Purification and Properties of NADP+-linked Glycerol Dehydrogenase from Neurospora crassa

Abstract

The properties of NADP+-linked glycerol dehydrogenase (EC 1.1.1.72) from N. crassa were studied following 505-fold purification, with 54% yield, by gel filtration, ion exchange chromatography and affinity chromatography. Specific staining for the purified enzyme after disc gel electrophoresis revealed a single band and after isoelectric focusing, 5 bands. No enzymically inactive bands were detected by general protein staining in control gels. Total MW was estimated to be about 160,000 with a subunit MW of about 43,000. The forward reaction from glycerol to D-glyceraldehyde had a pH optimum of 9.5 and was specific for glycerol as substrate, since no activity was detected with other polyols tested. The reverse reaction had a pH optimum of 6.5 and was maximal with D-glyceraldehyde as substrate. Km values for glycerol and D-glyceraldehyde were 1.43 .times. 10-1 M and 1.15 .times. 10-2 M, respectively. Dihydroxyacetone also served as substrate in the reverse reaction. The enzyme was NADP+-specific.