Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.6.1.1 (aspartate aminotransferase)
21,665 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The precursor protein of pig mitochondrial aspartate aminotransferase (pre-mAspAT) contains a 29-residue presequence (Joh, T., Nomiyama, H., Maeda, S., Shimada, K., and Morino, Y. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1-5). Pre-mAspAT produced in an in vitro transcription and translation system was avidly imported into pig and rat liver mitochondria to be processed to the mature form of the enzyme. The pre-mAspAT was also processed to the mature form upon incubation with mitochondrial extracts. We synthesized precursor proteins with alterations within the presequence and compared quantitatively the effects of these mutations on the rates of both import and processing. Single and multiple substitutions of four basic residues with neutral amino acids at positions 5, 8, 18, and 28 showed that each residue contributes differentially to import and processing. Substitutions of His5 and Arg8 with glycines abolished the import activity but did not appreciably affect the rate of processing. Substitution of Arg28 with leucine at the position adjacent to the cleavage site seriously impaired the processing without appreciably affecting the rate of import. Analysis of deletions revealed that the amino-terminal region from position 2 to 8 was essential for both the import and processing. Thus the positive charges in the amino-terminal region are critical for import while the amino-terminal peptide segment and the cleavage site region appear to be requisite for recognition by a processing protease.
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PMID:Import and processing of precursor to mitochondrial aspartate aminotransferase. Structure-function relationships of the presequence. 270 79

Fulicin (Phe-D-Asn-Glu-Phe-Val-NH2) is an endogenous neuropeptide containing a D-amino acid from ganglia of the African giant snail Achatina fulica. We have cloned a novel cDNA (1,995 nucleotides) encoding a fulicin precursor from the snail cerebral and subesophageal ganglia. The fulicin precursor protein (357 amino acids) contains one copy of fulicin and at least nine other putative alpha-amidated neuropeptides composed of four to six amino acid residues. Seven of the nine neuropeptides were novel, and the other two had the same structures as Mytilus inhibitory peptide-related peptides previously isolated from the ganglia of Helix pomatia. All sequences of 10 peptides are flanked by Lys-Arg(Lys) at the N-terminus and by Gly-Lys-Arg(Lys) at the C-terminus. Nucleotide sequence analysis revealed that D-Asn present in fulicin is encoded by the usual L-Asn codon (AAT). Although fulicin has as yet only been isolated from the central ganglia. RNA blot analysis revealed that single transcripts of approximately 2.0 kb in size also exist in the ventricles and atria. These results suggest that fulicin and related peptides are produced in neurons and the heart by the processing of a ribosomally made precursor, although the mechanism of in-chain epimerization remains unclear.
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PMID:A novel cDNA sequence encoding the precursor of the D-amino acid-containing neuropeptide fulicin and multiple alpha-amidated neuropeptides from Achatina fulica. 772 9

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.
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PMID:Acid-induced reversible unfolding of mitochondrial aspartate aminotransferase. 807 19

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

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.
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PMID:Refolding of the precursor and mature forms of mitochondrial aspartate aminotransferase after guanidine hydrochloride denaturation. 822 37

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

A clone encoding a plastid isoenzyme of aspartate amino-transferase (AAT5) was isolated from an Arabidopsis genomic library and its complete sequence determined. The gene for AAT5 (asp5) contains an open reading frame of 2447 bp comprising 11 exons separated by introns ranging in length from 74 to 207 bp. The upstream regulatory region contains a putative TATA box and multiple copies of two sequence motifs, CTCTT and AAAGAT, previously associated with nodule-specific gene activity in legumes. The deduced primary amino acid sequence of the protein product of asp5 was used to generate a three-dimensional structure of the AAT5 protein by using the computer program Sybyl: Biopolymer Composer and known AAT structures on the protein databases. Both the mature protein and its precursor protein containing a putative N-terminal transit peptide were modelled. The resulting structure of the precursor protein indicated that the transit peptide might also inhibit dimerization of the protein until after its translocation across the chloroplast membrane. The derived structure of the mature protein was then analysed in terms of its component elements of secondary structure, and the positions on the polypeptide back-bone corresponding to intron insertion sites were determined. It is observed that the introns tend to map to regions between structural subdomains of the protein and also map to sites on the surface of the molecule. The asp5 gene in Arabidopsis is thus consistent with Gilbert's exon-shuffling theory of gene evolution [Gilbert (1985) Science 228, 823-824]. A high degree of conservation of intron insertion sites between AAT genes from different plants and animals is observed, particularly within the part of the gene encoding a large beta-sheet structure that forms the structural and functional core of the protein. This beta-sheet structure is thus believed to compromise an ancient and very highly conserved moiety of the molecule.
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PMID:Chloroplastic aspartate aminotransferase from Arabidopsis thaliana: an examination of the relationship between the structure of the gene and the spatial structure of the protein. 892 Oct 7

Mitochondrial processing peptidase is a heterodimer consisting of alpha-mitochondrial processing peptidase (alpha-MPP) and beta-MPP. We investigated the role of alpha-MPP in substrate recognition using a recombinant yeast MPP. Disruption of amino acid residues between 10 and 129 of the alpha-MPP did not essentially impair binding activity with beta-MPP and processing activity, whereas truncation of the C-terminal 41 amino acids led to a significant loss of binding and processing activity. Several acidic amino acids in the region conserved among the enzymes from various species were mutated to asparagine or glutamine, and effects on processing of the precursors were analyzed. Glu353 is required for processing of malate dehydrogenase, aspartate aminotransferase, and adrenodoxin precursors. Glu377 and Asp378 are needed only for the processing of aspartate aminotransferase and adrenodoxin precursors, both of which have a longer extension peptide than the others studied. However, processing of the yeast alpha-MPP precursor, which has a short extension peptide of nine amino acids, was not affected by these mutations. Thus, effects of substitution of acidic amino acids on the processing differed with the precursor protein and depended on length of the extension peptides. alpha-MPP may function as a substrate-recognizing subunit by interacting mainly with basic amino acids at a region distal to the cleavage site in precursors with a longer extension peptide.
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PMID:Role of alpha-subunit of mitochondrial processing peptidase in substrate recognition. 973 75

We have determined the 2905 nucleotide sequence of the rhesus macaque factor IX complementary DNA (cDNA) and found it to be greater than 95% identical to that of the human factor IX cDNA. The cDNA has a large 3' untranslated region like the human cDNA, but unlike the human cDNA has two polyadenylation sites 224 nucleotides apart that are used for transcription of the messenger RNA. The deduced amino acid sequence is greater than 97% identical to that of human factor IX, differing in only 11 of 461 amino acids in the complete precursor protein. We found a single silent polymorphism in the nucleotide sequence at the third position of the codon for asparagine at position 167 in the secreted protein (AAC/AAT). All residues subject to posttranslational modifications in the human protein are also found in the rhesus factor IX sequence. The high degree of homology between the rhesus and human factor IX proteins suggested the possibility that the human factor IX protein might be nonimmunogenic in the rhesus. We tested the immunogenicity of human factor IX in three rhesus macaques by repeated intravenous injections of monoclonal antibody-purified, plasma-derived human factor IX over the course of more than a year and assessed the recovery and half-life of the infused protein, as well as in vitro indicators of antihuman factor IX antibodies. Human factor IX recovery and half-life remained unchanged over the course of a year in the three animals studied, and aPTT mixing studies showed no evidence for neutralizing antihuman factor IX antibodies. An outbred, nonhuman primate model that permits assessment of the level and duration of factor IX expression as well as vector safety would complement the use of other (mouse and canine) hemophilia B animal models in current use for the development of gene therapy for hemophilia B.
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PMID:The rhesus macaque as an animal model for hemophilia B gene therapy. 1006 60

The possible contribution of the mature portion of a mitochondrial precursor protein to its interaction with membrane lipids is unclear. To address this issue, we examined the interaction of the precursor to mitochondrial aspartate aminotransferase (pmAAT) and of a synthetic peptide corresponding to the 29-residue presequence peptide (mAAT-pp) with anionic phospholipid vesicles. The affinity of mAAT-pp and pmAAT for anionic vesicles is nearly identical. Results obtained by analyzing the effect of mAAT-pp or full-length pmAAT on either the permeability or microviscosity of the phospholipid vesicles are consistent with only a shallow insertion of the presequence peptide in the bilayer. Analysis of the quenching of Trp-17 fluorescence by brominated phospholipids reveals that this presequence residue inserts to a depth of approximately 9 A from the center of the bilayer. Furthermore, in membrane-bound pmAAT or mAAT-pp, both Arg-8 and Arg-28 are accessible to the solvent. These results suggest that the presequence segment lies close to the surface of the membrane and that the mature portion of the precursor protein has little effect on the affinity or mode of binding of the presequence to model membranes. In the presence of vesicles, mAAT-pp adopts considerable alpha-helical structure. Hydrolysis by trypsin after Arg-8 results in the dissociation of the remaining 21-residue C-terminal peptide fragment from the membrane bilayer, suggesting that the N-terminal portion of the presequence is essential for membrane binding. Based on these results, we propose that the presequence peptide may contain dual recognition elements for both the lipid and import receptor components of the mitochondrial membrane.
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PMID:Interaction of the precursor to mitochondrial aspartate aminotransferase and its presequence peptide with model membranes. 1093 77


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