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PubMed1995

Cumulative effects of mutations in newly synthesised mitochondrial aspartate aminotransferase on uptake into mitochondria.

Mutant genes were constructed which coded for the precursor form of mitochondrial aspartate aminotransferase in which residue cysteine 166 was mutated to either serine or alanine and for forms of the protein lacking both the presequence and residues 1-9 of the mature protein but carrying the same cysteine mutations. The protein products of all of these mutant genes were imported into mitochondria that had been added to the expression system but with varying degrees of efficiency. The results showed that the effects of mutation of cysteine 166 and of deletion of residues 1-9 of the mature protein on sequestration into mitochondria were essentially cumulative, suggesting that these parts of the protein are involved in distinct steps on the recognition/uptake pathway.

Alanine↗
PubMed1978

Isoenzymes of aspartate aminotransferase in chronic liver diseases.

The total activity and activity of the cytoplasmic and mitochondrial isoenzyme of aspartate aminotransferase was examined in blood plasma of 56 patients with chronic liver diseases (chronic hepatitis in 27, liver cirrhosis in 23, secondary neoplastic effection of the liver in 6). All the patients with biochemically active forms of liver disease manifested increased the total as well as cytoplasmic enzyme activity, as compared with control group, 57% of the patients manifested simultaneously also increased activity of the mitochondrial isoenzyme. In 13% of the patients with stabilised forms of liver diseases manifested isolated increase of the mitochondrial isoenzyme activity. This might be of importance for the evaluation of the course of the disease. In patients with tumorous metastases in the liver a strikingly high share and activity of mitochondrial isoenzyme was shown.

Aspartate Aminotransferases↗
PubMed1989

Aspartate aminotransferase isozymes in the genus Capsella (Brassicaceae): subcellular location, gene duplication, and polymorphism.

The subcellular location of aspartate aminotransferase isozymes (EC 2.6.1.1) in the genus Capsella (Brassicaceae) was studied. The diploid species C. grandiflora and C. rubella have three AAT isozymes, including one located in the plastids. Each locus is duplicated in the tetraploid Capsella bursa-pastoris. Variation at the plastid-coding locus exceeded that at the other loci. C. bursa-pastoris had some unique alleles not detected in the diploid species. Segregation in open-pollinated families revealed that Capsella grandiflora was outcrossing, whereas C. rubella was highly inbred, with most populations homozygous or uniform at all three loci. Inheritance in the tetraploid colonizer C. bursa-pastoris is disomic. This species was also predominantly selfing with outcrossing rates between 2% and 10%.

Alleles↗
PubMed1987

An antibody and anti-idiotypic antibody against the extra signal peptide of pre-aspartate aminotransferase.

A peptide (extra signal peptide) comprising amino acids 1-29 of pig liver pre-mitochondrial aspartate aminotransferase (p-mAAT) was synthesized chemically. The peptide was found to block the import of rat liver p-mAAT into rat liver mitochondria. An antibody raised against the peptide immunoprecipitated rat liver p-mAAT synthesized in a rabbit reticulocyte cell-free translation system. These results suggested that the extra signal peptide sequence of p-mAAT is essential for import of p-mAAT into the mitochondria and that there is structural homology between the extra signal peptides of pig and rat liver p-mAAT. An anti-idiotypic antibody against the peptide was also prepared and purified by affinity chromatography on an Affi-Gel 10 anti-peptide IgG column and was then characterized.

Animals↗
PubMed1976

[Histochemical study of the activity of aspartate aminotransferase in the spinal cord, the medulla oblongata and the central cerebellar nuclei of several vertebrates].

The aspartate aminotransferase activity (AAT) is reserched into the spinal cord, the medulla oblongata and the cerebellar nuclei of the rat, chicken, Lacerta lepida and Bufo calamitas. It's proved that the AAT activity shows in many locations, that are mainly: 1. In the nerve fibers 2. In the cytoplasmic membrane, and in the nuclear membrane of the neurons 3. In all neuronal cytoplasm, and 4. In the mitochondria of neurons and choroid plexus cells. The results base the idea that there's more than one pool of glutamic acid in relation to that AAT. It's suggested that the role that AAT plays is different in everyone of the described locations, and may be it's connected with transport phenomenons in the membrane, with energetic function on the mitochondria and with functions of the nerve impulse transmission in the synapsis. We remark, finally, the interest that the enzymatical works can have the time comming to establish homologies among similar structures of several animal's nervous system.

Animals↗
PubMed1990

Reversible dissociation and unfolding of aspartate aminotransferase from Escherichia coli: characterization of a monomeric intermediate.

The unfolding and dissociation of the dimeric enzyme aspartate aminotransferase (D) from Escherichia coli by guanidine hydrochloride have been investigated at equilibrium. The overall process was reversible, as judged from almost complete recovery of enzymic activity after dialysis of 0.7 mg of denatured protein/mL against buffer. Unfolding and dissociation were monitored by circular dichroism and fluorescence spectroscopy and occurred in three separate phases: D in equilibrium 2M in equilibrium 2M* in equilibrium 2U. The first transition at about 0.5 M guanidine hydrochloride coincided with loss of enzyme activity. It was displaced toward higher denaturant concentrations by the presence of either pyridoxal 5'-phosphate or pyridoxamine 5'-phosphate and toward lower denaturant concentrations by decreasing the protein concentration. Therefore, bound coenzyme stabilizes the dimeric state, and the monomer (M) is inactive because the shared active sites are destroyed by dissociation of the dimer. M was converted to M* and then to the fully unfolded monomer (U) in two subsequent transitions. M* was stable between 0.9 and 1.1 M guanidine hydrochloride and had the hydrodynamic radius, circular dichroism, and fluorescence of a monomeric, compact "molten globule" state.

Aspartate Aminotransferases↗
PubMed1976

Genetic analysis of aspartate aminotransferase isozymes from hybrids between Drosophila melanogaster and Drosophila simulans and mutagen-induced isozyme variants.

The aspartate aminotransferases (designated GOT1 and GOT2) are two enzymes of Drosophila melanogaster for which naturally occurring electrophoretic variants were not found. There is an electrophoretic difference between D. melanogaster and D. simulans. Since the F1 hybrid offspring of these species are sterile, a genetic analysis of the ordinary type cannot be done on differences between the two species. A method was devised to make "partial hybrids" in which one chromosome arm is homozygous for melanogaster genes in an otherwise hybrid background. By using this method, Got1 was localized to 2R and Got2 to 2L. Once a gene can be assigned to a chromosome, it may be followed in crossing schemes and mutations from mutagen treatments may be looked for. At the locus of Got1 a mutation with low activity was recovered and designated Got1lo. It was located at a genetic map position of 75 on 2R. A Got2 mutant with a greater migration to the anode was recovered and designated Got2J. It was located at a genetic map position of 3.0, and in the salivary chromosome was between 22B1 and 22B4 inclusive.

Animals↗
PubMed1982

Binding of substrates to aspartate aminotransferase. Evidence for a dissymmetrical binding.

The binding of substrates L-glutamate and 2-oxoglutarate to aspartate aminotransferase was studied by spectrophotometric titration according to Jenkins and D'Ari [J. Biol. Chem. 241 (1966) 2845-2854]. Our data, obtained over a wide range of substrate concentrations, were not consistent with the hypothesis of independent and equivalent binding sites. Two other possibilities were considered: (a) the sites are independent but not equivalent; (b) a negative cooperativity occurs between the sites. It is possible to distinguish between these two hypotheses because the substrate binding is complex, with a couple of substrates and two forms of enzyme in equilibrium with the enzyme-substrate complexes. Distinct equations delta A/Et=f[substrates], where delta A = change in absorbance upon addition of substrates to the enzyme and Et = total site concentration, were derived for each case. The data were directly fitted to these equations by an iterative multilinear regression analysis, and the equilibrium constants were computed. This analysis showed that the binding sites are independent and not equivalent, with dissociation constants for 2-oxoglutarate of 2 micro M and 280 micro M and dissociation constants for glutamate of 1.7 mM and 22 mM. The molar absorption coefficient of the binary complexes is 2250 M-1 cm-1.

Allosteric Regulation↗
PubMed1983

Postnatal changes in the activity of glutamate dehydrogenase and aspartate aminotransferase in the rat nervous system with special reference to the glutamate transmitter metabolism.

The activities of aspartate aminotransferase (AAT) and glutamate dehydrogenase (GIDH), the major glutamate metabolizing enzymes, were studied in hippocampal formation, cerebellar cortex, dorsal root ganglia, superior cervical ganglia and liver as a function of postnatal development. At birth, in all these nervous tissues the enzyme activities were quite low and showed similar levels (AAT 7-15 U/g wet weight; 0.18-0.23 U/mg protein; GIDH 3.4-13 U/g wet weight; 0.07-0.18 U/mg protein). Based on protein, AAT activity increased during the postnatal period studied 5.8 and 3.8 times in the hippocampal formation and cerebellar cortex, respectively, while the respective GIDH rise was 5.2 and 2.3 times. During postnatal maturation, enzyme activities in dorsal root ganglia showed only minor changes. In superior cervical ganglia, AAT and GIDH were remarkably constant. In liver the enzyme activities changed during postnatal development, but the activity curve profile was quite distinct from those obtained for brain regions. The steep rise of AAT and GIDH activities in brain regions is discussed as being a consequence of the maturation of preferably glutamatergic structures. Glutamatergic transmission processes obviously do not take place in superior cervical ganglia and dorsal root ganglia, and certainly not in liver. The present results suggest a quantitatively significant participation of glutamate transmitter metabolism in proportion to the whole glutamate metabolism of the CNS.

Aging↗
PubMed1999

Delineation of the insulin-responsive sequence in the rat cytosolic aspartate aminotransferase gene: binding sites for hepatocyte nuclear factor-3 and nuclear factor I.

Expression of the rat cytosolic aspartate aminotransferase gene is stimulated by glucocorticoids and repressed by insulin in the liver. The regulation by insulin and part of the glucocorticoid effect are mediated by a distal region in the promoter. A 142 bp fragment (-1844 to -1702) confers hormonal sensitivity to the heterologous thymidine kinase promoter in transient-transfection assays in H4IIEC3 hepatoma cells. Footprinting and gel-shift assays showed that several nuclear proteins bind to this region at conserved CCAAT-enhancer binding protein (C/EBP), activator protein (AP-1) and E-box sequences. Hepatocyte nuclear factor-3alpha (HNF-3)alpha and beta bind to sequences upstream of a glucocorticoid-responsive element (GRE) half-site as demonstrated by supershift experiments. Nuclear factor I (NFI)-like proteins bind downstream of the GRE half-site. These sites around the GRE motif overlap with five insulin responsive element (IRE) -like sequences (TG/ATTT). The effect of insulin was not prevented by any single mutation in the IRE-like sites. However, mutation of two IRE sites (namely IREc and d) prevented the insulin effect although only marginally affecting the glucocorticoid effect. The results suggest that the effect of insulin is due to a complex interplay of factors requiring the synergistic contribution of at least two sites and underline the contribution of HNF-3 and NFI-like proteins.

Animals↗
PubMed1999

Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate.

The three-dimensional structures of pyridoxal 5'-phosphate-type aspartate aminotransferase (AspAT) from Thermus thermophilus HB8 and pyridoxamine 5'-phosphate type one in complex with maleate have been determined by X-ray crystallography at 1.8 and 2.6 A resolution, respectively. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into one arm, one small domain, and one large domain. AspATs from many species were classified into aminotransferase subgroups Ia and Ib. The enzyme belongs to subgroup Ib, its sequence being less than 16% identical to the primary sequences of Escherichia coli, pig cytosolic, and chicken mitochondrial AspATs, which belong to subgroup Ia whose sequences are more than 40% identical and whose three-dimensional structures are quite similar with the active site residues almost completely conserved. The first X-ray analysis of AspAT subgroup Ib indicated that the overall and the active site structures are essentially conserved between the AspATs of subgroup Ia and the enzyme of subgroup Ib, but there are two distinct differences between them. (1) In AspAT subgroup Ia, substrate (or inhibitor) binding induces a large movement of the small domain as a whole to close the active site. However, in the enzyme of subgroup Ib, only the N-terminal region (Lys13-Val30) of the small domain approaches the active site to interact with the maleate. (2) In AspAT subgroup Ia, Arg292 recognizes the side chain carboxylate of the substrate; however, residue 292 of the enzyme in subgroup Ib is not Arg, and in place of Arg292, Lys109 forms a salt bridge with the side chain carboxylate. The thermostability of the enzyme is attained at least in part by the high content of Pro residues in the beta-turns and the marked increase in the number of salt bridges on the molecular surface compared with the mesophilic AspAT.

Amino Acid Sequence↗
PubMed1992

Effect of potassium and N-methyl-D-aspartate on the aspartate aminotransferase activity in cultured cerebellar granule cells.

The effect of potassium depolarization and N-methyl-D-aspartate (NMDA) on the activity of aspartate aminotransferase (AAT; EC 2.6.1.1), an enzyme suggested to be involved in neurotransmitter glutamate synthesis, was studied in cultured cerebellar granule neurons. Both KCl and NMDA increased AAT activity in a dose-dependent manner. When cells were treated 48-72 hr with 40 mM KCl or 150 microM NMDA the AAT was enhanced about 65-75%. The EC50 for NMDA and KCl were 25 microM and 17 mM, respectively. The effect of NMDA and KCl was specific for AAT without affecting the activity of other enzymes like lactate dehydrogenase or protein content and it was observed only in granule cells but not in astrocytes or cortical neurons. The effect of KCl was not mediated by an activation of excitatory amino acid receptors and was Ca(++)-dependent. The effect of NMDA was completely blocked by Mg++ and NMDA antagonists. The increase of AAT induced by AAT and KCl was blocked by cycloheximide and actinomycin D, suggesting an involvement of de novo synthesis of proteins and RNA. Kainic acid and quinolinic acid were also effective in increasing the AAT activity. The action of kainate was less effective than that of NMDA and it was observed only at relatively low concentrations (10 microM). Quinolinic acid raised the activity of AAT about 45% at a concentration of 500 microM. Other non-NMDA agonists did not modify the AAT activity. From these findings we can conclude that NMDA and KCl exert a trophic action on cerebellar granular neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1995

Decreasing the basicity of the active site base, Lys-258, of Escherichia coli aspartate aminotransferase by replacement with gamma-thialysine.

Alkylation of the K258C mutant of the wild-type aspartate aminotransferase (AATase) with bromoethylamine to give gamma-thialysine 258 was complicated by partial reaction with the five native cysteines [Planas, A., & Kirsch, J. F. (1991) Biochemistry 30, 8268-8276]. This problem is now overcome by carrying out the alkylation with K258CQ, in which Cys-258 is a unique cysteine residue in Quint, an engineered AATase in which the five cysteines have been converted to alanine [Gloss, L.M., et al. (1992) Biochemistry 31, 32-39]. The kinetics and spectral properties of the resulting enzyme, K258CQ-EA, have been examined and compared to those of WT and Quint. The replacement of Lys-258 by gamma-thia-Lys results in an acidic shift of 1.3 pH units in the pKa of the internal aldimine. The C alpha hydrogen kinetic isotope effects for Quint are 2.1 and 1.5 on D(kcat/KMAsp) and Dkcat, respectively. Replacement of Lys-258 by the weaker base, gamma-thia-Lys, increases these values to 3.3 and 2.6, respectively The changes of K258CQ-EA in ligand affinities and the keto acid half-reaction are minor; however, the kcat/KM values for amino acids are decreased by an order of magnitude. The KD values for PMP of K258CQ-EA and Quint are equal to each other (0.2 nM) and are 7-fold lower than that of WT. These combined effects are illustrated in the free energy diagrams of the reaction with L-Asp with K258CQ-EA, relative to WT (and Quint). The E.PLP and E.PMP complexes of Quint are 0.9 and 1.1 kcal/mol, respectively, more stable than those of WT.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1981

Demonstration of physical interactions between consecutive enzymes of the citric acid cycle and of the aspartate-malate shuttle. A study involving fumarase, malate dehydrogenase, citrate synthesis and aspartate aminotransferase.

By means of covalently immobilized fumarase and mitochondrial or cytoplasmic malate dehydrogenase we were able to detect physical interactions between different enzymes of the citric acid cycle (fumarase with malate dehydrogenase, malate dehydrogenase with citrate synthase and fumarase with citrate synthase) and between the enzymes of both mitochondrial and cytoplasmic halves of the aspartate-malate shuttle (aspartate amino-transferase and malate dehydrogenase). The interactions between fumarase and malate dehydrogenase were also investigated by immobilizing one enzyme indirectly through antibodies bound to Sepharose-protein A. Our results are consistent with a model in which maximally four molecules of malate dehydrogenase are bound to one fumarase molecule. This complex is able to bind either citrate synthase or aspartate aminotransferase. We propose that these enzymes bind alternatively, in order to allow the cell to perform citric acid cycle or shuttle reactions, according to its needs. The physiological meaning and implications on the regulation of metabolism of the existence of a large citric acid cycle/malate-aspartate shuttle multienzyme complex are discussed.

Animals↗
PubMed1990

Immunocytochemical localization of mitochondrial and cytosolic aspartate aminotransferase isozymes in the vestibular end-organs of rats.

The localization of mitochondrial (m-) and cytosolic (c-) aspartate aminotransferase (AAT) was examined in the vestibular ganglion neurons and sensory cells in the vestibular end-organs of rats by an indirect immunohistochemical method using antibodies specific for m- and c-AAT. Neurons in the vestibular ganglion were stained by both m- and c-AAT antibodies, but the vestibular sensory cells exhibited only m-AAT-like immunoreactivity and were not labeled by c-AAT. These findings suggested that aspartate is a neurotransmitter in the hair cells of the vestibular end-organs.

Animals↗
PubMed1994

Functional role of the amino-terminal mobile segment in catalysis by porcine cytosolic aspartate aminotransferase. Critical importance of Val17 and Phe18 for productive binding of substrates.

A notable feature of porcine cytosolic aspartate aminotransferase is the closure of the active site cleft by a mobile amino-terminal segment (residues 15-40) upon binding substrate. The functional roles of Val17 and Phe18, residues that are part of the mobile loop, have been studied in the site-directed mutants in which the size and hydrophobic nature of these residues have been changed. Absorption, circular dichroism spectra, susceptibility to protease 401, and thermal stability did not differ appreciably between wild type and mutant enzymes. In the overall transamination between aspartate and 2-oxoglutarate, V17A represented a typical Km mutant while V17I retained the substrate binding affinity fairly well. In contrast, replacement of Phe18 by Ala resulted in a large decrease in both catalytic rate and binding affinity for substrates. F18W, F18Y, and F18H showed a moderate decrease in kcat and a considerable increase in Km values. Single-turnover reactions with four individual substrates yielded analogous results to those obtained for the overall reaction and, in addition, revealed that k/Kd values of mutants F18A and F18H were over 10 times lower for C5 substrates (glutamate and 2-oxoglutarate) than those for C4 substrates (aspartate and oxalacetate). All mutant enzymes showed variously increased Kd values for substrate analogs such as 2-methylaspartate, succinate, and glutarate. 1H NMR observations of F18H, in which His18 served as a built-in probe, were in accord with the behavior that would be expected from the conformational transition. We conclude that, although Val17 and Phe18 may not be essential for catalysis, the presence of a bulky residue of appropriate size at each position is critical for productive binding of substrate.

Animals↗
PubMed1980

Complete amino acid sequence of mitochondrial aspartate aminotransferase from pig heart muscle. Tryptic peptides.

The amino acid sequences of 39 tryptic peptides from carboxymethylated mitochondrial aspartate aminotransferase from pig heart muscle were analyzed. The peptides were purified by gel filtration, ion exchange column chromatography, paper chromatography, and high voltage paper electrophoresis, and their sequences were examined by manual Edman degradation, carboxypeptidase digestion, and fragmentation with thermolysin or chymotrypsin. These peptides accounted for 318 of the total 401 amino acid residues in the protein subunit.

Amino Acid Sequence↗