Tombes R M, Shapiro B M
Department of Biochemistry, University of Washington, Seattle 98195.
J Biol Chem. 1987 Nov 25;262(33):16011-9.
Two isozymes of creatine kinase have been purified from sperm of the sea urchin, Strongylocentrotus purpuratus. One isozyme was purified from the sperm flagellum, and the other from the head. Both require nonionic detergent for extraction from sperm. The flagellar isozyme is a monomeric species with an Mr of 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126,000 from sucrose density gradient and gel filtration analyses. Creatine kinase from sperm heads was localized to the mitochondrion by an antibody raised against mouse muscle creatine kinase. This purified mitochondrial isozyme is multimeric, with an Mr of 47,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but 240,000 for the native enzyme. Peptide mapping indicates that the two isozymes are not related. The following kinetic characteristics were observed for the purified flagellar and mitochondrial isozymes, respectively. In the direction of ATP formation, at pH 6.6 and 25 degrees C, specific activities were 235 and 180 units/mg; pH optima were 6.7 and 6.9 and Michaelis constants were 0.13 and 0.055 mM for ADP and 5.8 and 2.7 mM for phosphocreatine. In the direction of phosphocreatine formation, at pH 7.5 and 25 degrees C, specific activities were 29 and 47 units/mg; pH optima were 7.5 and 7.7 and Michaelis constants were 0.89 and 0.31 mM for ATP and 39 and 62 mM for creatine. These unique isozymes constitute the termini of the phosphocreatine shuttle of sea urchin sperm that is responsible for energy transport from the mitochondrion to the distal flagellum (Tombes, R. M., and Shapiro, B. M. (1985) Cell 41, 325-334; Tombes, R. M., Brokaw, C. J., and Shapiro, B. M. (1987) Biophys. J., 52, 75-86).
已从紫海胆强壮柱头虫的精子中纯化出两种肌酸激酶同工酶。一种同工酶从精子鞭毛中纯化得到，另一种从精子头部纯化得到。从精子中提取这两种同工酶均需要非离子型去污剂。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析，鞭毛同工酶为单体，其相对分子质量为145,000，而通过蔗糖密度梯度离心和凝胶过滤分析，其相对分子质量为126,000。用针对小鼠肌肉肌酸激酶产生的抗体将精子头部的肌酸激酶定位到线粒体。这种纯化的线粒体同工酶是多聚体，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析，其相对分子质量为47,000，但天然酶的相对分子质量为240,000。肽图谱分析表明这两种同工酶没有相关性。分别观察到纯化的鞭毛同工酶和线粒体同工酶具有以下动力学特征。在ATP形成方向上，于pH 6.6和25℃时，比活性分别为235和180单位/毫克；最适pH分别为6.7和6.9，ADP的米氏常数分别为0.13和0.055毫摩尔，磷酸肌酸的米氏常数分别为5.8和2.7毫摩尔。在磷酸肌酸形成方向上，于pH 7.5和25℃时，比活性分别为29和47单位/毫克；最适pH分别为7.5和7.7，ATP的米氏常数分别为0.89和0.31毫摩尔，肌酸的米氏常数分别为39和62毫摩尔。这些独特的同工酶构成了海胆精子磷酸肌酸穿梭的两端，该穿梭负责能量从线粒体运输到鞭毛末端（汤姆斯，R.M.，和夏皮罗，B.M.（1985年）《细胞》41卷，325 - 334页；汤姆斯，R.M.，布罗考，C.J.，和夏皮罗，B.M.（1987年）《生物物理学杂志》52卷，75 - 86页）。
