Brokaw C J, Nagayama S M
J Cell Biol. 1985 Jun;100(6):1875-83. doi: 10.1083/jcb.100.6.1875.
Sea urchin spermatozoa demembranated with Triton X-100 in the presence of EGTA, termed potentially asymmetric, generate asymmetric bending waves in reactivation solutions containing EGTA. After they are converted to the potentially symmetric condition by extraction with Triton and millimolar Ca++, they generate symmetric bending waves in reactivation solutions containing EGTA. In the presence of EGTA, their asymmetry can be restored by addition of brain calmodulin or the concentrated supernatant obtained from extraction with Triton and millimolar Ca++. These extracts contain calmodulin, as assayed by gel electrophoresis, radioimmunoassay, activation of brain phosphodiesterase, and Ca++-dependent binding of asymmetry-restoring activity to a trifluorophenothiazine-affinity resin. Conversion to the potentially symmetric condition can also be achieved with trifluoperazine substituted for Triton during the exposure to millimolar Ca++, which suggests that the calmodulin-binding activity of Triton is important for this conversion. These observations suggest that the conversion to the potentially symmetric condition is the result of removal of some of the axonemal calmodulin and provide additional evidence for axonemal calmodulin as a mediator of the effect of Ca++ on the asymmetry of flagellar bending.
在乙二醇双乙醚二胺四乙酸(EGTA)存在的情况下,用曲拉通X-100去膜的海胆精子(称为潜在不对称精子),在含有EGTA的再激活溶液中会产生不对称弯曲波。在用曲拉通和毫摩尔钙离子提取使其转变为潜在对称状态后,它们在含有EGTA的再激活溶液中会产生对称弯曲波。在EGTA存在的情况下,通过添加脑钙调蛋白或用曲拉通和毫摩尔钙离子提取得到的浓缩上清液,其不对称性可以恢复。通过凝胶电泳、放射免疫测定、脑磷酸二酯酶的激活以及不对称性恢复活性与三氟吩噻嗪亲和树脂的钙离子依赖性结合分析,这些提取物含有钙调蛋白。在用毫摩尔钙离子处理期间,用三氟拉嗪替代曲拉通也能实现向潜在对称状态的转变,这表明曲拉通的钙调蛋白结合活性对这种转变很重要。这些观察结果表明,向潜在对称状态的转变是轴丝钙调蛋白部分去除的结果,并为轴丝钙调蛋白作为钙离子对鞭毛弯曲不对称性影响的介质提供了额外证据。