Zomer A W, de Weerd W F, Langeveld J, van den Bosch H
Centre for Biomembranes and Lipid Enzymology, Utrecht University, The Netherlands.
Biochim Biophys Acta. 1993 Oct 13;1170(2):189-96. doi: 10.1016/0005-2760(93)90070-p.
Alkyl-dihydroxyacetone phosphate synthase, the second enzyme involved in ether phospholipid biosynthesis from dihydroxyacetone phosphate and responsible for glycero-ether bond formation, has been purified from guinea-pig liver. Alkyl-dihydroxyacetone phosphate synthase was solubilized from a membrane fraction prepared from an enriched peroxisome fraction with Triton X-100 and potassium chloride. The solubilized enzyme was further purified by chromatography on QAE-Sephadex, Matrex Red, Phosphocellulose and Concanavalin A. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis alkyl-dihydroxyacetone phosphate synthase appears as a 65 kDa band. Chromatofocusing revealed an isoelectric point of pH 5.9 for the enzyme. The pH optimum of alkyl-dihydroxyacetone phosphate synthase was found to be between pH 7 and 8 in a 50 mM potassium phosphate buffer. The specific activity of the enzyme was estimated to be at least 350 nmol.min-1.mg-1, corresponding to a purification of at least 13,000-fold.
烷基二羟基丙酮磷酸合酶是参与从二羟基丙酮磷酸合成醚磷脂的第二种酶，负责甘油醚键的形成，已从豚鼠肝脏中纯化出来。烷基二羟基丙酮磷酸合酶用Triton X-100和氯化钾从富含过氧化物酶体的部分制备的膜部分中溶解出来。溶解的酶通过在QAE-葡聚糖凝胶、基质红、磷酸纤维素和伴刀豆球蛋白A上的色谱进一步纯化。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中，烷基二羟基丙酮磷酸合酶表现为一条65 kDa的条带。色谱聚焦显示该酶的等电点为pH 5.9。在50 mM磷酸钾缓冲液中，发现烷基二羟基丙酮磷酸合酶的最适pH在pH 7至8之间。该酶的比活性估计至少为350 nmol·min-1·mg-1，相当于至少13000倍的纯化。
