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Query: UNIPROT:P17174 (aspartate aminotransferase)
 14,872 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Structural features which control folding of homologous proteins in cell-free translation systems. The effect of a mitochondrial-targeting presequence on aspartate aminotransferase. 825 54

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.
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PMID:Protein folding in a cell-free translation system. The fate of the precursor to mitochondrial aspartate aminotransferase. 844 Jun 86

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.
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PMID:Interaction of a dimeric mitochondrial precursor with phospholipid vesicles: direct association of each subunit with the membrane is required for loss of functionality. 895 Oct 49

GroEL has a greater affinity for the mitochondrial isozyme (mAAT) of aspartate aminotransferase than for its cytosolic counterpart (cAAT) (Mattingly JR Jr, Iriarte A, Martinez-Carrion M, 1995, J Biol Chem 270:1138-1148), two proteins that share a high degree of sequence similarity and an almost identical spatial structure. The effect of detergents on the refolding of these large, dimeric isozymes parallels this difference in behavior. The presence of non-ionic detergents such as Triton X-100 or lubrol at concentrations above their critical micelle concentration (CMC) interferes with reactivation of mAAT unfolded in guanidinium chloride but increases the yield of cAAT refolding at low temperatures. The inhibitory effect of detergents on the reactivation of mAAT decreases progressively as the addition of detergents is delayed after starting the refolding reaction. The rate of disappearance of the species with affinity for binding detergents coincides with the slowest of the two rate-limiting steps detected in the refolding pathway of mAAT. Limited proteolysis studies indicate that the overall structure of the detergent-bound mAAT resembles that of the protein in a complex with GroEL. The mAAT folding intermediates trapped in the presence of detergents can resume reactivation either upon dilution of the detergent below its CMC or by adding beta-cyclodextrin. Thus, isolation of otherwise transient productive folding intermediates for further characterization is possible through the use of detergents.
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PMID:Opposite behavior of two isozymes when refolding in the presence of non-ionic detergents. 1008 79

Refolding of the acid-unfolded precursor to mitochondrial aspartate aminotransferase (pmAAT) is inhibited when cytosolic Hsc70 is included in the refolding reaction (Artigues, A., Iriarte, A., and Martinez-Carrion, M. (1997) J. Biol. Chem. 272, 16852-16861). At low molar excess of Hsc70 pmAAT is recovered in insoluble aggregates containing equal amounts of Hsc70. However, in the presence of a large excess of Hsc70, refolding of pmAAT is still arrested, but the enzyme remains in solution. Similar behavior was observed with two other cytosolic chaperones, bovine Hsp90 and yeast Ydj1. Coimmunoprecipitation of pmAAT using Hsc70 antibodies confirmed the formation of soluble Hsc70-pmAAT complexes at high concentrations of the chaperone. Data from analytical centrifugation, sedimentation in glycerol gradients, and partial purification of the soluble complexes indicate that multiple Hsc70 molecules bind per pmAAT polypeptide chain. The absence of catalytic activity together with the protease susceptibility of pmAAT bound to Hsc70, Hsp90, or Ydj1 suggest that these chaperones bind and maintain pmAAT in a partially unfolded state, analogous to the import-competent conformation of the protein synthesized in cell-free extracts. Remarkably, the purified pmAAT bound to Hsc70 or Ydj1, but not to Hsp90, is imported by isolated mitochondria in a reticulocyte lysate-dependent manner. Thus, both Hsc70 and Ydj1 can trap an import-competent folding intermediate of pmAAT, but productive binding and import into mitochondria require the collaboration of additional cytosolic factors from the lysate.
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PMID:Binding to chaperones allows import of a purified mitochondrial precursor into mitochondria. 1198 13

Several halogenated alkenes are metabolized in part to cysteine S-conjugates, which are mitochondrial toxicants of kidney and, to a lesser extent, other organs. Toxicity is due to cysteine S-conjugate beta-lyases, which convert the cysteine S-conjugate into pyruvate, ammonia and a reactive sulphur-containing fragment. A section of the human population is exposed to halogenated alkenes. To understand the health effects of such exposure, it is important to identify cysteine S-conjugate beta-lyases that contribute to mitochondrial damage. Mitochondrial aspartate aminotransferase [Cooper, Bruschi, Iriarte and Martinez-Carrion (2002) Biochem. J. 368, 253-261] and mitochondrial branched-chain aminotransferase [Cooper, Bruschi, Conway and Hutson (2003) Biochem. Pharmacol. 65, 181-192] exhibit beta-lyase activity toward S -(1,2-dichlorovinyl)-L-cysteine (the cysteine S-conjugate of trichloroethylene) and S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene). Turnover leads to eventual inactivation of these enzymes. Here we report that mitochondrial L-alanine-glyoxylate aminotransferase II, which, in the rat, is most active in kidney, catalyses cysteine S-conjugate beta-lyase reactions with S -(1,1,2,2-tetrafluoroethyl)-L-cysteine, S -(1,2-dichlorovinyl)-L-cysteine and S -(benzothiazolyl-L-cysteine); turnover leads to inactivation. Previous workers showed that the reactive-sulphur-containing fragment released from S -(1,1,2,2-tetrafluoroethyl)-L-cysteine and S -(1,2-dichlorovinyl)-L-cysteine is toxic by acting as a thioacylating agent - particularly of lysine residues in nearby proteins. Toxicity, however, may also involve 'self-inactivation' of key enzymes. The present findings suggest that alanine-glyoxylate aminotransferase II may be an important factor in the well-established targeting of rat kidney mitochondria by toxic halogenated cysteine S-conjugates. Previous reports suggest that alanine-glyoxylate aminotransferase II is absent in some humans, but present in others. Alanine-glyoxylate aminotransferase II may contribute to the bioactivation (toxification) of halogenated cysteine S-conjugates in a subset of individuals exposed to halogenated alkenes.
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PMID:L-alanine-glyoxylate aminotransferase II of rat kidney and liver mitochondria possesses cysteine S-conjugate beta-lyase activity: a contributing factor to the nephrotoxicity/hepatotoxicity of halogenated alkenes? 1285 50


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