Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae.
Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae.
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DOI：
10.1016/s0021-9258(18)47833-7
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发表时间：
1987-10
期刊：
影响因子：
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通讯作者：
Michael J. Kelley;G. Carman
中科院分区：
文献类型：
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作者：
Michael J. Kelley;G. Carman
从酿酒酵母中纯化了膜相关磷脂生物合成酶CDP-二酰甘油合成酶(CTP：磷脂酸胞苷转移酶，EC 2.7.7.41)，纯化倍数为2300倍。纯化步骤包括线粒体膜Triton X-100增溶、CDP-甘油-琼脂糖亲和层析和羟基磷灰石层析。通过天然和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定，该过程得到了几乎均一的酶制剂。线粒体结合和纯化的CDP-二酰甘油合成酶的辐射灭活表明，该天然酶的相对分子质量为114,000。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法纯化的酶制剂产生两个亚基，分子量分别为56,000和54,000。纯化的酶制备抗体免疫沉淀CDP-二酰甘油合成酶活性和亚基。在pH 6.5时，CDP-甘油二酯合成酶的活性依赖于镁离子和Triton X-100。硫代反应剂抑制活性。该酶的反应活化能为9kcal/mol，在30℃以上不稳定，CTP和磷脂酸盐的Km值分别为1和0.5 mm，Vmax为4,700nmol/min/mg。动力学和同位素交换反应的结果表明，该酶催化了一系列的铋铋反应机理。
The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) was purified 2,300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4,700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism.