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The nature of the multiple forms of cytoplasmic aspartate aminotransferase from pig and sheep heart.

ABSTRACT: Starch-gel electrophoresis of sheep heart aspartate aminotransferase was carried out over the range pH7.0-8.5. The enzyme separates into three subforms in the same way as the pig heart enzyme. As the pH was increased the distance migrated by each subform increased by the same amount, so that they remained the same distance apart. Titration of the enzyme over the appropriate pH range was used to calculate the difference in charge between the subforms and it was concluded that they differ by one charged group per dimer from their nearest neighbour on the electrophoretogram over the whole pH range studied. It was also shown that the pig-heart alpha and beta subforms differ by almost one charged group per dimer in the range pH5.5-5.7 and that the spacing between the subforms on starch-gel electrophoresis at pH8.0 is the same as that for the sheep enzyme. Since the charge difference between the subforms is maintained over such a wide range of pH, it is concluded that they probably differ from each other in covalent structure, because of the improbability that conformational differences can give rise to such behaviour. The relationship between the subforms and inactive binding of the coenzyme is also examined.

SUBMITTER: John R

PROVIDER: S-EPMC1168092 | biostudies-other | 1974 Aug

REPOSITORIES: biostudies-other

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The unfolding and refolding of cytoplasmic aspartate aminotransferase from pig heart.

Project description:The unfolding of cytoplasmic aspartate aminotransferase from pig heart in solutions of guanidinium chloride (GdnHCl) was studied. Data from protein fluorescence, c.d. and thiol-group reactivity indicated that the enzyme was unfolded in 6 M-GdnHCl. Spectroscopic studies showed that this unfolding was accompanied by dissociation of the pyridoxal 5'-phosphate cofactor. On dilution of the GdnHCl, re-activation of the enzyme occurred in reasonable yield, provided that dithiothreitol and pyridoxal 5'-phosphate were present. The regain of activity obeyed second-order kinetics. In the absence of added dithiothreitol and pyridoxal 5'-phosphate, substantial formation of high-Mr aggregates occurred.

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Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver.

Project description:1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s(20,w) of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25 degrees C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25 degrees C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.

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Two forms of aspartate aminotransferase in rat liver and kidney mitochondria.

Project description:1. Butan-1-ol solubilizes that portion of rat liver mitochondrial aspartate aminotransferase (EC 2.6.1.1) that cannot be solubilized by ultrasonics and other treatments. 2. A difference in electrophoretic mobilities, chromatographic behaviour and solubility characteristics between the enzymes solubilized by ultrasonic treatment and by butan-1-ol was observed, suggesting the occurrence of two forms of this enzyme in rat liver mitochondria. 3. Half the aspartate aminotransferase activity of rat kidney homogenate was present in a high-speed supernatant fraction, the remainder being in the mitochondria. 4. A considerable increase in aspartate aminotransferase activity was observed when kidney mitochondrial suspensions were treated with ultrasonics or detergents. 5. All the activity after maximum activation was recoverable in the supernatant after centrifugation at 105000g for 1hr. 6. The electrophoretic mobility of the kidney mitochondrial enzyme was cathodic and that of the supernatant enzyme anodic. 7. Cortisone administration increased the activities of both mitochondrial and supernatant aspartate aminotransferases of liver, but only that of the supernatant enzyme of kidney.

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The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with thermolysin and elastase.

Project description:Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.

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The primary structure of aspartate aminotransferase from pig heart muscle. Digestion with a proteinase having specificity for lysine residues.

Project description:Carboxymethylated aspartate aminotransferase was digested with a proteinase claimed to be specific for lysine residues. Complete cleavage occurred at 12 of the 19 lysine residues in the protein, but at the remaining seven residues cleavage was either restricted or absent. In addition, cleavage was observed at three of the 26 arginine residues. These results are discussed with reference to the amino acid residues adjacent to points of complete or restricted cleavage. The complete primary structure of aspartate aminotransferase, based on these and other studies, is given. Evidence for the assignment of some acid and amide side chains has been deposited as Supplementary Publication SUP 50050 (11 pp.) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975) 145, 5. The evidence for the assignment of residue 366 was less conclusive than for the other acid and amide side chains and is, therefore, given in the main paper.

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Purification and general properties of aspartate aminotransferase of ox heart.

Project description:1. A five-step procedure for preparing highly purified aspartate aminotransferase from ox heart is described. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis in starch gel and in polyacrylamide gel. 3. The pure enzyme has an isoelectric point of about pH5, and E(1%) (1cm.) 14.40 at 278mmu. 4. The molecular weight of the pure enzyme was determined as 96000 by sedimentation equilibrium. 5. The pH optimum for the pure enzyme was about 8. It was determined by a new assay technique. 6. A difference in the electrophoretic migration rate between the enzyme from ox heart and brain and the enzyme from pig heart and brain suggests a species specificity rather than an organ specificity. 7. A new effect of deionization on the visible-absorption spectrum of the enzyme was observed.

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