Simultaneous purification of mitochondrial and cytoplasmic isozymes of aspartate aminotransferase from beef kidney.
Mitochondrial and cytoplasmic isozymes of aspartate transaminase are separated from beef kidney homogenates by ammonium sulfate fractionation. The mitochondrial isozyme is purified essentially as described earlier (Eur. J. Biochem., 1972, 26, 196-206) with slight modification in order to increase the yield. The cytoplasmic isozyme is purified by heat treatment followed by ion exchange cellulose chromatography and gel chromatography. The enzyme is pure in the ultracentrifuge and in polyacrylamide gel electrophoresis; it shows only one anionic band and no subforms. It has a molecular weight of 93,000 +/- 2000 and is composed of two subunits of 46,000 M.W. The enzyme has a specific activity of 49 micronmoles of oxalacetate x min-1 x mg-1. It contains 5 SH groups per subunit; three are directly titratable with p-mercuribenzoate and the other two only after addition of 0.2% SDS; there is no evidence of S-S groups. Km values for aspartate, glutamate, alpha-ketoglutarate and oxalacetate are in the order 1.25, 3.2, 0.06 and 0.41 mM in the cytoplasmic isozyme and 0.7, 5.0, 1.25 and 0.12 mM in the mitochondrial one.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001219:Aspartate Aminotransferases / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D003593:Cytoplasm / Q000201:enzymology*; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000032:analysis
D000596:Amino Acids; D007527:Isoenzymes; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D001219:Aspartate Aminotransferases