PURIFICATION OF NAD MALIC ENZYME FROM POTATO AND INVESTIGATION OF SOME PHYSICAL AND KINETIC-PROPERTIES
PURIFICATION OF NAD MALIC ENZYME FROM POTATO AND INVESTIGATION OF SOME PHYSICAL AND KINETIC-PROPERTIES
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DOI：
10.1016/0003-9861(81)90297-6
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发表时间：
1981-01-01
影响因子：
3.9
通讯作者：
WEDDING, RT
中科院分区：
文献类型：
--
作者：
GROVER, SD;CANELLAS, PF;WEDDING, RT
描述了从马铃薯块茎线粒体中纯化NAD苹果酸酶（EC 1.1.1.39）至接近同质的方法。纯化的酶对NAD或NADP具有活性，并且对Mg 2+或Mn 2+起作用。当用Mg 2+和NAD测定酶时，Vapp [表观速度]最大。当Mn ~（2+）取代Mg ~（2+）时，NAD-连接反应的Vapp降低，但所有底物的Km值均大幅下降。当使用NADP代替NAD时，Mg 2+连接的反应的Vapp降低，并且大多数底物的Km值增加。酶的最适pH值取决于所用的金属离子和辅因子，并且在6.4和6.8之间变化。在pH 6.8，饱和水平的Mg 2+和NAD，该酶的周转数为37，000 min-1。pH曲线的形状表明2至3个质子参与酶的活化，仅1个质子参与失活过程。在5 mM二硫苏糖醇和2 mM MgCl 2存在下酶的MW为490，000，如通过凝胶过滤测定的。较低MW形式的酶在较低水平的二硫苏糖醇的凝胶过滤和天然凝胶电泳中占主导地位。酶的十二烷基硫酸钠凝胶电泳显示MW为61，000和58，00的2个主带，表明高MW形式的亚基化学计量可能是α 4 β 4。由于较小的亚基可能是蛋白水解产物，因此该酶可能是相同亚基的八聚体。
A procedure is described for purification of NAD malic enzyme (EC 1.1.1.39) to near homogeneity from potato tuber mitochondria. The purified enzyme is active with either NAD or NADP, and functions with either Mg2+ or Mn2+. Vapp [apparent velocity] is greatest when the enzyme is assayed with Mg2+ and NAD. When Mn2+ replaces Mg2+ the Vapp of the NAD-linked reaction decreases but the Km values for all substrates drop substantially. When NADP is used in place of NAD, the Vapp of the Mg2+-linked reaction decreases and the Km values for most substrates increase. The pH optimum of the enzyme depends on the metal ion and cofactor used and varies between 6.4 and 6.8. At pH 6.8, with saturating levels of Mg2+ and NAD, the turnover number of the enzyme is 37,000 min-1. The shape of the pH profile indicates the involvement of 2 to 3 protons in the activation of the enzyme only 1 proton is involved in the inactivation process. The MW of the enzyme in the presence of 5 mM dithiothreitol and 2 mM MgCl2 is 490,000 as determined by gel filtration. A lower MW form of the enzyme predominates in gel filtration at lower levels of dithiothreitol and in native gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis of the enzyme reveals 2 main bands with MW of 61,000 and 58,00, suggesting that the subunit stoichiometry of the high-MW form may be .alpha.4.beta.4. Since the possibility that the smaller subunit may be a proteolytic artifact, the enzyme may prove to be an octamer of identical subunits.