Purification and characterization of a novel NADPH(NADH)-dependent hydroxypyruvate reductase from spinach leaves. Comparison of immunological properties of leaf hydroxypyruvate reductases.
A novel hydroxypyruvate reductase preferring NADPH to NADH as a cofactor was purified over 1500-fold from spinach leaf extracts. The enzyme was an oligomer of about 70 kDa, composed of two subunits of 38 kDa each. The Km for hydroxypyruvate (with NADPH) was about 0.8 mM in the pH range 5.5-6.5, and 0.3 mM at pH 8.2. The Vmax. was highest in the pH range 5.5-6.5 and decreased by about 65% at pH 8.2. Above pH 6.0, the enzyme was prone to a strong substrate inhibition by hydroxypyruvate. The reductase could use glyoxylate as an alternative substrate, with rates up to one-quarter of those with hydroxypyruvate. This glyoxylate-dependent activity preferred NADPH to NADH as a cofactor. Rabbit antibodies prepared against NADPH(NADH)-hydroxypyruvate reductase were highly specific for this enzyme and did not cross-react with peroxisomal NADH(NADPH)-dependent hydroxypyruvate reductase, as found by Western immunoblots of proteins from leaf extracts of spinach, pea and wheat. Antibodies raised against purified NADH(NADPH)-hydroxypyruvate reductase were also highly specific, recognizing only their own antigen. To our knowledge, this is the first report in the literature of the occurrence of NADPH(NADH)-hydroxypyruvate reductase in leaves, and the first to provide immunological comparison of leaf hydroxypyruvate reductases. Because of the relatively high rates of the novel reductase in leaf extracts (at least 20 mumol/h per mg of chlorophyll), this enzyme might be an important side-component of the glycollate pathway (photorespiration), possibly utilizing hydroxypyruvate 'leaked' from peroxisomes, and thus contributing to the glycerate pool derived from glycollate. Because of the glyoxylate-dependent activity, the enzyme may also contribute to glycollate formation in leaves.
Kleczkowski
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LA
Department of Biochemistry, University of Missouri, Columbia 65211.
Randall
D D
DD
D000429:Alcohol Oxidoreductases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D050537:Hydroxypyruvate Reductase; D007122:Immunoelectrophoresis; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity
D009243:NAD; D009249:NADP; D000429:Alcohol Oxidoreductases; D050537:Hydroxypyruvate Reductase