Isolation and characterization of adipose tissue glycerol-3-phosphate dehydrogenase.
Recent studies on the differentiation of human preadipocytes have extensively used GPDH as a convenient marker enzyme to follow the development of the cells. Since the properties of human adipose tissue GPDH is largely unknown, it was considered of interest to characterize the purified enzyme. Glycerol-3-phosphate dehydrogenase was purified to homogeneity using blue Sepharose and hydroxylapatite chromatography. Monomeric molecular mass of GPDH was estimated using SDS-PAGE while the dimeric mass was estimated using non-denaturing PAGE. Fluorometric titrations were used to measure the binding of NADH to the enzyme. Inactivation experiments with proteolytic enzymes, urea and heat treatment were used to investigate a possible conformational change due to NADH binding. The purified enzyme displayed a monomeric molecular mass of 35,000 Da, a dimeric molecular mass of 74,000 Da and an isoelectric point (pI) of 5.85. The enzyme exhibited a pH optimum of 7.5 for the reduction of DHAP and 9.0 for G-3-P oxidation. Glycerol (50%) was found to stabilize the enzyme activity during storage, but altered the kinetic properties of the enzyme, acting as a competitive activator with respect to DHAP reduction. GPDH was inhibited by sulfhydryl modifying reagents and fatty acids. The effectiveness of inhibition by saturated fatty acids increased proportionately with chain length from decanoate to stearate. In addition preincubation of the enzyme, in the presence of oleate, resulted in a time dependent inactivation. Time dependent inactivation of GPDH by both iodoacetate and oleate was prevented by the presence of NADH but not NAD+.(ABSTRACT TRUNCATED AT 250 WORDS)
Koekemoer
T C
TC
Department of Biochemistry, University of Port Elizabeth, South Africa.
Litthauer
D
D
Oelofsen
W
W
D000273:Adipose Tissue / Q000201:enzymology*; D002846:Chromatography, Affinity; D004099:Dihydroxyacetone Phosphate / Q000378:metabolism; D004789:Enzyme Activation; D005227:Fatty Acids / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D005994:Glycerophosphates / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000737:chemistry; D019807:Iodoacetic Acid; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation
D005227:Fatty Acids; D005994:Glycerophosphates; D007461:Iodoacetates; D009243:NAD; D004099:Dihydroxyacetone Phosphate; D005993:Glycerolphosphate Dehydrogenase; D005990:Glycerol; D019807:Iodoacetic Acid