Mitochondrial aspartate transaminase. II. Isolation and characterization of the multiple forms

Michuda, Catherine M.; Martinez‐Carrion, Marino

doi:10.1021/bi00831a041

Cited by 65 publications

(21 citation statements)

References 23 publications

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“…N-terminal amino acid analysis by the dansyl method gave serine as the sole N-terminal residue. In addition, measurements of absorbance due to the protein and to the cofactor at pH 5.4 and pH 8.0 gave values similar to those reported previously for the pure enzyme [24].…”

Section: Purity Of the Enzymesupporting

confidence: 87%

“…These represent the so-called subforms of the enzyme [lo]. The origin of the subforms is not clear, but Michuda and Martinez-Carrion [24] have found no chemical differences between them and suggest that they may arise by minor conformational changes. A similar suggestion was made previously [26] to account for the origin of subforms of the cytoplasmic isozyme, although more recently [27] it has been claimed that the subforms differ from one another in the state of amidation of one or more amino acid side chains.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Large-Scale Purification and Some Properties of the Mitochondrial Aspartate Aminotransferase from Pig Heart

Barra

Bossa

Doonan³

et al. 1976

Eur J Biochem

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A method has been developed which allows isolation of 0.3 -0.5 g of mitochondrial aspartate aminotransferase in five days starting from 10 pig hearts; the method does not involve initial preparation of mitochondria. Mitochondrial malate dehydrogenase and the cytoplasmic aspartate aminotransferase may conveniently be recovered from side fractions. The product mitochondrial aspartate aminotransferase is homogeneous as judged by various electrophoretic techniques and by N-terminal analysis. Crystals of the enzyme have been obtained both from concentrated, essentially salt-free, solutions and from solutions of ammonium sulphate. The amino acid composition, N and C-terminal amino acid sequences and subunit molecular weight have been determined; these characteristic properties are compared with those of the cytoplasmic isozyme from the same source.Aspartate aminotransferase is one of a small group of enzymes the members of which exist in tissues of higher organisms in two distinct forms, one associated with the soluble fraction and the other with mitochondria [ l -31. The isozymes of aspartate aminotransferase differ from one another in chemical, physical, catalytic and immunochemical properties (for a review, see [4]). The primary structure of the cytoplasmic enzyme from pig heart has now been established [5,6] and it therefore seemed of interest to carry out similar studies on the mitochondrial form to establish whether the structures are related and if so, the degree of homology between them; this information is not available for any other pair of cytoplasmic and mitochondrial isozymes. Our preliminary results showed that the structures are indeed related [7] and this conclusion has been independently confirmed [8,9].Existing methods for isolation of the mitochondrial isozyme [4,10] started from preparations of intact mitochondria and were not suitable for large scale work. We have developed a new method, starting from whole-cell homogenates, which exploits the cationic properties of the mitochondrial isozyme and allows rapid isolation of the protein in quantities sufficient Ahbreviufion. Dansyl, 5-dimethylaminonaphthalene-l-sulphonyl.Enzymes. Aspartate aminotransferase (EC 2.6

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Section: Purity Of the Enzymesupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Large-Scale Purification and Some Properties of the Mitochondrial Aspartate Aminotransferase from Pig Heart

Barra

Bossa

Doonan³

et al. 1976

Eur J Biochem

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“…In both tryptophanase (43,44) and tyrosine phenol-lyase (45) from E. coli, cations like K ϩ were shown to induce and stabilize active conformations of these enzymes. With aspartate aminotransferase, at alkaline pH, it was shown that chloride anions (38) as well as dicarboxylic acids (46) can act as competitive inhibitors, possibly by ion pairing with positively charged residues in the active site that serve to bind the ␣-carboxylate group of the substrate.…”

Section: Fig 5 Sds-page Of Human Bcatc Andmentioning

confidence: 99%

Overexpression and Characterization of the Human Mitochondrial and Cytosolic Branched-chain Aminotransferases

Davoodi¹,

Drown²,

Bledsoe³

et al. 1998

Journal of Biological Chemistry

116

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We have developed overexpression systems for the human branched-chain aminotransferase isoenzymes. The enzymes function as dimers and have substrate specificity comparable with the rat enzymes. The human cytosolic enzyme appears to turn over 2-5 times faster than the mitochondrial enzyme, and there may be anion and cation effects on the kinetics of both enzymes. The two proteins demonstrate similar absorption profiles, and the far UV circular dichroism spectra show that no global structural changes occur when the proteins are converted from the pyridoxal to pyridoxamine form. On the other hand, the near UV circular dichroism spectra suggest differences in the local environment surrounding tyrosines within these proteins. Both enzymes require a reducing environment for maximal activity, but the mitochondrial enzyme can be inhibited by nickel ions in the presence of reducing agents, while the cytosolic enzyme is unaffected. Chemical denaturation profiles of the proteins show that there are differences in structural stability. Titration of -SH groups with 5,5-dithiobis(2-nitrobenzoic acid) suggests that no disulfide bonds are present in the mitochondrial enzyme and that at least two disulfide bonds are present in the cytosolic enzyme. Two -SH groups are titrated in the native form of the mitochondrial enzyme, leading to complete inhibition of activity, while only one -SH group is titrated in the cytosolic enzyme with no effect on activity. Although these proteins share 58% identity in primary amino acid sequence, the local environment surrounding the active site appears unique for each isoenzyme.

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“…(d) The concentrations of substrates used throughout these studies were not high enough to form abortive complexes [12]. Further, the isozyme-substrate complexes formed in the preliminary incubation mixtures were as catalytically active as the free isozyme in the assay of residual activity.…”

Section: Isozyrne Preparation and Assaymentioning

confidence: 97%

Kinetics of the Inhibition of Aspartate Aminotransferase Isozymes by dl‐Glyceraldehyde 3‐Phosphate

Kopelovich

Sweetman

Nisselbaum

1971

European Journal of Biochemistry

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The inhibitor dissociation constants and types of inhibition of the isozymes of aspartate aminotransferase from rat liver by the time-dependent inhibitor DL-glyceraldehyde 3-phosphate were determined from residual enzyme activity after equilibration of each isozyme with the inhibitor and one substrate in the preliminary incubation mixtures.The results showed that the inhibition of the cationic isozyme was completely competitive with respect to a-ketoglutarate and completely noncompetitive with respect to L-aspartate. The pyridoxamine form ofthe cationic isozyme was approximately 10 times more sensitive to inhibition by DL-glyceraldehyde 3-phosphate (Ki = 0.084 mM) than the pyridoxal form (Ki = 0.98 mM). Inhibition of the anionic isozyme was mixed partially competitive-partially noncompetitive with respect to a-ketoglutarate and partially noncompetitive with respect to L-aspartate. The pyridoxamine form of the anionic isozyme was approximately 5 times more sensitive to inhibition by DL-glyceraldehyde 3-phosphate (Ki = 0.39 mM) than the pyridoxal form (Ki = 1.9 mM). Both forms of the cationic isozyme were more sensitive t o inhibition than the corresponding forms of the anionic isozyme.The degree to which inhibition of the anionic isozyme was partial was assessed by determining the interaction constants a and /3. These were a = 6.9 and /3 = 0.32, and indimted that the anionic isozyme, when fully combined with DL-glyceraldehyde 3-phosphate, retained 32 ", , of its catalytic capacity. The cationic isozyme was completely inhibited with a = 00 and /3 = 0.Comparison of the Ki values for the pyridoxamine and pyridoxal forms of the isozymes of aspartate amhotransferase with the concentrations of D-glyceraldehyde %phosphate reported for perfused liver and for incubated erythrocytes suggests that inhibition may occur in vivo.I n a previous communication [i], we demonstrated that DL-glyceraldehyde 3-phosphate is a time-dependent inhibitor of the cationic and anionic isozymes of aspartate aminotransferase purified from rat liver. Regulation of the activity of these isozymes has been implicated in the control of gluconeogenesis in vivo [1-51. I n the present study, the inhibitor dissociation constants and types of inhibition have been determined in order to evaluate the possibility of inhibition of the isozymes of aspartate aminotransferase in vivo by the glycolytic intermediate, D-glyceraldehyde 3-phosphate. Since the inhibition is time-dependent [i], the Ki values and the types of inhibition with respect to the substrates a-ketoglutarate and L-aspartate were determined from Unuaual Abbreviations. m-Glyceraldehyde 3-phosphate, glyceraldehyde-3-P ; pyridoxal 5'-phosphate, pyridoxal-5-P ; a-ketoglutarate, ketoglutarate or Kg ; L-aspartate, aspartate or Asp.Enzymes. Aspartate aminotransferase or L-aspartate : 2-oxoglutarate aminotransferase (EC 2.6.1.1). 249measurements of residual enzyme activity after equilibration of each isozyme with various concentrations of inhibitor and one substrate. Rate equations were derived for thes...

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Mitochondrial aspartate transaminase. II. Isolation and characterization of the multiple forms

Cited by 65 publications

References 23 publications

Large-Scale Purification and Some Properties of the Mitochondrial Aspartate Aminotransferase from Pig Heart

Large-Scale Purification and Some Properties of the Mitochondrial Aspartate Aminotransferase from Pig Heart

Overexpression and Characterization of the Human Mitochondrial and Cytosolic Branched-chain Aminotransferases

Kinetics of the Inhibition of Aspartate Aminotransferase Isozymes by dl‐Glyceraldehyde 3‐Phosphate

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