The localization of alpha-hydroxy acid oxidase in renal microbodies.

The enzymatic properties and cellular localization of a-hydroxy acid oxidase have been studied in renal tissue of the rat. The enzyme was most active in the presence of D, La-hydroxy valeric acid and D, La-hydroxy butyric acid and was inactive in the presence of glycolic acid and D-alanine. It appeared to have no cofactor requirement. When subjected to electrophoresis in acrylamide gels, followed by cytochemical development, the enzyme was visualized as a major form accompanied by a minor component. The properties of the major electrophoretic form as determined by cytochemical reaction and densitometric scanning were similar to those determined by quantitative biochemical assay. When subjected to differential centrifugation and density equilibrium centrifugation, particles containing a-hydroxy acid oxidase sedimented with particles containing D-amino acid oxidase; a-hydroxy acid oxidase, therefore, is associated with renal microbodies. The fact that a-hydroxy acid oxidase transfers electrons to Nitro blue tetrazolium or to Tetra nitro blue tetrazolium made it possible to localize the enzyme by a light microscopic cytochemical procedure. The enzyme was found by this method to be localized predominantly in cells of the proximal tubule. I t was present in particles approximately 0.5 p to 1.0 p in diameter mainly situated in the basal portions of these cells. These cytochemical preparations probably accurately reflect the cellular distribution of a-hydroxy acid oxidase and, hence, of those microbodies which contain the enzyme. Microbodies are subcellular particles characterized by their content of D-amino acid oxidase, urate oxidase, and catalase (Beaufay et al., ’64). While the metabolic function of microbodies is unknown, it appears significant that two of these enzymes, D-amino acid oxidase and urate oxidase, produce hydrogen peroxide while the third, catalase, breaks down this product. Recently, a fourth enzyme, a-hydroxy acid oxidase, has been identified in microbodies derived from renal tissue of the rat and from Tetrahymena (Allen and Beard, ’65; Baudhuin et al., ’65), which like Damino acid oxidase and urate oxidase, produces hydrogen peroxide. Thus the suspicion that microbodies are engaged in the metabolism of hydrogen peroxide is further strengthened. Our studies of the properties of a-hydroxy acid oxidase from renal tissue of the rat, the sedimentation behavior of particles containing this enzyme, and its localization by a microscopic cytochemical method form the subject of this paper. MATERIALS AND METHODS All work was carried out using the kidneys of 90-180-day-old male rats. J. EXP. ZOOL., 160: 329-344. The following methods were used in biochemical characterization and identification studies : x-Hydroxy acid oxidase by 2, 6-dichlorophenolindophenol (DIP) reduction, modified from Robinson et al. (’62); 0.05 M Sorenson’s phosphate buffer, pH 7.5; 0.5 M sodium L-lactate or 0.05 M L-isomer of any other a-hydroxy acid; 33 uM DIP; 33 VM N-methylphenazonium methosulfate (“phenazine methosulfate,” PMS). The change in optical density was measured at 600 mw at 25°C. a-Hydroxy acid oxidase by determination of a-keto acid formation, modified from Robinson et al. (’62); 0.05 M Sorenson’s phosphate buffer, pH 7.5; 0.5 M L-lactic acid or 0.05 M L-isomer of any other a-hydroxy acid; 33 UM PMS. After incubation at 37°C the reaction was stopped by the addition of one-half volume of 15% trichloroacetic acid. a-Keto acid in the supernatant fluid obtained by lowspeed centrifugation was determined by the formation of 2,4-dinitrophenylhydra1 Research supported by grants from the U. S. Public Health Service, Institute of Arthritis. and Metabolic Diseases (AM 5731) and The Unlverslty of Michlgan Cancer Research Institute, American Cancer Society, Michigan Division Account.