Localization and Properties of Hydroxypyruvate and Glyoxylate Reductases in Spinach Leaf Particles

Abstract

A NADH-hydroxypyruvate reductase was located in peroxisomes isolated from spinach leaves. The enzyme was precipitated by between 15 to 23 g of (NH4)2SO4 per 100 ml and had a pH optimum at 6.4. It was also active with NADPH at a pH optimum of 5.1, and the NADH:NADPH ratio of maximum activity was about 13:1. The reductase with either NADH or NADPH was about 4.3-fold more active with hydroxypyruvate than with glyoxylate. Through separation of proteins by electrofocusing, by Sephadex G200 column chromatography, and by starch gel and polyacrylamide gel electrophoresis, it was shown that a single peroxisomal protein catalyzed these reactions. The enzymic activity with NADH was not significantly affected by salts, except for nitrate, which was inhibitory. Activity with NADPH was inhibited by salts. A NADPH-glyoxylate reductase was located in the chloroplasts, which were separated from other particles by isopycnic sucrose density gradient centrifugation. It reduced glyoxylate 16-fold faster than hydroxypyruvate and was highly specific for NADPH. The enzyme was precipitated by between 30 to 45 g of (NH4)2SO4 per 100 ml and had a pH optimum of 6.2. Maximum activity per g of tissue of the chloroplast reductase, as measured with NADPH and glyoxylate, was about 1/20 that of the peroxisomal enzyme measured with NADH and hydroxypyruvate. Peroxisomes may function for disposal of excess reducing equivalents from photosynthesis via a glycolate-glyoxylate shuttle. Excess NADPH formed in the chloroplasts is consumed in the reduction of glyoxylate to glycolate, which is then oxidized in the peroxisome. Glyoxylate may return to the chloroplast to complete the cycle or be further metabolized in the peroxisomes. The peroxisomal reductase functions in this latter process for the reduction of hydroxypyruvate to glycerate.