Primary structure of aspartate aminotransferase from horse heart and comparison with that of other homotopic and heterotopic isoenzymes

doi:10.1016/0305-0491(83)90280-8

Comparative Biochemistry and Physiology Part B: Comparative Biochemistry

Volume 76, Issue 3, 1983, Pages 483-487

https://doi.org/10.1016/0305-0491(83)90280-8 Get rights and content

Abstract

1.
1. Sulphydryl groups of mitochondrial aspartate aminotransferase from horse heart were titrated with 5,5′-dithiobis (2-nitrobenzoic acid).
2.
2. From analysis of peptic peptides, 378 amino acid residues (94.3% of the total) in the protein were identified.
3.
3. The results of amino acid sequence analysis are compared with those of cytosolic and mitochondrial aspartate aminotransferases from other sources.

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The complete amino acid sequence of cytoplasmic aspartate aminotransferase from pig heart

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Cited by (12)

Isolation and partial characterization of a broad specificity aminotransferase from Leishmania mexicana promastigotes
1998, Molecular and Biochemical Parasitology
A broad specificity aminotransferase (BSAT), with high activity with both, aromatic amino acids and aspartate as substrates, was purified to homogeneity from promastigotes of Leishmania mexicana by a method involving chromatography on DEAE-cellulose, Red-120-Sepharose and Mono Q, and gel filtration on Sephacryl S-200. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, was 90 kDa, the native enzyme is a dimer of similar subunits. The amino acid composition was determined, as well as the sequence of four internal peptides obtained by tryptic digestion. Two of these peptides, consisting of 49 amino acid residues in total, showed high similarity (57%) with corresponding sequences of plant aspartate aminotransferases, whereas they had only 33% identity with the aromatic aminotransferase of Escherichia coli, and 16% identity with the tyrosine aminotransferase from the related parasite Trypanosoma cruzi. The BSAT contained only one 1/2 Cys residue per monomer. The optimal pH for the enzyme reaction, with tyrosine and α-oxoglutarate as substrates, was 7.0. The apparent Km values for tyrosine, phenylalanine, tryptophan and glutamate, with oxaloacetate as co-substrate, were 1.3, 0.9, 0.9 and 171.8 mM, respectively; the value for aspartate with α-oxoglutarate as co-substrate was 2.5 mM, and that for alanine with α-oxoglutarate as co-substrate was 216 mM. The values for pyruvate, α-oxoglutarate and oxaloacetate, with tyrosine as co-substrate, were 5.6, 0.71 and 0.12 mM, respectively. These results suggest that the enzyme is a broad-specificity aminotransferase, able to transaminate the aromatic amino acids, aspartate, and to a lower extent alanine, with high sequence similarity to aspartate aminotransferases.
Modulation of amino acid and 2-oxo acid pools in Trichomonas vaginalis by aspartate aminotransferase inhibitors
1986, Molecular and Biochemical Parasitology
The amino acid pool sizes of Trichomonas vaginalis are reported. Alanine, glutamic acid, proline and leucine account for 72% of the measured amino acids. Growth of T. vaginalis was unaffected by gostatin, an irreversible inhibitor of aspartate aminotransferase, when the enzyme activity within the cell had been completely inhibited and a specific elevation of the aspartate pool had occurred. In media lacking aspartate and glutamate, the amino acid substrates of the aspartate aminotransferase reaction, gostatin caused a larger increase in the aspartate pool. During incubation of cells with or without gostatin, aspartate and glutamate were produced in the medium, presumably by proteolysis of medium proteins. Hence any requirement for the aspartate aminotransferase reaction might have been bypassed. Glutamate-γ-hydroxamate and aminooxyacetate inhibited growth of T. vaginalis but caused large changes in the pool-sizes of aspartate, glutamate, pyruvate plus oxaloacetate and 2-oxoglutarate, suggesting a more general interference with amino acid metabolism.
Aspartate: 2-Oxoglutarate aminotransferase from Trichomonas vaginalis: Comparison with pig heart cytoplasmic enzyme
1986, Comparative Biochemistry and Physiology -- Part B: Biochemistry and
- 1.
  1. The aspartate:2-oxoglutarate aminotransferase from the protozoon Trichomonas vaginalis exists as a mixture of sub-forms of identical Mr and amino acid composition, and of similar catalytic properties. The amino acid composition closely resembles that of aspartate aminotransferase from prokaryotic and vertebrate sources.
- 2.
  2. Some molecular and catalytic properties of the T. vaginalis aspartate aminotransferase are compared with those of the cytoplasmic pig heart enzyme. A major difference is in the ability of the trichomonal enzyme to transaminate aromatic amino acids and 2-oxo acids.
- 3.
  3. A range of inhibitors have been used to compare the active-site regions of the T. vaginalis and cytoplasmic pig heart aspartate aminotransferases.
The primary structure of mitochondrial aspartate aminotransferase from human heart
1985, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
The complete amino acid sequence of the mitochondrial asparate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from human heart has been determined based mainly on analysis of peptides obtained by digestion with trypsin and by chemical cleavage with cyanogen bromide. Comparison of the sequence with those of the isotopic isoenzymes from pig, rat and chicken showed 27, 29 and 55 differences, respectively, out of a total of 401 amino acid residues. Evidence for structural microheterogeneity at position 317 has also been obtained.
Partial amino-acid sequence and cysteine reactivities of cytosolic aspartate aminotransferase from horse heart
1984, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Cytosolic aspartate aminotransferase (l-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) from horse heart has five cysteine residues, two of which can be titrated with 5,5′-dithiobis(2-nitrobenzoid acid) in the native enzyme with no impairment of catalytic activity. The rate of modification is unaffected by the presence of substrates. Reaction with N-ethylmaleimide leads to loss of catalytic activity, the rate of inactivation being increased by the presence of substrates. Peptides containing 361 amino-acid residues (about 88% of the total number in the protein) have been isolated and aligned by comparison with the known sequence of the isotopic isoenzyme from pig heart. In the regions compared, 342 of the residues are identical. Hence, assuming that those regions are representative of the whole, then the cytosolic isoenzymes from horse and from pig have about 95% identity of structure. Uniquely among the mammalian cytosolic aspartate aminotransferase so far examined, the enzyme from horse heart is acetylated at the N-terminus.
The complete amino acid sequence of aspartate aminotransferase from Escherichia coli: Sequence comparison with pig isoenzymes
1984, Biochemical and Biophysical Research Communications
The amino acid sequence of aspartate aminotransferase from $E. coli$ B was determined by the alignment of seven cyanogen bromide peptides. The established sequence of the subunit was composed of 396 amino acid residues, and the molecular weight was calculated to be 43,573. The sequence was compared with those of the pig cytoplasmic and mitochondrial isoenzymes, showing that nearly 30% of all residues were invariant and that the $E. coli$ enzyme exhibited the same degree of homology (about 40%) with either of them. Although majority of the residues were substituted, the functional residues constituting the active site structure were conserved.

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^☆: This paper is dedicated to the memory of the late Professor Eraldo Antonini.

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Comparative Biochemistry and Physiology Part B: Comparative Biochemistry

Abstract

References (30)

Preliminary crystallographic study of aspartate: 2-oxoglutarate aminotransferase from pig heart

J. molec. Biol.

Primary structure of mitochondrial aspartate aminotransferase from turkey liver

Primary structure of haemoglobin from trout (Salmo trideus)

Interspecies comparisons of aspartate aminotransferases based on terminal and active site sequences

Comp. Biochem. Physiol.

Mitochondrial bovine aspartate aminotransferase

Interspecies comparisons of aspartate aminotransferases based on amino acid compositions

Comp. Biochem. Physiol.

Syncatalytic sulphydryl group modification in mitochondrial aspartate aminotransferases from chicken and pig heart

Biochem. biophys. Res. Commun.

Syncatalytic conformational changes in mitochondrial aspartate aminotransferases

Primary structure of mitochondrial glutamic oxaloacetic transaminase from rat liver: comparison with that of the pig heart isozyme

Biochem. biophys. Res. Commun.

Aspartate transaminase from E. coli amino acid sequence of the NH₂-terminal 33 residues and chymotryptic pyridoxyl peptide

Biochem. biophys. Res. Commun.

The effect of sulphydryl group reagents on the permeation of mitochondrial aspartate aminotransferase into mitochondria in vitro

Archs. Biochem. Biophys.

The complete amino acid sequence of cytoplasmic aspartate aminotransferase from pig heart

FEBS Lett.

Purification and basic properties of the asparate aminotransferases from a variety of sources

Comp. Biochem. Physiol.

Interspecies comparisons of aspartate aminotransferases based on immunochemical methods

Comp. Biochem. Physiol.

Cytosolic and mitochondrial isoenzymes of glutamic-oxalacetic transaminase from human heart

J. biol. Chem.

Cited by (12)

Isolation and partial characterization of a broad specificity aminotransferase from Leishmania mexicana promastigotes

Modulation of amino acid and 2-oxo acid pools in Trichomonas vaginalis by aspartate aminotransferase inhibitors

Aspartate: 2-Oxoglutarate aminotransferase from Trichomonas vaginalis: Comparison with pig heart cytoplasmic enzyme

The primary structure of mitochondrial aspartate aminotransferase from human heart

Partial amino-acid sequence and cysteine reactivities of cytosolic aspartate aminotransferase from horse heart

The complete amino acid sequence of aspartate aminotransferase from Escherichia coli: Sequence comparison with pig isoenzymes

General paperPrimary structure of aspartate aminotransferase from horse heart and comparison with that of other homotopic and heterotopic isoenzymes☆

Abstract

J. molec. Biol.

Comp. Biochem. Physiol.

Comp. Biochem. Physiol.

Biochem. biophys. Res. Commun.

Biochem. biophys. Res. Commun.

Biochem. biophys. Res. Commun.

Archs. Biochem. Biophys.

FEBS Lett.

Comp. Biochem. Physiol.

Comp. Biochem. Physiol.

J. biol. Chem.

General paper
Primary structure of aspartate aminotransferase from horse heart and comparison with that of other homotopic and heterotopic isoenzymes☆