Purification and characterization of glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae.
The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD+ oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saccharomyces cerevisiae strain H44-3D by affinity- and ion-exchange chromatography. SDS-PAGE indicated that the enzyme had a molecular mass of approximately 42,000 (+/- 1,000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer. The pH optimum for the reduction of dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pI of 7.4. NADPH will not substitute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent Km values obtained were 0.023 and 0.54 mM for NADH and DHAP, respectively. NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity. Ki values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.
Albertyn
J
J
Department of Microbiology and Biochemistry, University of the Orange Free State, Bloemfontein, South Africa.
van Tonder
A
A
Prior
B A
BA
D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004099:Dihydroxyacetone Phosphate / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005993:Glycerolphosphate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D009243:NAD; D009249:NADP; D004099:Dihydroxyacetone Phosphate; D005993:Glycerolphosphate Dehydrogenase