UNIPROT:P17174 - FACTA Search

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

Tyrosine, added to the growth medium of a strain of Escherichia coli K-12 lacking transaminase B, repressed the tyrosine, phenylalanine, and tryptophan aminotransferase activities while leaving the aspartate aminotransferase activity unchanged. This suggested that the aspartate and the aromatic aminotransferase activities, previously believed to reside in the same protein, viz. transaminase A, are actually nonidentical. Further experiments showed that, upon incubation at 55 C, the aspartate aminotransferase of crude extracts was almost completely stable, whereas the tyrosine and phenylalanine activities were rapidly inactivated. Apoenzyme formation was faster, and apoenzyme degradation proceeded more slowly with aspartate aminotransferase than with tyrosine aminotransferase. Electrophoresis in polyacrylamide gels separated the aminotransferases. A more rapidly moving band contained tyrosine, phenylalanine, and tryptophan aminotransferases, and a slower band contained aspartate aminotransferase. A mutant of E. coli K-12 with low levels of aspartate aminotransferase exhibited unchanged levels of tyrosine aminotransferase. Thus, transaminase A appears to be made up of at least two proteins: one of broad specificity whose synthesis is repressed by tyrosine and another, specific for aspartate, which is not subject to repression by amino acids. The apparent molecular weights of both the aspartate and the aromatic aminotransferases, determined by gel filtration, were about 100,000.
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PMID:Nonidentity of the aspartate and the aromatic aminotransferase components of transaminase A in Escherichia coli. 440 56

Data from six primary hybrids and twenty-two subclones have confirmed the assignment of the mitochondrial form of glutamate oxaloacetate transaminase to chromosome 16. Family studies have provided independent confirmation of this and have suggested the gene order PGP-16qh-GOT2-HP. These studies were made easier by the development of a new stain for the detection of GOT activity.
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PMID:Mapping studies on human mitochondrial glutamate oxaloacetate transaminase. 711 92

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-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.
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PMID:Structural features of the precursor to mitochondrial aspartate aminotransferase responsible for binding to hsp70. 755 89

To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake.
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PMID:3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake. 756 34

Panicum miliaceum has at least three isozymes of aspartate aminotransferase (AspAT); the cytosolic and mitochondrial isozymes (cAspAT and mAspAT) are major components and the third is a minor isozyme. Fractionation of leaf subcellular components showed that the minor isozyme was localized in plastids (pAspAT). We purified the three isozymes from green leaves of P. miliaceum. Both cAspAT and pAspAT consisted of triple subforms having the same molecular size but different isoelectric points. No substantial difference in enzymatic properties was observed among these isozymes besides the pH profiles. We isolated a full-length cDNA clone for pAspAT. This clone contains an open reading frame that encodes 457 amino acids. The amino-terminal region of the pAspAT precursor shares common features of plastid transit peptides. The amino acid sequence of P. miliaceum pAspAT shows higher similarity with other plant pAspATs than P. miliaceum cAspAT and mAspAT. The mRNA levels of the three isozymes were high in leaves compared with roots and mesocotyls. The three isozymes showed different expression patterns against environmental stimuli such as light and nitrate. The activities and protein levels of cAspAT and mAspAT increased during greening in accordance with those of phosphoenolpyruvate carboxylase and NAD-malic enzyme involved in the C4 pathway, primarily as a consequence of the increase in the levels of their mRNAs. By contrast, pAspAT was constitutively expressed during greening. The activity and protein levels of cAspAT and mAspAT selectively increased during recovery from an nitrogen deficit, primarily as a consequence of increase in the levels of their mRNAs while those of pAspAT remained unchanged.
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PMID:Aspartate aminotransferase isozymes in Panicum miliaceum L., an NAD-malic enzyme-type C4 plant: comparison of enzymatic properties primary structures, and expression patterns. 773 57

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.
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PMID:Homologous proteins with different affinities for groEL. The refolding of the aspartate aminotransferase isozymes at varying temperatures. 783 72

Physicochemical studies have suggested that the 43-kDa plasma membrane fatty acid binding protein (FABPpm) is closely related to the mitochondrial isoform of aspartate aminotransferase (mAspAT). In the present studies, mAspAT was not detected immunohistochemically or by immunoblotting in plasma membranes of proliferating 3T3-L1 fibroblasts. During controlled differentiation to an adipocyte phenotype, mAspAT became detectable by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated 3T3-L1 adipocytes. The pattern of expression paralleled the previously reported expression both of FABPpm and of the Vmax for saturable uptake of long chain free fatty acids. As with anti-FABPpm, antibodies to mAspAT selectively inhibited the uptake of [3H]-oleate in 3T3-L1 adipocytes but not in fibroblasts, while having no effect on uptake of either 2-deoxyglucose or the medium chain fatty acid octanoate. Preabsorption of anti-FABPpm with mAspAT, or of anti-mAspAT with FABPpm, abolished immunopositivity in immunohistochemical and immunoblotting studies, as well as the ability of either antibody to inhibit [3H]-oleate uptake. These studies provide strong biologic evidence for the identity of FABPpm and mAspAT, and for the hypothesis that FABPpm/mAspAT mediates the uptake of long chain free fatty acids.
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PMID:Mitochondrial aspartate aminotransferase expressed on the surface of 3T3-L1 adipocytes mediates saturable fatty acid uptake. 787 64

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.
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PMID:Binding to phospholipid vesicles impairs substrate-mediated conformational changes of the precursor to mitochondrial aspartate aminotransferase. 807 48

A relationship between plasma membrane fatty acid binding protein (FABPpm), a putative membrane transporter for long-chain fatty acids, and the mitochondrial isoform of aspartate aminotransferase (m-AspAT) has been reported. Accordingly, we have compared the chemical and immunological properties of rat liver m-AspAT with those of rat liver FABPpm isolated by two procedures: 1) detergent solubilization of the membranes followed by purification via fatty acid affinity chromatography (FABP-1) or 2) salt extraction of the membranes and subsequent purification by high-performance liquid chromatography (HPLC; FABP-2). Comparison of the three protein preparations revealed no differences with respect to NH2-terminal amino acid sequence, amino acid composition, peptides from tryptic digests, AspAT enzymatic activity, isoelectric point, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), retention on five different HPLC columns, and immunoprecipitation and immunoblotting of SDS-PAGE separated proteins with polyclonal antisera. Examination of the proteins by nondenaturing PAGE showed a consistent second band in FABP-1 and FABP-2 not always present in m-AspAT. However, whenever present, this band was immunoreactive with antibodies to both m-AspAT and FABP-1. Hence, FABP-1 and FABP-2 are indistinguishable from one another. They are also at least closely related, if not identical, to m-AspAT.
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PMID:Comparison of plasma membrane FABP and mitochondrial isoform of aspartate aminotransferase from rat liver. 823 19

Cytosolic aspartate aminotransferase (cAspAT) participates in gluconeogenesis in the liver and is expected to exert a glyceroneogenic function in the adipose tissue when the supply of glucose is limited. Here we demonstrate that adipose cAspAT messenger RNA (mRNA) is increased when rats are fed a low carbohydrate diet. In the 3T3-F442A, BFC-1 adipocyte cell lines and differentiated adipocytes in primary culture, a 24 h glucose deprivation induces approximately a 4-fold increase in cytosolic AspAT (cAspAT) mRNA, whereas mitochondrial AspAT mRNA remains unchanged. cAspAT activity is also increased in a weaker but reproducible manner. Addition of glucose within a physiological range of concentrations reverses the increase of cAspAT mRNA in 8 h (EC50 = 1.25 g/liter). Such a regulation requires protein synthesis and is specific for adipocytes differentiated in culture. It does not occur in Fao or H4IIE hepatoma cells, in C2 muscle cells, or in 293 kidney cells. 2-deoxyglucose mimicks glucose, while 3-orthomethyl-glucose has no effect, suggesting that glucose-6-phosphate is the effector. cAspAT mRNA stability is not affected by glucose deprivation. To ascertain the transcriptional nature of the glucose effect, we have stably transfected 3T3-F442A adipoblasts with constructs containing the chloramphenicol acetyltransferase reporter gene under the control of either 5'-deletions of the cAspAT gene promoter or internal fragments in an heterologous context. We demonstrate that a glucose response element(s) is present in the region between -1838 and -1702 bp relative to the translation start site. In this region, three DNA sequences bind nuclear proteins from adipocytes as shown by footprinting experiments. Our results indicate that cAspAT gene transcription is repressed by glucose selectively in adipocytes.
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PMID:Identification of an adipocyte-specific negative glucose response region in the cytosolic aspartate aminotransferase gene. 983 31

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