Michels S, Scagliarini S, Della Seta F, Carles C, Riva M, Trost P, Branlant G
Laboratoire d'Enzymologie et de Génie Génétique, Université de Nancy I, URA CNRS 457, Vandoeuvre-lès-Nancy, France.
FEBS Lett. 1994 Feb 14;339(1-2):97-100. doi: 10.1016/0014-5793(94)80393-5.
Non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.9) from spinach leaves was purified to homogeneity using an improved purification procedure. Thus, a major contaminant with molecular mass and ion-exchange properties similar to non-phosphorylating GAPDH was eliminated. Using this pure non-phosphorylating GAPDH, cofactor stereospecificity was determined by 1H NMR. Analysis of the NADPH formed from the hydride transfer from glyceraldehyde-3-phosphate to [4-2H]NADP showed that the enzyme belongs to the A-stereospecific dehydrogenase family. This stereospecificity is the same as that described for the aldehyde dehydrogenase (ALDH) superfamily and opposite to that of the phosphorylating GAPDH. Moreover, results from peptide sequencing analysis suggest a similarity in sequence between the non-phosphorylating GAPDH and ALDHs. Thus, the results taken all together strongly suggest that non-phosphorylating GAPDH belongs to the ALDH family and has no close relationship to the phosphorylating GAPDH class.
采用改进的纯化方法，从菠菜叶中纯化出了非磷酸化的NADP依赖型甘油醛-3-磷酸脱氢酶（GAPDH）（EC 1.2.1.9），使其达到了同质状态。这样，就消除了一种分子量和离子交换特性与非磷酸化GAPDH相似的主要污染物。使用这种纯的非磷酸化GAPDH，通过1H NMR确定了辅因子的立体特异性。对从甘油醛-3-磷酸向[4-2H]NADP进行氢化物转移所形成的NADPH的分析表明，该酶属于A-立体特异性脱氢酶家族。这种立体特异性与醛脱氢酶（ALDH）超家族所描述的相同，与磷酸化GAPDH的相反。此外，肽测序分析结果表明，非磷酸化GAPDH与ALDHs在序列上具有相似性。因此，综合所有结果强烈表明，非磷酸化GAPDH属于ALDH家族，与磷酸化GAPDH类别没有密切关系。
