Isolation of a sn-glycerol 3-phosphate: NAD oxidoreductase from spinach leaves

Abstract

An NAD-dependent glycerol 3-phosphate dehydrogenase ( sn-glycerol 3-phosphate: NAD oxidoreductase; EC 1.1.1.8) has been purified from spinach leaves by a three-step procedure involving ion-exchange, gel filtration, and affinity chromatography. The enzyme has been purified over 10,000-fold to a specific activity of 38. It has a molecular weight of approximately 63,500. The pH optimum for the reduction of dihydroxyacetone phosphate is 6.8 and for glycerol 3-phosphate oxidation it is 9.5. During dihydroxyacetone phosphate reduction hyperbolic kinetics were observed when either NADH or dihydroxyacetone phosphate was the variable substrate, but concentrations of NADH greater than 150 μ m were inhibitory. Michaelis constants were 0.30–0.35 m m for dihydroxyacetone phosphate and 0.01 m m for NADH. Glycerol 3-phosphate oxidation obeyed Michaelis-Menten kinetics with a K m of 0.19 m m for NAD and 1.6 m m for glycerol 3-phosphate. The enzyme was specific for those substrates, and dihydroxyacetone, glyceraldehyde, glyceraldehyde 3-phosphate, NADPH, NADP, and glycerol were not utilized. The spinach leaf enzyme appears to be in the cytoplasm and probably functions for the production of glycerol 3-phosphate from dihydroxyacetone phosphate.