolic cDNA probe labeled one major (2.1 kb) and two minor (1.8 and 4 kb) mRNAs. The 1.8- kb and the 2.1-kb cytosolic aspartate aminotransferase mRNAs differ in their 3' ends and probably result from the use of either of the two polyadenylation signals present in the 3' noncoding region of the major cytosolic aspartate aminotransferase mRNA. Glucocorticoid hormones increased the activity of cytosolic but not mitochondrial aspartate aminotransferase in both liver and kidney. The increase in the enzyme activity was accompanied by an increase in the amount of the three corresponding mRNAs, while the mitochondrial enzyme mRNA was not significantly modified. AD - Institut National de la Sante et de la Recherche Medicale, Hopital Henri Mondor, Creteil, France. PMID- 0003182856 CU - 1997 SI - GENBANK/J04171 SO - J Biol Chem 1988 Nov 25;263(33):17459-66 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [206 medline neighbors] UI - 88213399 AU - Roberts WJ AU - Hubert E AU - Iriarte A AU - Martinez-Carrion M TI - Site-specific methylation of a strategic lysyl residue in aspartate aminotransferase. LA - Eng MH - Amino Acid Sequence MH - Animal MH - Aspartate Transaminase/*metabolism MH - Circular Dichroism MH - Isoenzymes/metabolism MH - *Lysine MH - Methylation MH - Myocardium/*enzymology MH - Oxidation-Reduction MH - Peptide Fragments/isolation & purification MH - Peptide Mapping MH - Protein Conformation MH - Support, U.S. Gov't, P.H.S. MH - Swine MH - Trypsin RN - EC 2.6.1.1 (Aspartate Transaminase) RN - EC 3.4.21.4 (Trypsin) RN - 0 (Isoenzymes) RN - 0 (Peptide Fragments) RN - 56-87-1 (Lysine) PT - JOURNAL ARTICLE ID - HL-38412/HL/NHLBI ID - GM-38184/GM/NIGMS DA - 19880620 DP - 1988 May 25 IS - 0021-9258 TA - J Biol Chem PG - 7196-202 SB - M SB - X CY - UNITED STATES IP - 15 VI - 263 JC - HIV AA - Author EM - 198808 AB - Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar ratio of formaldehyde to protein (40:1); and, upon reduction with sodium cyanoborohydride, 85% of the formaldehyde is incorporated at Lysine 258 and 15% at the amino-terminal alanyl residue. The modified protein, characterized after tryptic hydrolysis, separation of the peptides by high performance liquid chromatography procedures and subsequent amino acid analysis, shows that lysine 258 is preferentially modified as a dimethylated derivative. Modified apoenzyme can accept and tightly bind added coenzyme pyridoxal phosphate, as measured by circular dichroism procedures. The methylated enzyme is essentially catalytically inactive when measured by standard enzymatic assays. On the other hand, addition of the substrate, glutamate, produces the characteristic absorption spectral shifts for conversion of the active site-bound pyridoxal form of the coenzyme (absorbance at 400 nm) to its pyridoxamine form (absorbance at 330 nm). Such a half-transamination- like process occurs as in native enzyme, albeit at several orders of magnitude lower rate. This event takes place even though the characteristic internal holoenzyme Schiff's base between Lys-258 and aldehyde of bound pyridoxal phosphate does not exist in methylated, reconstituted holoenzyme. It is concluded that this chemically transformed enzyme can undergo a half-transamination reaction with conversion of active site-bound coenzyme from a pyridoxal to a pyridoxamine form, even when overall catalytic turnover transamination cannot be detected. AD - Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298. PMID- 0003130380 CU - 1997 SO - J Biol Chem 1988 May 25;263(15):7196-202 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [199 medline neighbors] UI - 88269528 AU - Sanchez-Ruiz JM AU - Martinez-Carrion M TI - A Fourier-transform infrared spectroscopic study of the phosphoserine residues in hen egg phosvitin and ovalbumin. LA - Eng MH - Animal MH - Chickens MH - *Egg Proteins MH - Egg White MH - Fourier Analysis MH - Nuclear Magnetic Resonance/methods MH - *Ovalbumin MH - *Phosphoserine MH - *Phosvitin MH - Protein Conformation MH - *Serine/analogs & derivatives MH - Spectrophotometry, Infrared/methods MH - Support, Non-U.S. Gov't MH - Support, U.S. Gov't, P.H.S. RN - 0 (Egg Proteins) RN - 17885-08-4 (Phosphoserine) RN - 56-45-1 (Serine) RN - 9006-59-1 (Ovalbumin) RN - 9008-96-2 (Phosvitin) PT - JOURNAL ARTICLE ID - GM-38184/GM/NIGMS ID - HL-38412/HL/NHLBI DA - 19880819 DP - 1988 May 3 IS - 0006-2960 TA - Biochemistry PG - 3338-42 SB - M CY - UNITED STATES IP - 9 VI - 27 JC - A0G AA - Author EM - 198810 AB - A Fourier-transform infrared spectroscopic study of hen egg phosvitin and ovalbumin has been carried out. Bands arising from monoanionic and dianionic phosphate monoester [Shimanouchi, T., Tsuboi, M., & Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498] can be identified easily in the 1300-930 cm-1 region in spectra of solutions of O-phosphoserine and phosvitin, a highly phosphorylated protein. On the other hand, spectra of ovalbumin show a relatively strong absorption above 1000 cm-1 arising from the protein moiety. Below 1000 cm-1, a single band at 979 cm-1 is observed; this band is not present in spectra of dephosphorylated ovalbumin, and therefore, it has been assigned to the symmetric stretching of the phosphorylated Ser-68 and Ser-344 in the dianionic ionization state. In addition, bands arising from symmetric and antisymmetric stretchings of the monoanionic ionization state, and from the antisymmetric stretching of the dianionic state, can be detected above 1000 cm-1 in difference spectra of ovalbumin minus dephosphorylated ovalbumin. The effect of pH on the infrared spectra of O-phosphoserine, phosvitin, and ovalbumin is consistent with the phosphoserine residues undergoing ionization with pK values about 6. This study demonstrates that Fourier-transform infrared spectroscopy can be a useful technique to assess the ionization state of phosphoserine residues in proteins in solution. AD - School of Basic Life Sciences, Division of Molecular Biology and Biochemistry, University of Missouri, Kansas City 64110. PMID- 0003134047 CU - 1996 SO - Biochemistry 1988 May 3;27(9):3338-42 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [111 medline neighbors] UI - 94160305 AU - Martinez-Carrion M AU - Clarke J AU - Gonzalez-Ros JM AU - Garcia-Borron JC TI - Fluorescent probes for the acetylcholine receptor surface environments. LA - Eng MH - Azides MH - Cell Membrane/chemistry MH - *Fluorescent Dyes MH - Lipid Bilayers MH - Maleimides MH - Receptors, Cholinergic/*analysis/chemistry MH - Surface Properties RN - 0 (Azides) RN - 0 (Fluorescent Dyes) RN - 0 (Lipid Bilayers) RN - 0 (Maleimides) RN - 0 (Receptors, Cholinergic) RN - 42189-56-0 (N-(3-pyrene)maleimide) RN - 69489-90-3 (pyrenesulfonyl azide) PT - JOURNAL ARTICLE PT - REVIEW PT - REVIEW, ACADEMIC DA - 19940330 DP - 1988 IS - 0306-0225 TA - Subcell Biochem PG - 363-92 CY - ENGLAND VI - 13 JC - V72 EM - 199406 RF - 99 PMID- 0002577860 NP - X SO - Subcell Biochem 1988;13:363-92 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [3 protein links] [1 nucleotide link] [202 medline neighbors] UI - 88106546 AU - Mattingly JR Jr AU - Rodriguez-Berrocal FJ AU - Gordon J AU - Iriarte A AU - Martinez-Carrion M TI - Molecular cloning and in vivo expression of a precursor to rat mitochondrial aspartate aminotransferase. LA - Eng MH - Amino Acid Sequence MH - Animal MH - Aspartate Transaminase/biosynthesis/*genetics MH - Base Sequence MH - DNA/genetics MH - Escherichia coli/genetics MH - Mitochondria, Liver/enzymology MH - Molecular Sequence Data MH - Protein Precursors/biosynthesis/*genetics MH - Rats MH - Recombinant Proteins/biosynthesis RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Protein Precursors) RN - 0 (Recombinant Proteins) RN - 9007-49-2 (DNA) PT - JOURNAL ARTICLE DA - 19880220 DP - 1987 Dec 31 IS - 0006-291X TA - Biochem Biophys Res Commun PG - 859-65 SB - M SB - X CY - UNITED STATES IP - 3 VI - 149 JC - 9Y8 AA - Author EM - 198804 AB - A 2.4 kilobase cDNA for rat mitochondrial aspartate aminotransferase (E.C. 2.6.1.1.) was isolated and sequenced. The predicted presequence is 93% homologous to the presequences of the enzyme from pig and mouse. The predicted amino acid sequence of the mature enzyme differs from that determined directly by amino acid sequencing (Huynh, Q.K., Sakakibara, R., Watanabe, T., and Wada, H. (1981) J. Biochem. (Tokyo) 90, 863-875) at 13 amino acids residues. The most important difference is at position 140 where the cDNA encodes a tryptophanyl residue rather than the previously reported glycine. This critical residue is now seen to be conserved in all aspartate aminotransferases. The coding region of this cDNA was inserted into the plasmid cloning vector pKK233-2 and used to stably express an unfused precursor in Escherichia coli JM105. AD - Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri-Kansas City 64110. PMID- 0003322287 CU - 1997 SI - GENBANK/M18467 SO - Biochem Biophys Res Commun 1987 Dec 31;149(3):859-65 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [164 medline neighbors] UI - 88022796 AU - Artigues A AU - Villar MT AU - Ferragut JA AU - Gonzalez-Ros JM TI - Thermal perturbation studies of membrane-bound acetylcholine receptor from Torpedo: effects of cholinergic ligands and membrane perturbants. LA - Eng MH - Animal MH - Bungarotoxins/metabolism MH - Calorimetry, Differential Scanning MH - Cell Membrane/drug effects/metabolism MH - Cholic Acids/pharmacology MH - Deoxycholic Acid/pharmacology MH - Detergents/*pharmacology MH - Glucosides/pharmacology MH - *Heat MH - Macromolecular Systems MH - Parasympathomimetics/*pharmacology MH - Receptors, Cholinergic/drug effects/*metabolism MH - Support, Non-U.S. Gov't MH - Surface-Active Agents/*pharmacology MH - Torpedo/*metabolism RN - 0 (Bungarotoxins) RN - 0 (Cholic Acids) RN - 0 (Detergents) RN - 0 (Glucosides) RN - 0 (Macromolecular Systems) RN - 0 (Parasympathomimetics) RN - 0 (Receptors, Cholinergic) RN - 0 (Surface-Active Agents) RN - 29836-26-8 (octyl-beta-D-glucoside) RN - 81-25-4 (cholic acid) RN - 83-44-3 (Deoxycholic Acid) PT - JOURNAL ARTICLE DA - 19871112 DP - 1987 Oct IS - 0003-9861 TA - Arch Biochem Biophys PG - 33-41 SB - M SB - X CY - UNITED STATES IP - 1 VI - 258 JC - 6SK AA - Author EM - 198801 AB - Thermal perturbation techniques have been used to probe structural features of the nicotinic acetylcholine receptor (AcChR). The information obtained from differential scanning calorimetry (DSC) of AcChR membranes (M.C. Farach and M. Martinez-Carrion (1983) J. Biol. Chem. 258, 4176) in the absence and in the presence of cholinergic ligands and local anesthetics, is comparable to that obtained from a simpler technique of heat inactivation of the alpha-bungarotoxin (alpha- Bgt) binding sites on the AcChR protein in similar samples. When AcChR membranes are heated at approximately 1 degree C/min, heat inactivation of toxin binding sites has a characteristic T50 value (temperature at which 50% of the initial capacity to bind alpha-Bgt remains) of approximately 60 degrees C. When heated at a constant temperature during increasing periods of time, the rate at which heat inactivation occurs is also characteristic of the temperature chosen for the experiment. The above thermal parameters are also sensitive to perturbation of the AcChR membrane matrix by the presence of subsolubilizing concentrations of detergents. Moreover, elimination of detergents by dialysis allows us to evaluate the reversibility or irreversibility of AcChR thermal destabilization induced by detergents or other membrane perturbants. Under the experimental conditions used, structural destabilization induced by octylglucoside or cholate can be fully reversed by detergent dialysis, while that exerted by deoxycholate cannot. "Thermal gel" analysis of the aggregation of AcChR subunits induced by heat (G. Soler, J. R. Mattingly, and M. Martinez- Carrion (1984) Biochemistry 23, 4630) has also been used to assess the effects of detergent presence on the AcChR protein. When deoxycholate is used as the perturbing agent, there is a particularly effective sulfhydryl-mediated aggregation of the gamma-delta subunit group, which appears to correlate with the irreversible destabilization of alpha-Bgt binding sites induced by that detergent. AD - Department of Biology, School of Sciences, University of the Balearic Islands, Palma de Mallorca, Spain. PMID- 0003662540 CU - 1992 SO - Arch Biochem Biophys 1987 Oct;258(1):33-41 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [200 medline neighbors] UI - 88024942 AU - Garcia-Borron JC AU - Bieber AL AU - Martinez-Carrion M TI - Reductive methylation as a tool for the identification of the amino groups in alpha-bungarotoxin interacting with nicotinic acetylcholine receptor. LA - Eng MH - Amines MH - Amino Acid Sequence MH - Animal MH - Bungarotoxins/*metabolism MH - Cell Membrane/metabolism MH - Electric Organ/metabolism MH - Kinetics MH - Methylation MH - Protein Conformation MH - Receptors, Cholinergic/isolation & purification/*metabolism MH - Receptors, Nicotinic/isolation & purification/*metabolism MH - Support, U.S. Gov't, Non-P.H.S. MH - Support, U.S. Gov't, P.H.S. MH - Torpedo RN - 0 (alpha-bungarotoxin receptor) RN - 0 (Amines) RN - 0 (Bungarotoxins) RN - 0 (Receptors, Cholinergic) RN - 0 (Receptors, Nicotinic) PT - JOURNAL ARTICLE ID - GM-24885/GM/NIGMS ID - GM-34677/GM/NIGMS DA - 19871207 DP - 1987 Jul 14 IS - 0006-2960 TA - Biochemistry PG - 4295-303 SB - M CY - UNITED STATES IP - 14 VI - 26 JC - A0G AA - Author EM - 198802 AB - alpha-Bungarotoxin (alpha Bgt) is a postsynaptic neurotoxin which blocks cholinergic transmission at the neuromuscular junction by binding tightly to the acetylcholine receptor (AcChR). The number of methylation sites in alpha Bgt has been shown to decrease significantly upon binding of the toxin to the AcChR [Soler, G., Farach, M. C., Farach, H. A., Mattingly, J. R., & Martinez-Carrion, M. (1983) Arch. Biochem. Biophys. 225, 872-878]. We have compared the chemical reactivities of amino groups in free and AcChR-bound alpha Bgt in an attempt to identify the regions in the alpha Bgt molecule that become masked upon binding to the AcChR. Free alpha Bgt and AcChR-bound alpha Bgt were reductively methylated with formaldehyde and sodium cyanoborohydride, and the rate of modification of each one of the available amino groups was followed by cleaving the methylated toxin with V8 protease and resolving the resulting peptides by reversed- phase, high-performance liquid chromatography. Under conditions of limited reagent availability, five of seven amino groups in free alpha Bgt reacted readily, whereas two other amino groups, probably those corresponding to Lys-51 and Lys-70, displayed lower reactivity. Upon binding to the AcChR, the rates of reductive methylation of residues Ile-1, Lys-26, and Lys-38 were considerably reduced (although to differing extents). The degree of protection was most pronounced for Lys-26. The rates of methylation of the amino groups in all other positions remained unchanged. These results allow further definition of the minimal binding surface of a representative neurotoxin. AD - Department of Biochemistry, Virginia Commonwealth University, Richmond 23298. PMID- 0003663591 CU - 1996 SO - Biochemistry 1987 Jul 14;26(14):4295-303 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [193 medline neighbors] UI - 87269616 AU - Clarke J AU - Garcia-Borron JC AU - Martinez-Carrion M TI - (1-Pyrene)sulfonyl azide: a fluorescent probe for measuring the transmembrane topology of acetylcholine receptor subunits. LA - Eng MH - Animal MH - *Azides MH - Cell Membrane/analysis MH - *Fluorescent Dyes MH - Photochemistry MH - Receptors, Cholinergic/*analysis MH - Spectrometry, Fluorescence MH - Support, U.S. Gov't, Non-P.H.S. MH - Support, U.S. Gov't, P.H.S. MH - Torpedo RN - 0 (Azides) RN - 0 (Fluorescent Dyes) RN - 0 (Receptors, Cholinergic) RN - 69489-90-3 (pyrenesulfonyl azide) PT - JOURNAL ARTICLE ID - GM-24885/GM/NIGMS ID - GM-34677/GM/NIGMS DA - 19870827 DP - 1987 Jul IS - 0003-9861 TA - Arch Biochem Biophys PG - 101-9 SB - M SB - X CY - UNITED STATES IP - 1 VI - 256 JC - 6SK AA - Author EM - 198710 AB - (1-Pyrene)sulfonyl azide (PySA), a fluorescent, lipophilic photolabel, was used as a probe for the transmembrane topology of the acetylcholine receptor (AchR) subunits. Photolabeling of native, alkaline-extracted, and reconstituted AchR membrane preparations resulted in the labeling of all the AchR subunits. However the reconstituted AchR membrane preparations incorporated twice as much PySA into each mole of the AchR complex. Photolabeling of all subunits of the AchR does not appear to alter the agonist concentration response of AchR-mediated cation translocation. PMID- 0003606117 CU - 1996 SO - Arch Biochem Biophys 1987 Jul;256(1):101-9 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [192 medline neighbors] UI - 86278051 AU - Clarke JH AU - Martinez-Carrion M TI - Labeling of functionally sensitive sulfhydryl-containing domains of acetylcholine receptor from Torpedo californica membranes. LA - Eng MH - Alkylation MH - Animal MH - Binding, Competitive MH - Bungarotoxins/metabolism MH - Carbachol/pharmacology MH - Cell Membrane/metabolism MH - Cell Membrane Permeability/drug effects MH - Cysteine/metabolism MH - Electric Organ/*metabolism MH - Ethylmaleimide/metabolism MH - Fluorescent Dyes MH - Human MH - Hydrogen-Ion Concentration MH - Maleimides/pharmacology MH - Receptors, Cholinergic/drug effects/*metabolism MH - Spectrometry, Fluorescence MH - Sulfhydryl Compounds/*metabolism MH - Sulfhydryl Reagents MH - Support, U.S. Gov't, Non-P.H.S. MH - Torpedo/*metabolism RN - 0 (Bungarotoxins) RN - 0 (Fluorescent Dyes) RN - 0 (Maleimides) RN - 0 (Receptors, Cholinergic) RN - 0 (Sulfhydryl Compounds) RN - 0 (Sulfhydryl Reagents) RN - 128-53-0 (Ethylmaleimide) RN - 42189-56-0 (N-(3-pyrene)maleimide) RN - 4371-52-2 (Cysteine) RN - 51-83-2 (Carbachol) PT - JOURNAL ARTICLE DA - 19860917 DP - 1986 Aug 5 IS - 0021-9258 TA - J Biol Chem PG - 10063-72 SB - M SB - X CY - UNITED STATES IP - 22 VI - 261 JC - HIV AA - Author EM - 198611 AB - N-(1-Pyrene)maleimide, a fluorescent, lipophilic, alkylating agent, was used as a probe for the nicotinic acetylcholine receptor (AChR). Preincubation with N-(1-pyrene)maleimide under nonreducing conditions inhibits agonist-induced cation permeability of AChR-enriched membranes. This inhibition is dependent on the concentration of N-(1- pyrene)maleimide used. This correlation was also exhibited by resonance energy transfer of tryptophan fluorescence to N-(1-pyrene)maleimide and by the labeling stoichiometries. However, agonist-induced desensitization, as based on the time-dependent inhibition of alpha- bungarotoxin binding upon preincubation with the agonist carbamylcholine, was unaffected by N-(1-pyrene)maleimide. Alkylation of the AChR by N-(1-pyrene)maleimide is pH-dependent with an apparent pKa of 7.5 and is unaffected by preincubation with carbamylcholine, alpha- bungarotoxin, tubocurarine, or decamethonium. Preincubation with a 25- fold molar excess of N-ethylmaleimide partially protects against N-(1- pyrene)maleimide, yet simultaneous incubation with an equimolar concentration does not protect. In contrast, simultaneous incubation with equimolar concentrations of phenylmaleimide or naphthylmaleimide inhibited N-(1-pyrene)maleimide alkylation by 52 and 67%, respectively. Each AChR subunit is labeled by N-(1-pyrene)maleimide. Prior alkylation with N-ethylmaleimide does not alter the labeling profile but lowers the amount of labeling of all subunits. Reductive methylation of membranes under conditions which dimethylate all or most protein amino groups does not inhibit alkylation by N-(1-pyrene)maleimide. The above results, as well as amino acid analysis of N-(1-pyrene)maleimide- alkylated receptor, indicate that a homologous class of cysteines, which reside in each subunit within the AChR domain embedded in the membrane, are involved in the reaction with N-(1-pyrene)maleimide. PMID- 0003733702 CU - 1992 SO - J Biol Chem 1986 Aug 5;261(22):10063-72 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [176 medline neighbors] UI - 86243290 AU - Sanchez-Ruiz JM AU - Martinez-Carrion M TI - Ionization state of the coenzyme 5'-phosphate ester in cytosolic aspartate aminotransferase. A Fourier transform infrared spectroscopic study. LA - Eng MH - Animal MH - Arsenates/pharmacology MH - Aspartate Transaminase/*metabolism MH - Cytosol/enzymology MH - Fourier Analysis MH - Hydrogen-Ion Concentration MH - Myocardium/enzymology MH - Phosphates/metabolism MH - Protein Binding MH - Pyridoxal Phosphate/*metabolism MH - Spectrophotometry, Infrared/methods MH - Support, Non-U.S. Gov't MH - Support, U.S. Gov't, Non-P.H.S. MH - Support, U.S. Gov't, P.H.S. MH - Swine RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Arsenates) RN - 0 (Phosphates) RN - 54-47-7 (Pyridoxal Phosphate) PT - JOURNAL ARTICLE ID - HL22265/HL/NHLBI ID - GM24885/GM/NIGMS DA - 19860801 DP - 1986 May 20 IS - 0006-2960 TA - Biochemistry PG - 2915-20 SB - M CY - UNITED STATES IP - 10 VI - 25 JC - A0G AA - Author EM - 198610 AB - In order to determine the ionization state of the 5'-phosphate of bound pyridoxal phosphate, a Fourier transform infrared spectroscopic study of cytosolic aspartate aminotransferase has been carried out. Dianionic and monoanionic phosphate monoesters give rise to two bands each in the infrared spectrum [Shimanouchi, T., Tsuboi, M., & Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498]. These bands can be identified in infrared spectra of the free coenzyme in solution. Due to interfering bands arising from the protein, only the band assigned to the symmetric stretching of the dianionic phosphate is observed in holoenzyme solutions. The integrated intensity of this band does not change with pH in the range 5.3-8.6, while for free pyridoxal phosphate, the integrated intensity of the same band changes with pH according to the pK value expected for the 5'-phosphate group in solution. Moreover, the value of the integrated intensity for the bound cofactor is close to the value given by free cofactor at pH 8-9. These results suggest that the 5'-phosphate of the bound cofactor remains mostly dianionic throughout the investigated pH range and disfavor other interpretations in terms of ionization of the phosphate group on the basis of the nuclear magnetic resonance 31P chemical shift-pH titration curve of holoenzyme [Schnackerz, K. D. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, E. A., Ed.) Part A, pp 195- 208, Alan R. Liss, New York].(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 0003718929 CU - 1997 SO - Biochemistry 1986 May 20;25(10):2915-20 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [123 medline neighbors] UI - 87092929 AU - Toro-Goyco E AU - Cora EM AU - Kessler MJ AU - Martinez-Carrion M TI - Induction of experimental myasthenia gravis in rhesus monkeys: a model for the study of the human disease. LA - Eng MH - Agglutination Tests MH - Animal MH - Antibody Formation MH - Macaca mulatta/immunology MH - Myasthenia Gravis/*immunology MH - Receptors, Cholinergic/immunology MH - Support, U.S. Gov't, P.H.S. MH - Torpedo/immunology RN - 0 (Receptors, Cholinergic) PT - JOURNAL ARTICLE ID - RR-7-2115/RR/NCRR ID - RR-01293/RR/NCRR DA - 19870128 DP - 1986 Apr IS - 0738-0658 TA - P R Health Sci J PG - 13-8 SB - M CY - PUERTO RICO IP - 1 VI - 5 JC - QJF AA - Author EM - 198704 AB - Experimental autoimmune myasthenia gravis (EAMG) was induced in rhesus monkeys using purified acetylcholine receptor (AChR) from Torpedo california. A single dose of 80 micrograms induced antibody formation two weeks after injection. Two subsequent doses at two-week intervals caused clinical signs (anorexia, fatigability, weight loss, ptosis and dysphagia) which initially responded to treatment with neostigmine. Histologic examination of post-mortem tissues revealed lesions characteristic of myasthenia gravis in man: muscular atrophy, fibrous degeneration and lymphocytic infiltration. Antibodies were quantitated in the sera of three other monkeys which received only 60 micrograms of purified AChR. Abnormally high titers persisted for two years (60-200 micrograms /ml versus 0-10 micrograms/ml for controls). A monkey injected with 60 micrograms AChR as part of reconstituted membrane vesicles had lower titers (30-50 micrograms/ml) than those which received purified receptor. Only those monkeys with antibody titers exceeding 800 micrograms/ml developed overt disease. These titers were 4-100 times higher than those reported for myasthenic humans. The antibody-antigen molar ratios were higher for monkeys with disease than for asymptomatic animals. These data suggest that the diversity of antibody molecules synthesized by the sensitized monkeys determined the appearance of clinical signs, and that the cross reaction of anti- torpedo antibodies with monkey receptor was primarily responsible for the development of EAMG. PMID- 0003797623 CU - 1996 SO - P R Health Sci J 1986 Apr;5(1):13-8 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [196 medline neighbors] UI - 85207786 AU - Iriarte A AU - Kraft K AU - Martinez-Carrion M TI - The separate effects of coenzyme components may not be additive. Roles of pyridoxal and inorganic phosphate in aspartate aminotransferase apoenzymes. LA - Eng MH - Animal MH - Apoenzymes/*metabolism MH - Apoproteins/*metabolism MH - Aspartate Transaminase/*metabolism MH - Calorimetry, Differential Scanning MH - Cytosol/enzymology MH - Hydrogen-Ion Concentration MH - Isoenzymes/metabolism MH - Kinetics MH - Mitochondria, Heart/*enzymology MH - Myocardium/*enzymology MH - Nuclear Magnetic Resonance MH - Phosphates/*pharmacology MH - Pyridoxal/*pharmacology MH - Support, U.S. Gov't, P.H.S. MH - Swine RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Apoenzymes) RN - 0 (Apoproteins) RN - 0 (Isoenzymes) RN - 0 (Phosphates) RN - 66-72-8 (Pyridoxal) PT - JOURNAL ARTICLE ID - HL-22265/HL/NHLBI ID - GM 24885/GM/NIGMS DA - 19850725 DP - 1985 Jun 25 IS - 0021-9258 TA - J Biol Chem PG - 7457-63 SB - M SB - X CY - UNITED STATES IP - 12 VI - 260 JC - HIV AA - Author EM - 198509 AB - Both cytosolic and mitochondrial aspartate transaminase can be resolved of pyridoxal phosphate. The resulting apoenzymes still bind individual structural components of the coenzyme. The separate contributions of coenzyme components to protein thermal stability have been independently assessed for phosphate ions (Pi) and for the pyridoxal or pyridoxamine components of the coenzyme. 31P NMR and differential scanning calorimetry reveal that the thermodynamic contributions of binding are not additive and are dissimilar for the two isozymes. High and low affinity sites for Pi binding are present in both apoenzymes with only the low affinity site being present in the holoenzyme forms. The contribution of both bound phosphates to increasing temperatures (Tm) and enthalpies (delta Hd) of denaturation differ between the isozymes and within sites. In either isozyme occupancy of the high affinity site by Pi produces only a 4- or 5- degree increase in the Tm value with respect to Pi-free apoenzyme. By contrast, in the mitochondrial apoenzyme, the presence of Pi at the second low affinity site increases the calorimetric parameters from Tm = 47 degrees C and delta Hd = 4.7 cal g-1 to Tm = 62 degrees C and delta Hd = 7 cal g-1. For cytosolic apoenzyme the respective changes are from 66 to 69.5 degrees C and 5.2 to 5.8 cal g-1. Addition of pyridoxal, but not pyridoxamine, displaces the high affinity Pi in both apoenzymes. This shows that the pyridine ring and Pi groups of pyridoxal-P bind exclusive of each other when they are not covalently linked as an ester, as in the coenzyme. The observation has been exploited as a method to prepare completely dephosphorylated mitochondrial apoenzyme. Electrostatic effects, structural differences in the phosphate binding pockets, and steric effects can be invoked to account for the Pi and pyridine binding behavior in the two proteins. PMID- 0003997881 CU - 1997 SO - J Biol Chem 1985 Jun 25;260(12):7457-63 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [179 medline neighbors] UI - 85095833 AU - Ferragut JA AU - Gonzalez-Ros JM AU - Peterson DL AU - Weir DL AU - Franson RC AU - Martinez-Carrion M TI - Rapid purification of a phospholipase-free alpha-bungarotoxin: maintenance of cation barriers of acetylcholine receptor membranes upon preincubation with purified toxin. LA - Eng MH - Bungarotoxins/*isolation & purification/pharmacology MH - Cations/*metabolism MH - Cell Membrane Permeability/drug effects MH - Chemistry MH - Chromatography, High Pressure Liquid MH - Elapid Venoms/analysis MH - Electrophoresis, Polyacrylamide Gel MH - Molecular Weight MH - Phospholipases/*isolation & purification MH - Receptors, Cholinergic/*drug effects MH - Support, U.S. Gov't, Non-P.H.S. MH - Support, U.S. Gov't, P.H.S. RN - EC 3.1.- (Phospholipases) RN - 0 (Bungarotoxins) RN - 0 (Cations) RN - 0 (Elapid Venoms) RN - 0 (Receptors, Cholinergic) PT - JOURNAL ARTICLE ID - NS-17029/NS/NINDS DA - 19850131 DP - 1984 Dec IS - 0003-9861 TA - Arch Biochem Biophys PG - 628-35 SB - M SB - X CY - UNITED STATES IP - 2 VI - 235 JC - 6SK AA - Author EM - 198504 AB - The purification of highly homogeneous, phospholipase-free alpha- bungarotoxin (alpha-Bgt) from the venom of the elapid Bungarus multicinctus or from commercial samples of alpha-Bgt is described. The method combines a conventional procedure for the purification of alpha- Bgt [D. Mebs, K. Narita, S. Iwanaga, Y. Samejima, and C. Y. Lee (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 243-262] with high-resolution gel- filtration and cation-exchange chromatography steps to remove membrane- damaging, contaminating phospholipase activity. The procedure also removes contaminating radioactive peptides from commercial preparations of 125I-alpha-Bgt. Apparent homogeneity of the purified alpha-Bgt (referred to as fraction D in the text), as well as the absence of contaminating phospholipase A2 activity, is assessed by (i) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, (ii) gel-filtration and cation-exchange high-performance liquid chromatography, (iii) direct measurements of phospholipase A2 activity under conditions where very low enzymatic levels should be detected, (iv) lack of interference with the passive cation permeability properties of acetylcholine receptor membranes, (v) competitive inhibition of 125I-alpha-Bgt binding to the acetylcholine receptor membranes, and (vi) amino acid analysis and end-group (C- and N-terminus) determination. alpha-Bgt preparations subjected to these criteria do not exert the increase in membrane passive permeability to cations detected with other laboratory or commercial samples of alpha- Bgt. Availability of the new alpha-Bgt preparation allows for an assessment of the inertness of alpha-Bgt on lipid membrane properties while preventing cholinergic ligand binding to nicotinic acetylcholine receptor-rich membranes. These conditions are necessary for experiments requiring maintenance of the physical and phospholipid integrity of membranes. PMID- 0006517603 CU - 1996 SO - Arch Biochem Biophys 1984 Dec;235(2):628-35 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [157 medline neighbors] UI - 85088536 AU - Donnelly D AU - Mihovilovic M AU - Gonzalez-Ros JM AU - Ferragut JA AU - Richman D AU - Martinez-Carrion M TI - A noncholinergic site-directed monoclonal antibody can impair agonist- induced ion flux in Torpedo californica acetylcholine receptor. LA - Eng MH - Animal MH - Antibodies, Monoclonal/*diagnostic use MH - Antigen-Antibody Complex MH - Cell Membrane/physiology MH - Ion Channels/*immunology/physiology MH - Kinetics MH - Receptors, Cholinergic/*immunology/physiology MH - Spectrometry, Fluorescence MH - Support, Non-U.S. Gov't MH - Support, U.S. Gov't, Non-P.H.S. MH - Support, U.S. Gov't, P.H.S. MH - Thallium/diagnostic use MH - Torpedo RN - 0 (Antibodies, Monoclonal) RN - 0 (Antigen-Antibody Complex) RN - 0 (Ion Channels) RN - 0 (Receptors, Cholinergic) RN - 7440-28-0 (Thallium) PT - JOURNAL ARTICLE ID - NS15462/NS/NINDS ID - NS17029/NS/NINDS DA - 19850221 DP - 1984 Dec IS - 0027-8424 TA - Proc Natl Acad Sci U S A PG - 7999-8003 SB - M SB - X CY - UNITED STATES IP - 24 VI - 81 JC - PV3 AA - Author EM - 198504 AB - We have employed several monoclonal antibodies (mAbs) directed against several regions of the acetylcholine receptor (AcChoR) to assist in the determination of the antigenic structure of this multisubunit glycoprotein and to better understand molecular events involved in the impairment of neuromuscular transmission in the autoimmune disease myasthenia gravis. Among three mAbs shown to block agonist-induced ion fluxes, mAb 371A is a putative probe of an ion channel domain(s) of the AcChoR. It appears to bind to an antigenic determinant whose structure is maintained upon treatment with sodium dodecyl sulfate, the stoichiometry of binding being of one mAb per alpha-bungarotoxin binding site. Binding of mAb 371A to the AcChoR does not affect binding of cholinergic agonists or antagonists (carbamoylcholine and d- tubocurarine) or neurotoxins (alpha-bungarotoxin) or the ability of membrane-bound AcChoR to undergo reversible sensitization- desensitization affinity transitions. However, this mAb inhibits agonist-induced thallium (T1+) influx into AcChoR-rich membrane vesicles, as measured on a millisecond time scale by means of a rapid kinetics "stopped-flow/fluorescence quenching" technique. The stoichiometry of inhibition by bound mAb 371A coincides with that for maximal binding. PMID- 0006096872 CU - 1996 SO - Proc Natl Acad Sci U S A 1984 Dec;81(24):7999-8003 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [200 medline neighbors] UI - 85047162 AU - Soler G AU - Mattingly JR Jr AU - Martinez-Carrion M TI - Effects of heating on the ion-gating function and structural domains of the acetylcholine receptor. LA - Eng MH - Animal MH - Carbachol/pharmacology MH - Cell Membrane/metabolism MH - Electric Organ/*metabolism MH - Heat MH - Ion Channels/drug effects/*metabolism MH - Kinetics MH - Macromolecular Systems MH - Molecular Weight MH - Receptors, Cholinergic/drug effects/*metabolism MH - Support, U.S. Gov't, Non-P.H.S. MH - Support, U.S. Gov't, P.H.S. MH - Thermodynamics MH - Torpedo RN - 0 (Ion Channels) RN - 0 (Macromolecular Systems) RN - 0 (Receptors, Cholinergic) RN - 51-83-2 (Carbachol) PT - JOURNAL ARTICLE ID - GM-24885/GM/NIGMS DA - 19841227 DP - 1984 Sep 25 IS - 0006-2960 TA - Biochemistry PG - 4630-6 SB - M CY - UNITED STATES IP - 20 VI - 23 JC - A0G AA - Author EM - 198503 AB - The ion-gating ability and the protein electrophoretic band patterns of the acetylcholine receptor from Torpedo californica electroplax were examined after receptor-enriched membrane vesicles were progressively heated. The ion translocation function was lost over a temperature range of 40-55 degrees C. Previous results have shown that the stoichiometry of alpha-bungarotoxin binding is not affected by these temperatures, although bound toxin reversibly dissociates within this temperature range, and that toxin binding is irreversibly lost at somewhat higher temperatures [Soler, G., Farach, M.C., Farach, H. A., Jr., Mattingly, J.R., Jr., & Martinez-Carrion, M. (1983) Arch. Biochem. Biophys. 225, 872]. Thermal gel analysis [Lysko, K. A., Carlson, R., Taverna, R., Snow, J., & Brandts, J.F. (1981) Biochemistry 20, 5570], a sodium dodecyl sulfate-polyacrylamide gel electrophoretic procedure which detects thermally induced aggregation of the components of multimeric systems, was applied to heated acetylcholine receptor enriched membranes. This technique suggests two structural domains susceptible to thermal perturbation within the receptor molecule, one consisting of the Mr 50 000 and the two Mr 40 000 subunits and the other consisting of the Mr 60 000 and 65 000 subunits. Heat disrupts molecular events linking agonist binding with ion-channel opening in the acetylcholine receptor molecule. PMID- 0006093861 CU - 1996 SO - Biochemistry 1984 Sep 25;23(20):4630-6 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [137 medline neighbors] UI - 84212549 AU - Iriarte A AU - Farach HA Jr AU - Martinez-Carrion M TI - Coenzyme active site occupancy as an indicator of independence of the subunits of mitochondrial aspartate aminotransferase. LA - Eng MH - Animal MH - Aspartate Transaminase/*metabolism MH - Binding Sites MH - Calorimetry, Differential Scanning MH - Heat MH - Isoelectric Focusing MH - Isoenzymes/*metabolism MH - Macromolecular Systems MH - Mitochondria, Heart/*enzymology MH - Spectrophotometry MH - Support, U.S. Gov't, P.H.S. MH - Swine RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Isoenzymes) RN - 0 (Macromolecular Systems) PT - JOURNAL ARTICLE ID - HL-22265/HL/NHLBI ID - GM-24885/GM/NIGMS DA - 19840716 DP - 1984 Jun 10 IS - 0021-9258 TA - J Biol Chem PG - 7003-10 SB - M SB - X CY - UNITED STATES IP - 11 VI - 259 JC - HIV AA - Author EM - 198409 AB - The enzyme, aspartate aminotransferase, is a dimer consisting of two identical subunits which contain overlapping subunit regions ( Eichele , G., Ford, G.C., Glor , M., Jansonius , J.N., Mavrides , C., and Christen , P. (1979) J. Mol. Biol. 133, 161-180), suggesting the possibility of subunit interactions. The structurally similar cytosolic isozyme exhibits noncooperative binding of pyridoxal 5'-phosphate ( Boettcher , M., and Martinez -Carrion, M. (1975) Biochemistry 14, 4528- 4531; Relimpio , A., Iriarte , A., Chlebowski , J.F., and Martinez - Carrion, M. (1981) J. Biol. Chem. 256, 4478-4488) in which the apoenzyme/holoenzyme hybrid dimer shows a distinctive thermal stability. Using a nonequilibrium isoelectric focusing technique, it can be shown that mitochondrial aspartate aminotransferase also binds cofactor in a noncooperative random fashion. However, differential scanning calorimetry (DSC) thermograms show different characteristics from the cytosolic form. These differences are interpreted in terms of unique subunit interactions in this isozyme. Heating to the various DSC transition temperatures shows that the anomalous DSC thermograms in partially coenzyme-saturated apoenzyme preparations are due to a selective dissociation of apoenzyme subunits into monomers which are irreversibly denatured. The remaining holoenzyme monomers reassociate and form stable holoenzyme dimers. The net result is retention of the initial concentration of holoenzyme subunits present in any given mixture. Random occupancy of active sites and similar electrophoretic and DSC patterns upon heating of partially saturated apoenzyme preparations is observed whether the coenzyme, pyridoxal phosphate or pyridoxamine phosphate alone, or borohydride-reduced Schiff's bases of coenzyme-substrate analogue derivatives are used as active site directed ligands. The latter resemble covalent enzyme-substrate intermediates. PMID- 0006725280 CU - 1997 SO - J Biol Chem 1984 Jun 10;259(11):7003-10 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [157 medline neighbors] UI - 84231225 AU - Gonzalez-Ros JM AU - Ferragut JA AU - Martinez-Carrion M TI - Binding of anti-acetylcholine receptor antibodies inhibits the acetylcholine receptor mediated cation flux. LA - Eng MH - Animal MH - Autoantibodies/*physiology MH - *Cations MH - Cell Membrane Permeability MH - Electric Organ/*analysis MH - Epitopes MH - Fluorescent Dyes MH - Immunoglobulins, Fab MH - Pyrenes/metabolism MH - Receptors, Cholinergic/*immunology/metabolism MH - Thallium/metabolism MH - Torpedo/*immunology RN - 0 (Autoantibodies) RN - 0 (Cations) RN - 0 (Epitopes) RN - 0 (Fluorescent Dyes) RN - 0 (Immunoglobulins, Fab) RN - 0 (Pyrenes) RN - 0 (Receptors, Cholinergic) RN - 6528-53-6 (1,3,6,8-pyrene tetrasulfonate) RN - 7440-28-0 (Thallium) PT - JOURNAL ARTICLE DA - 19840622 DP - 1984 Apr 30 IS - 0006-291X TA - Biochem Biophys Res Commun PG - 368-75 SB - M SB - X CY - UNITED STATES IP - 2 VI - 120 JC - 9Y8 AA - Author EM - 198409 AB - A fast kinetics, spectroscopic technique has been applied to the study of the transient cation flux associated to the binding of cholinergic agonist to native acetylcholine receptor (AcChR)-rich membrane vesicles in presence of anti-AcChR antibodies. The technique is based on the collisional quenching of an intravesicularly trapped fluorophore by externally added T1+ which substitutes for physiologically occurring cations. Presence of polyclonal Fab fragments from goat anti-AcChR antibodies bound to the membrane AcChR promotes a 80-90% inhibition on the observed rate constants of T1+ influx. The observed inhibition process appears to follow a non-competitive pattern between antibody and cholinergic ligand binding, suggesting that in the AcChR protein the antigenic sites responsible for ion translocation may be other than those involved in ligand binding. RO - M:BDS PMID- 0006203520 LR - 19841219 CU - 1995 SO - Biochem Biophys Res Commun 1984 Apr 30;120(2):368-75 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [154 medline neighbors] UI - 84111547 AU - Iriarte A AU - Hubert E AU - Kraft K AU - Martinez-Carrion M TI - Selective tryptic cleavage of native cytoplasmic aspartate transaminase holoenzyme. LA - Eng MH - Amino Acid Sequence MH - Animal MH - Aspartate Transaminase/*metabolism MH - Aspartic Acid/analogs & derivatives/metabolism MH - Calorimetry, Differential Scanning MH - Chromatography, High Pressure Liquid MH - Cytoplasm/enzymology MH - Isoenzymes/*metabolism MH - Myocardium/enzymology MH - Support, U.S. Gov't, P.H.S. MH - Swine MH - Trypsin/*metabolism RN - EC 2.6.1.1 (Aspartate Transaminase) RN - EC 3.4.21.4 (Trypsin) RN - 0 (Isoenzymes) RN - 56-84-8 (Aspartic Acid) RN - 6384-92-5 (N-Methylaspartate) PT - JOURNAL ARTICLE ID - HL-22265/HL/NHLBI DA - 19840305 DP - 1984 Jan 25 IS - 0021-9258 TA - J Biol Chem PG - 723-8 SB - M SB - X CY - UNITED STATES IP - 2 VI - 259 JC - HIV AA - Author EM - 198405 AB - The cytoplasmic isozyme of aspartate transaminase is inactivated by trypsin due to loss of a 19-residue peptide from the NH2-terminal region. A second peptide bond at Arg-25 is then cleaved by trypsin leaving a residual core protein, transaminase 26-412. Inactivation by trypsin resembles that for the mitochondrial enzyme (Sandmeier, E., and Christen, P. (1980) J. Biol. Chem. 255, 10284-10289), yet occurs 10 times faster for the cytoplasmic isozyme. In the mitochondrial enzyme, trypsin cleavage produces equal concentrations of proteins missing the first 26 and 31 amino acids. Sequence variation in the NH2-terminal regions can explain such differences. Specifically, the mitochondrial NH2 terminus has no trypsin-susceptible residue at position 19 and is stabilized by an electrostatic interaction between Asp-15 and Arg-292, whereas position 15 is a valyl residue in the cytoplasmic enzyme. Calorimetric data reveal both a decreased transition temperature (Td) and enthalpy (delta Hd) of denaturation in transaminases 20-412 and 26- 412. Interaction of substrates with the active site chromophore and differential scanning calorimetry (DSC) reveal that catalytically inactive transaminases 20-412 and 26-412 can bind amino acid substrates and produce spectroscopically detectable conversion of the pyridoxal to the pyridoxamine form of the protein. By contrast, substrate analogs only form enzymatic Michaelis-type complexes. PMID- 0006363406 CU - 1997 SO - J Biol Chem 1984 Jan 25;259(2):723-8 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [150 medline neighbors] UI - 84194239 AU - Iriarte A AU - Hubert H AU - Kraft K AU - Martinez-Carrion M TI - Small molecular regulators of the specific trypsin cleavages in aspartate transaminase isozymes. LA - Eng MH - Adenine Nucleotides/pharmacology MH - Amino Acid Sequence MH - Animal MH - Aspartate Transaminase/*metabolism MH - Isoenzymes/*metabolism MH - Kinetics MH - Peptide Fragments/metabolism MH - Phosphates/pharmacology MH - Trypsin/*metabolism RN - EC 2.6.1.1 (Aspartate Transaminase) RN - EC 3.4.21.4 (Trypsin) RN - 0 (Adenine Nucleotides) RN - 0 (Isoenzymes) RN - 0 (Peptide Fragments) RN - 0 (Phosphates) PT - JOURNAL ARTICLE DA - 19840618 DP - 1984 IS - 0361-7742 TA - Prog Clin Biol Res PG - 269-76 SB - M CY - UNITED STATES VI - 144B JC - PZ5 EM - 198408 PMID- 0006718408 CU - 1997 SO - Prog Clin Biol Res 1984;144B:269-76 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [149 medline neighbors] UI - 84194223 AU - Iriarte A AU - Relimpio AM AU - Chlebowski JF AU - Martinez-Carrion M TI - Differences in thermodynamic stabilities among enzyme and coenzyme ligand complexes in aspartate aminotransferase. LA - Eng MH - Animal MH - Apoenzymes/metabolism MH - Aspartate Transaminase/*metabolism MH - Binding Sites MH - Calorimetry, Differential Scanning MH - Dicarboxylic Acids/pharmacology MH - Drug Stability MH - Ligands MH - Protein Binding MH - Protein Conformation MH - Pyridoxal Phosphate/*pharmacology MH - Thermodynamics RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Apoenzymes) RN - 0 (Dicarboxylic Acids) RN - 0 (Ligands) RN - 54-47-7 (Pyridoxal Phosphate) PT - JOURNAL ARTICLE DA - 19840618 DP - 1984 IS - 0361-7742 TA - Prog Clin Biol Res PG - 107-15 SB - M CY - UNITED STATES VI - 144B JC - PZ5 EM - 198408 PMID- 0006718393 CU - 1997 SO - Prog Clin Biol Res 1984;144B:107-15 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [139 medline neighbors] UI - 84194256 AU - Martinez-Carrion M AU - Mattingly J AU - Iriarte A TI - Phosphate as a probe of structure and function of aspartate transaminase isozymes. LA - Eng MH - Affinity Labels/pharmacology MH - Animal MH - Aspartate Transaminase/*metabolism MH - Binding Sites MH - Comparative Study MH - Cytosol/enzymology MH - Hydrogen-Ion Concentration MH - Isoenzymes/*metabolism MH - Kinetics MH - Lysine MH - Mitochondria/enzymology MH - Nuclear Magnetic Resonance MH - Phosphates/*pharmacology MH - Protein Binding MH - Pyridoxal Phosphate/metabolism MH - Thermodynamics RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Affinity Labels) RN - 0 (Isoenzymes) RN - 0 (Phosphates) RN - 54-47-7 (Pyridoxal Phosphate) RN - 56-87-1 (Lysine) PT - JOURNAL ARTICLE DA - 19840618 DP - 1984 IS - 0361-7742 TA - Prog Clin Biol Res PG - 97-106 SB - M CY - UNITED STATES VI - 144B JC - PZ5 EM - 198408 PMID- 0006425860 CU - 1997 SO - Prog Clin Biol Res 1984;144B:97-106 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [166 medline neighbors] UI - 83283471 AU - Paraschos A AU - Gonzalez-Ros JM AU - Martinez-Carrion M TI - Absorption filtration. A tool for the measurement of ion tracer flux in native membranes and reconstituted lipid vesicles. LA - Eng MH - Absorption MH - Animal MH - Carbachol/pharmacology MH - Cell Membrane/metabolism MH - Electric Organ/metabolism MH - Ion Channels/drug effects/*metabolism MH - Kinetics MH - Liposomes MH - Receptors, Cholinergic/*metabolism MH - Sodium/*metabolism MH - Support, U.S. Gov't, Non-P.H.S. MH - Support, U.S. Gov't, P.H.S. MH - Torpedo RN - 0 (Ion Channels) RN - 0 (Liposomes) RN - 0 (Receptors, Cholinergic) RN - 51-83-2 (Carbachol) RN - 7440-23-5 (Sodium) PT - JOURNAL ARTICLE ID - NS 17029/NS/NINDS DA - 19831021 DP - 1983 Sep 7 IS - 0006-3002 TA - Biochim Biophys Acta PG - 223-33 SB - M SB - X CY - NETHERLANDS IP - 2 VI - 733 JC - A0W AA - Author EM - 198312 AB - A rapid, simple and reproducible method has been developed for the measurement of ion tracer flux with both native membrane vesicles and reconstituted lipid vesicle systems. Following the absorption of vesicles onto glass fiber filters, tracer flux is performed directly upon the deposited samples. In contrast to the more conventional vacuum and gel filtration techniques, absorption filtration exhibits comprehensive data retrieval whereby the removal of extravesicular ions, the retention of intravesicular ions and the amount of ions fluxed can be accurately analyzed. Both influx and efflux assays have been designed to measure the carbamylcholine-induced flux of 22Na+ which is characteristic of acetylcholine receptor-enriched membranes from Torpedo californica electroplax. The flux signal-to-background noise ratio is maximized in the efflux assay, since agonist activation is performed subsequent to the exhaustive removal of extravesicular tracer. An interesting feature of the influx assay is that the agonist- induced uptake of 22Na+ can be repeated with the original vesicles which additionally maximizes the flux signal. With either approach, the inactivation of ionophoric activity due to prolonged exposure to agonists ('desensitization') can be reversed upon removal of agonist without dilution of the deposited samples. Due to the large array of glass fiber filters and ion-exchange disks, the absorption filtration technique should be able to accommodate the transport and binding of soluble molecules to a variety of intact cells, membranes and reconstituted lipid vesicles. PMID- 0006309229 CU - 1996 SO - Biochim Biophys Acta 1983 Sep 7;733(2):223-33 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [200 medline neighbors] UI - 84022591 AU - Soler G AU - Farach MC AU - Farach HA Jr AU - Mattingly JR Jr AU - Martinez-Carrion M TI - Reductive methylation as a probe of the heat-labile alpha-bungarotoxin- acetylcholine receptor membrane complex: evidence for surface interactions. LA - Eng MH - Animal MH - Bungarotoxins/*metabolism MH - Cell Membrane/metabolism MH - Drug Stability MH - Electric Organ/*metabolism MH - Heat MH - Kinetics MH - Methylation MH - Oxidation-Reduction MH - Receptors, Cholinergic/*metabolism MH - Support, U.S. Gov't, Non-P.H.S. MH - Torpedo RN - 0 (Bungarotoxins) RN - 0 (Receptors, Cholinergic) PT - JOURNAL ARTICLE DA - 19831123 DP - 1983 Sep IS - 0003-9861 TA - Arch Biochem Biophys PG - 872-8 SB - M SB - X CY - UNITED STATES IP - 2 VI - 225 JC - 6SK AA - Author EM - 198401 AB - alpha-Bungarotoxin (alpha-Bgt) is a potent postsynaptic neurotoxin which blocks neurotransmission by binding very tightly to the acetylcholine-receptor (AcChR) protein. We have previously shown (P. Calvo-Fernandez, and M. Martinez-Carrion (1981) Arch. Biochem. Biophys. 208, 154-159) that alpha-Bgt free in its native solution conformation incorporates 12 methyl groups when reductively methylated using formaldehyde and sodium cyanoborohydride. We now show that when the alpha-Bgt molecule is bound to the AcChR contained in native membranes prepared from Torpedo californica electroplax, the number of accessible methylation sites is significantly reduced. This favors a model of alpha-Bgt-AcChR interaction involving significant numbers of lysyl moieties distributed over a reasonably large surface of the toxin molecule. In addition, this paper presents a novel procedure for the rapid and nondestructive dissociation of the toxin-AcChR membrane complex which takes advantage of the thermal instability of the complex. PMID- 0006625612 CU - 1992 SO - Arch Biochem Biophys 1983 Sep;225(2):872-8 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [166 medline neighbors] UI - 84000392 AU - Gonzalez-Ros JM AU - Farach MC AU - Martinez-Carrion M TI - Ligand-induced effects at regions of acetylcholine receptor accessible to membrane lipids. LA - Eng MH - Animal MH - Azides MH - Carbachol/*pharmacology MH - Cell Membrane/metabolism MH - Electric Organ/*metabolism MH - Kinetics MH - Ligands MH - Membrane Lipids/*metabolism MH - Methane/analogs & derivatives/pharmacology MH - Nitroparaffins/pharmacology MH - Receptors, Cholinergic/drug effects/*metabolism MH - Spectrometry, Fluorescence MH - Support, U.S. Gov't, Non-P.H.S. MH - Torpedo RN - 0 (Azides) RN - 0 (Ligands) RN - 0 (Membrane Lipids) RN - 0 (Nitroparaffins) RN - 0 (Receptors, Cholinergic) RN - 51-83-2 (Carbachol) RN - 69489-90-3 (pyrenesulfonyl azide) RN - 74-82-8 (Methane) RN - 75-52-5 (nitromethane) PT - JOURNAL ARTICLE DA - 19831123 DP - 1983 Aug 2 IS - 0006-2960 TA - Biochemistry PG - 3807-11 SB - M CY - UNITED STATES IP - 16 VI - 22 JC - A0G AA - Author EM - 198401 AB - The effectiveness of fluorescence quenching of pyrene-1-sulfonyl azide, a hydrophobic probe used to photo-label acetylcholine receptor (AcChR)- rich electroplax membranes [Sator, V., Gonzalez-Ros, J. M., Calvo- Fernandez, P., & Martinez-Carrion, M. (1979) Biochemistry 18, 1200], is used to study the accessibility of the covalently attached fluorophore to extramembranous quenchers as a function of occupancy of cholinergic receptor binding sites. In these membranes, binding of water-soluble cholinergic ligands to specific sites at the extracellular side affects the fluorophore located in a distant topographical area of the AcChR molecule. When a neurotransmitter analogue (carbamylcholine) is present, the susceptibility of the covalently attached fluorophore to quenching with externally added nitromethane decreases in comparison with that of the same membranes in the absence of carbamylcholine. This neurotransmitter agonist effect is, however, reversible as removal of carbamylcholine by dialysis restores the quenching effectiveness to that of resting nonliganded membranes. The presence of bound alpha- bungarotoxin produces an opposite effect to that of carbamylcholine and induces an increase in susceptibility to quenching agent. These results are interpreted in terms of long-range effects induced by occupancy of cholinergic sites which are detected by covalently bound fluorophore located at regions of the AcChR protein accessible through the lipid matrix of the Torpedo membrane. Such effects are presumably due to molecular rearrangements within the membrane-bound AcChR structure. PMID- 0006615803 CU - 1992 SO - Biochemistry 1983 Aug 2;22(16):3807-11 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [169 medline neighbors] UI - 83213317 AU - Mattingly JR Jr AU - Farach HA Jr AU - Martinez-Carrion M TI - Properties of the active site lysyl residue of mitochondrial aspartate aminotransferase in solution. LA - Eng MH - Affinity Labels/pharmacology MH - Animal MH - Apoenzymes/*antagonists & inhibitors MH - Apoproteins/*antagonists & inhibitors MH - Aspartate Transaminase/*antagonists & inhibitors MH - Binding Sites/drug effects MH - Hydrogen-Ion Concentration MH - Kinetics MH - *Lysine MH - Mitochondria, Heart/*enzymology MH - Pyridoxamine/*analogs & derivatives/pharmacology MH - Support, U.S. Gov't, P.H.S. MH - Swine RN - EC 2.6.1.1 (Aspartate Transaminase) RN - 0 (Affinity Labels) RN - 0 (Apoenzymes) RN - 0 (Apoproteins) RN - 54522-09-7 (bromoacetylpyridoxamine) RN - 56-87-1 (Lysine) RN - 67731-58-2 (bromoacetylpyridoxamine phosphate) RN - 85-87-0 (Pyridoxamine) PT - JOURNAL ARTICLE ID - HL-22265/HL/NHLBI DA - 19830708 DP - 1983 May 25 IS - 0021-9258 TA - J Biol Chem PG - 6243-9 SB - M SB - X CY - UNITED STATES IP - 10 VI - 258 JC - HIV AA - Author EM - 198309 AB - Two vitamin B6 derivatives, N-bromoacetylpyridoxamine (BAPM) and its phosphate ester have been found to be affinity-labeling reagents for mitochondrial aspartate aminotransferase (EC 2.6.1.1). These derivatives were first shown to react with a critical sulfhydryl group in tryptophan synthase (Higgins, W., and Miles, E. W. (1978) J. Biol. Chem. 253, 4648-4652). In the apoaminotransferase, BAPM has now been found to inactivate by covalently modifying a critical lysyl residue, preventing reconstitution of the apoenzyme by pyridoxal 5'-phosphate. The dependence of the rate of inactivation upon the concentration of the reagent is consistent with a rapid equilibrium binary complex formation prior to the inactivation reaction. Both the dissociation constant for this complex and the rate of the reaction leading to inactivation are dependent on pH. BAPM binds best from pH 7.5 to 8.5. The rate of inactivation increases from pH 6 to 9. Succinate and phosphate competitively bind to the apoenzyme, protecting against BAPM inactivation. The C-5'-phosphorylated derivative is rapidly and tightly bound by the apotransaminase to form an inactive, noncovalent adduct. This bound reagent subsequently alkylates Lys-258. The rate of this covalent incorporation increases from pH 6 to 9 and is greater than the rate of BAPM modification at all pH values. The effect of pH on the reaction rates of both pyridoxal derivatives is interpreted to indicate protonation of Lys-258 at neutral pH values. These derivatives may also be analogs to a reaction intermediate different from those observed in other affinity-labeling studies. The ionization states of the Lys-258 epsilon-amino group apparently vary with the nature of the affinity label. These variations can be explained in terms of changing ionization states of Lys-258 in the steps of catalysis as well as in terms of the occupancy of charged sites on the protein by active site- directed substrates or inhibitory compounds. PMID- 0006406477 CU - 1997 SO - J Biol Chem 1983 May 25;258(10):6243-9 ------------------------------------------------------------------------ Other Formats: [Citation Format] Links: [202 medline neighbors] UI - 83160978 AU - Farach MC AU - Martinez-Carrion M TI - A differential scanning calorimetry study of acetylcholine receptor- rich membranes from Torpedo californica. LA - Eng MH - Animal MH - Binding, Competitive MH - Bungarotoxins/metabolism MH - Calorimetry, Differential Scanning MH - Cell Membrane/physiology MH - Electric Organ/physiology MH - Kinetics MH - Receptors, Cholinergic/*physiology MH - Support, U.S. Gov't, Non-P.H.S. MH - Temperature MH - Torpedo RN - 0 (Bungarotoxins) RN - 0 (Receptors, Cholinergic) PT - JOURNAL ARTICLE DA - 19830505 DP - 1983 Apr 10 IS - 0021-9258 TA - J Biol Chem PG - 4166-70 SB - M CY - UNITED STATES IP - 7 VI - 258 JC - HIV AA - Author EM - 198307 AB - Various acetylcholine receptor-rich membrane preparations from Torpedo californica electroplax tissue were examined using the techniques of differential scanning calorimetry coupled with gel electrophoretic analysis of heat-denaturing material and functional assays following passage through discrete transitions. In unfractionated membranes, four irreversible calorimetric transitions were observed, one of which (Td = 59 degrees C) could be assigned to a complete loss of acetylcholine receptor function. A second lower temperature transition apparently corresponds to loss of certain peripheral membrane proteins including the Mr = 43,000 polypeptide and the acetylcholinesterase activity. Membrane preparations highly enriched in acetylcholine receptor polypeptides contained a major transition at 59 degrees C which could be shown to be sensitive to the presence of added ligands of the acetylcholine receptor, supporting its assignment to structural alterations of the receptor protein or its arrangement in the membrane. PMID- 0006833247 CU - 1992 SO - J Biol Chem 1983 Apr 10;258(7):4166-70 ------------------------------------------------------------------------