The localization of α‐hydroxy acid oxidase in renal microbodies

The enzymatic properties and cellular localization of α‐hydroxy acid oxidase have been studied in renal tissue of the rat. The enzyme was most active in the presence of D, L‐ α‐hydroxy valeric acid and D, L‐ α‐hydroxy butyric acid and was inactive in the presence of glycolic acid and D‐alanine. It appeared to have no co‐factor 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 α‐hydroxy acid oxidase sedimented with particles containing D‐amino acid oxidase; α‐hydroxy acid oxidase, therefore, is associated with renal microbodies. The fact that α‐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. It was present in particles approximately 0.5 μ to 1.0 μ in diameter mainly situated in the basal portions of these cells. These cytochemical preparations probably accurately reflect the cellular distribution of α‐hydroxy acid oxidase and, hence, of those microbodies which contain the enzyme.