On the determination of isozyme levels in preparations containing cytoplasmic and mitochondrial aspartate aminotransferase.
A spectrophotometric assay has been developed for the determination of the content of each isozyme of aspartate transaminase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) in physiological fluids or tissue extracts. The methods relies on the ability of adipate, at low pH and ionic strength to inhibit the cytoplasmic isozyme but not the one from mitochondria. Two assays are necessary, one at pH 8.0 which measures the content of both isozymes and another at low pH which measures primarily the amount of mitochondrial isozyme. Results obtained by this simple procedure match those in which each isozyme is inhibited by its antibody. The validity of the results obtained by the new method was tested at different ratios of cytoplasmic:mitochondrial isozyme and with tissue extracts. Since the amounts of each isozyme determined by radial immunodiffusion match those values gathered by following enzymatic activity, it is concluded that the quantity of each isozyme obtained from its respective catalytic activity must represent the total protein content of each isozyme in a given sample.
D000818:Animals; D001219:Aspartate Aminotransferases / Q000276:immunology / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008931:Mitochondria, Muscle / Q000201:enzymology*; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D009928:Organ Specificity; D009994:Osmolar Concentration; D013552:Swine
D007527:Isoenzymes; D001219:Aspartate Aminotransferases