Aminotransferases in peroxisomes from spinach leaves

Organelles in spinach leaves were isolated in sucrose gradients by isopycnic centrifugation. The peroxisomal fraction was the only cellular site for two different and irreversible aminotransferases which utilized glyoxylate as the amino acceptor in the formation of glycine. These were a serine:glyoxylate aminotransferase with a specific activity of 1.54 µmoles x min -1 x mg -1 peroxisomal protein and a glutamate:glyoxylate aminotransferase with a specific activity of 2.4. Both enzymes also catalyzed an alanine:glyoxylate reaction, and the serine:glyoxylate aminotransferase catalyzed a serine:pyruvate reaction. These activities of the peroxisomal serine:glyoxylate aminotransferase coincided in fractions isolated on ion exchange or isoelectric focusing columns. For serine:glyoxylate aminotransferase reaction the K m (glyoxylate) was 0.15 m m , the K m (pyruvate) was 2.82 m m , and the K m (serine) was 2.72 m m . The pH optimum was about 7. This enzyme was inhibited by d -serine, and phosphate buffer at 70 m m inhibited it 80%. Aspartate:α-ketoglutarate aminotransferase activity was located in peroxisomes, mitochondria, and chloroplasts. By ion exchange chromatography and polyacrylamide gel electrophoresis these were separated into three isoenzymes. Isoenzyme 1 was by far the most active, and it was the only form in mitochondria and chloroplasts. The combined specific activity of the peroxisomal aspartate aminotransferases was a low 0.15 µmole x min -1 x mg -1 protein. Isoenzyme 1 represented about half of this activity in the peroxisomes, and the peroxisomes contained in addition small amounts of Isoenzymes 2 and 3. The metabolic sequence for glycolate metabolism in leaves was modified to include the three different peroxisomal aminotransferases for glutamate:glyoxylate, serine:glyoxylate, and aspartate:α-ketoglutarate.