Koekemoer T C, Litthauer D, Oelofsen W
Department of Biochemistry, University of Port Elizabeth, South Africa.
Int J Biochem Cell Biol. 1995 Jun;27(6):625-32. doi: 10.1016/1357-2725(95)00012-E.
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)
近期关于人前脂肪细胞分化的研究广泛使用甘油磷酸脱氢酶（GPDH）作为一种方便的标记酶来追踪细胞的发育过程。由于人脂肪组织GPDH的特性在很大程度上尚不明确，因此对纯化后的该酶进行特性描述被认为是有意义的。使用蓝色琼脂糖凝胶和羟基磷灰石色谱法将甘油-3-磷酸脱氢酶纯化至同质。利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）估算GPDH的单体分子量，同时利用非变性聚丙烯酰胺凝胶电泳估算其二聚体分子量。采用荧光滴定法测量还原型辅酶Ⅰ（NADH）与该酶的结合。使用蛋白水解酶、尿素和热处理进行失活实验，以研究由于NADH结合可能导致的构象变化。纯化后的酶单体分子量为35,000道尔顿，二聚体分子量为74,000道尔顿，等电点（pI）为5.85。该酶在还原磷酸二羟丙酮（DHAP）时最适pH值为7.5，在氧化甘油-3-磷酸（G-3-P）时最适pH值为9.0。发现甘油（50%）在储存期间可稳定酶活性，但会改变酶的动力学特性，在还原DHAP方面作为竞争性激活剂起作用。GPDH受到巯基修饰试剂和脂肪酸的抑制。饱和脂肪酸的抑制效果随链长从癸酸盐到硬脂酸盐成比例增加。此外，在油酸盐存在的情况下对该酶进行预孵育会导致时间依赖性失活。碘乙酸盐和油酸盐对GPDH的时间依赖性失活可被NADH而非NAD⁺的存在所阻止。（摘要截选至250词）
