Glyoxylate aminotransferase in peroxisomes from rat liver and kidney.

Abstract

An aminotransferase was isolated from peroxisomes that had been separated by isopycnic centrifugation of homogenates from rat liver or kidney. The enzyme was located only in the peroxisomes and in the soluble fraction, presumably from broken peroxisomes. Within the peroxisomes, this aminotransferase was in the soluble matrix. This specific aminotransferase was not found in spinach leaves. The enzyme was relatively specific for glyoxylate as the amino group acceptor. L-Leucine and L-phenylalanine were the preferred amino donors; other amino acids were less efficiently utilized. Rates were 181 nmol X min-1 of peroxisomal protein with leucine, and 134 with phenylalanine. The rate with serine was only 28% as fast and there was no reaction with glutamate. The reactions were essentially irreversible. Treatment of peroxisomes with 0.04% Triton X-100 increased enzyme activity 80%. The enzyme in the peroxisomes was stable at 50 degrees. The enzyme was purified 100-fold. Activities with leucine, phenylalanine, and histidine could not be separated by gel filtration and DEAE-cellulose chromatography. Its molecular weight was estimated to be 72,000. Reaction kinetics were ping-pong. The Km (glyoxylate) was 0.5 mM with leucine and 0.67 mM with phenylalanine. Km (leucine) was 2.5 mM and Km (phenylalanine) was 2.8 mM. Substrate inhibition occurred at over 4 mM glyoxylate but did not occur with the amino donors. pH optima were 8.5 for leucine and phenylalanine and 6.2 for histidine. There was no requirement for exogenous pyridoxal phosphate, but activity was inhibited by phenylhydrazine and isonicotinic acid hydrazide. The glyoxylate aminotransferase developed postnatally and increased with age until rats were 40 days old. There was more activity in female than male rats. About 50% of the activity disappeared if rats were starved overnight. Clofibrate treatment of male rats increased this enzyme activity in isolated peroxisomes. Rats on a high casein diet had slightly higher enzyme activity.