Purification and properties of the major isozymic form of cytoplasmic glycerol-3-phosphate dehydrogenase from rabbit liver.

The major isozymic form of sn-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) has been purified from rabbit liver using a simplified three-step chromatographic procedure involving an ion exchange and two affinity chromatography steps. The 1200-fold purified enzyme is electrophoretically homogeneous, nucleotide-free, and possesses a specific activity of 295 units/mg and an isoelectric point of 6.5. A steady-state kinetic analysis was applied to both the forward and reverse reactions. The NADH oxidation reaction was found to adhere to Michaelis-Menten behavior with Km values of 22 micrometer and 75 micrometer for NADH and dihydroxyacetone phosphate, respectively. In the NAD reduction reaction, sigmoidal kinetic patterns were observed when NAD was the variable substrate whereas with sn-glycerol-3-phosphate as the variable substrate, strictly hyperbolic kinetics were observed. The apparent Km values for NAD and glycerol-3-phosphate were 83 and 909 micrometer, respectively. By comparison with published reports, these results demonstrate that the rabbit muscle and liver isozymes of sn-glycerol-3-phosphate dehydrogenase have different kinetic properties and suggest that the liver isozyme is better adapted to participation in glyconeogenesis in vivo.