Purification and properties of rat kidney and liver ornithine aminotransferase.

Abstract

Ornithine aminotransferase was purified from rat kidney by sonication of the isolated kidney mitochondria followed by ammonium sulfate fractionation of the solubilized protein. The ammonium sulfate precipitate was washed with distilled water and the insoluble material was extracted with 0.01 M-phosphate buffer containing 0.016 mMpyridoxal phosphate. This treatment preferentially solubilized the ornithine aminotransferase. The final preparation was purified 550-fold with a specific activity of 23.7 u/mg protein and a yield of 56%. One protein band was demonstrated by polyacrylamide gel disc electrophoresis. The molecular weight of the purified enzyme was estimated to be 165,000 by gel filtration on Sephadex G-200 column. The purified kidney enzyme was specific for L-ornithine as an amino group donor and a-oxoglutarate as an amino group acceptor. The Km for L-ornithine and aoxoglutarate was 5.9 mmol/1 and 1.0 mmol/1 respectively. The pH optimum was between 7.6 and 7.8. Kidney ornithine aminotransferase was induced by estrogen treatment. The increase in activity was due to an increase in enzyme amount as indicated by the augmentation in the specific activity of the mitochondrial preparation and in the yield of the purified enzyme. Liver ornithine aminotransferase was not affected by estrogen. The enzyme from liver could not be distinguished from the kidney enzyme by chemical or physical methods. Therefore, the variable response to estrogen seems to be organ-specific.