The extraction and purification of two isoenzymes of L-aspartate:2-oxoglutarate aminotransferase.

Abstract

Summary 1. The isoenzymes of aspartate transaminase ( L -aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) in extracts of rat heart, liver, kidney, and skeletal muscle were investigated using a differential assay method and photoautographs of electrophoresis gels. 2. Both soluble isoenzyme I and particle-bound isoenzyme II were present in all four tissues examined. 3. Water and sucrose extractions were only partially effective in removing mitochondrial transaminase (isoenzyme II) from these tissues whereas butan-1-ol extraction was found to be very effective. 4. Following (NH 4 ) 2 SO 4 fractionation of butan-1-ol extracts from whole rat liver the peak of total transaminase activity was found in the 60–70% saturated fraction. The peak of supernatant transaminase (isoenzyme I) was in the 50–60% saturated fraction. The peak of the mitochondrial transaminase (isoenzyme II) was in the 70–80% saturated fraction. 5. Isoenzyme I was obtained from rat liver sucrose supernatant after butan-1-ol treatment by precipitation between 50 and 70% satd. (NH 4 ) 2 SO 4 . 6. Isoenzyme II was precipitated from butan-1-ol extracts of rat liver mitochondria by 70–90% satd. (NH 4 ) 2 SO 4 . 7. Further purification of these isoenzymes was achieved by DEAE-cellulose chromatography.