Purification and some properties of cytoplasmic aspartate aminotransferase from sheep liver.
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.
D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000032:analysis / Q000302:isolation & purification*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003593:Cytoplasm / Q000201:enzymology; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009928:Organ Specificity; D011817:Rabbits / Q000276:immunology; D012756:Sheep; D013045:Species Specificity; D013552:Swine; D014461:Ultracentrifugation
D007527:Isoenzymes; D001219:Aspartate Aminotransferases