Substrate specificity and activities of glyoxylate and hydroxyypyruvate reductases from leaf extracts: differentiation by ammonium sulfate fractionation and by immunoprecipitation

Abstract

Leaf extracts from seven monocotyledonous and dicotyledonous species contained considerable levels of NADPH‐dependent glyoxylate‐ and hydroxypyruvate reductase activities. These activities ranged from 0.02 to 0.22 μmol (mg protein)−1 min−1. For all plants tested, the glyoxylate reductase (GR) activity, assayed with either NADPH or NADH, was sensitive to inhibition by acetohydroxamate, a glycine analogue. Hydroxypyruvate reductase (HPR) activities were unaffected by acetohydroxamate. Differential precipitation of soluble leaf proteins of spinach, pea and barley by ammonium sulfate (0–45% and 45–60% saturation) indicated the presence of at least three distinct reductases, which differed in their specificities for glyoxylate, hydroxypyruvate and NAD(P)H. For all species, the NADH‐dependent HPR‐activity was almost completely precipitated by low ammonium sulfate concentration (45%), while precipitation of the NADPH‐GR, NADH‐GR and, to some extent, NADPH‐HPR activities required 60% ammonium sulfate. The NADPH‐dependent GR and HPR activities had high affinity for glyoxylate and hydroxypyruvate, respectively, as indicated by low apparent Km values of 40–120 μM. The occurrence of at least three distinct reductases utilizing hydroxypyruvate and/or glyoxylate as substrate was supported by antibody‐precipitation studies using antibodies prepared against NADH(NADPH)‐HPR, the well‐known peroxisomal enzyme that also shows non‐specific GR activity. These data are discussed with respect to recent reports on the purification and characterization of NADPH(NADH)‐GR, and NADPH (NADH)‐HPR, two cytosolic reductases, and the role is assessed for these enzymes in reducing hydroxypyruvate and glyoxylate that may be leaked from peroxisomes.