Gibbons B H, Gibbons I R
J Biol Chem. 1987 Jun 15;262(17):8354-9.
Irradiation of demembranated flagella of sea urchin sperm at 365 nm in the presence of 0.05-1 mM MgATP and 5-10 microM vanadate (Vi) cleaves the alpha and beta heavy chains of the outer arm dynein at the same site and at about the same rate as reported previously for the solubilized dynein (Gibbons, I. R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J. Y., and Gibbons, B. H. (1987) J. Biol. Chem. 262, 2780-2786). The decrease in intact alpha and beta heavy chain material is biphasic, with about 80% being lost with a half-time of 8-10 min, and the remainder more slowly. Five other axonemal polypeptides of Mr greater than 350,000 are lost similarly, concomitant with the appearance of at least 9 new peptides of Mr 150,000-250,000. The motility of irradiated sperm flagella upon subsequent dilution into reactivation medium containing 1 mM ATP and 2.5 mM catechol shows a progressive decrease in flagellar beat frequency for irradiation times that produce up to about 50% cleavage of the dynein heavy chains; more prolonged irradiation causes irreversible loss of motility. Competition between photocleaved and intact outer arm dynein for rebinding to dynein-depleted sperm flagella shows that cleavage has little effect upon the ability for rebinding, although the cleaved dynein partially inhibits subsequent motility. Substitution of MnATP for the MgATP in the irradiation medium prevents the loss of all of the axonemal polypeptides during irradiation for up to 60 min and also protects the potential for subsequent flagellar motility. It is concluded that loss of the five axonemal polypeptides upon irradiation results from a Vi-sensitized photocleavage similar to that which occurs in the alpha and beta heavy chains of outer arm dynein and that these polypeptides represent Vi-inhibitable ATPase subunits of dyneins located in the inner arms and possibly elsewhere in the flagellar axoneme.
在0.05 - 1 mM MgATP和5 - 10 microM钒酸盐（Vi）存在的情况下，用365 nm波长的光照射海胆精子的去膜鞭毛，其外臂动力蛋白的α和β重链在同一位置被切割，切割速率与之前报道的溶解态动力蛋白的切割速率大致相同（吉本斯，I.R.，李 - 艾福德，A.，莫茨，G.，菲利普森，C.A.，唐，W.-J.Y.，以及吉本斯，B.H.（1987年）《生物化学杂志》262卷，2780 - 2786页）。完整的α和β重链物质的减少呈双相性，约80%在8 - 10分钟的半衰期内丢失，其余部分丢失得较慢。另外五种分子量大于350,000的轴丝多肽也以类似方式丢失，同时出现至少9种分子量为150,000 - 250,000的新肽段。将照射后的精子鞭毛随后稀释到含有1 mM ATP和2.5 mM儿茶酚的再激活培养基中，对于导致动力蛋白重链切割达约50%的照射时间，鞭毛摆动频率会逐渐降低；照射时间延长会导致不可逆的运动能力丧失。光切割的外臂动力蛋白和完整的外臂动力蛋白在与动力蛋白缺失的精子鞭毛重新结合方面存在竞争，结果表明切割对重新结合能力影响不大，尽管切割后的动力蛋白会部分抑制后续的运动能力。在照射培养基中用MnATP替代MgATP，可防止轴丝多肽在长达60分钟的照射过程中丢失，还能保护后续鞭毛运动的潜力。结论是，照射时这五种轴丝多肽的丢失是由类似于外臂动力蛋白α和β重链中发生的Vi敏化光切割导致的，并且这些多肽代表位于内臂以及可能位于鞭毛轴丝其他部位的动力蛋白的Vi抑制性ATP酶亚基。
