Kinukawa Masashi, Nomura Mamoru, Vacquier Victor D
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92093-0202, USA.
J Biol Chem. 2007 Feb 2;282(5):2947-55. doi: 10.1074/jbc.M607972200. Epub 2006 Dec 4.
The mitochondrion of sea urchin sperm is located at the base of the sperm head, and the flagellum extends from the mitochondrion for approximately 40 microM. These sperm have two known flagellar, non-mitochondrial, enzymatic systems to rephosphorylate ADP. The first involves the phosphocreatine shuttle, where flagellar creatine kinase (Sp-CK) uses phosphocreatine to rephosphorylate ADP. The second system, studied in this report, is adenylate kinase (Sp-AK), which uses 2 ADP to make ATP + AMP. Cloning of Sp-AK shows that, like Sp-CK, Sp-AK has three catalytic domains. Sp-AK localizes along the entire flagellum, and most of it is tightly bound to the axoneme. Sp-AK activity and flagellar motility were studied using demembranated sperm. The specific Sp-AK inhibitor Ap5A blocks enzyme activity with an IC50 of 0.41 microM. In 1 mm ADP, flagella reactivate motility in 5 min; 1 microM Ap5A completely inhibits this reactivation. No inhibition of motility occurs in Ap5A when 1 mm ATP is added to the reactivation buffer. The pH optimum for Sp-AK is 7.7, an internal pH at which sperm are fully motile. The pH optimum for Sp-CK is 6.7, an internal pH at which sperm are immotile. In isolated, detergent-permeabilized flagella, assayed at pH 7.6, the Km for Sp-AK is 0.32 mm and the Vmax is 2.80 microM ATP formed/min/mg of protein. When assayed at pH 7.6, the Sp-CK Km is 0.25 mm and the Vmax 5.25. At the measured in vivo concentrations of ADP of 114 microM, at pH 7.6, the axonemal Sp-AK could contribute approximately 31%, and Sp-CK 69%, of the total non-mitochondrial ATP synthesis associated with the demembranated axoneme. Thus, Sp-AK could contribute substantially to ATP synthesis utilized for motility. Alternatively, Sp-AK could function in the removal of ADP, which is a potent inhibitor of dynein ATPase.
海胆精子的线粒体位于精子头部的基部，鞭毛从线粒体延伸约40微米。这些精子有两种已知的鞭毛非线粒体酶系统来使ADP重新磷酸化。第一种涉及磷酸肌酸穿梭，其中鞭毛肌酸激酶（Sp-CK）利用磷酸肌酸使ADP重新磷酸化。本报告中研究的第二种系统是腺苷酸激酶（Sp-AK），它利用2个ADP生成ATP + AMP。Sp-AK的克隆表明，与Sp-CK一样，Sp-AK有三个催化结构域。Sp-AK沿整个鞭毛定位，并且大部分紧密结合在轴丝上。使用去膜精子研究了Sp-AK活性和鞭毛运动性。特异性Sp-AK抑制剂Ap5A以0.41 microM的IC50阻断酶活性。在1 mM ADP中，鞭毛在5分钟内重新激活运动性；1 microM Ap5A完全抑制这种重新激活。当向重新激活缓冲液中加入1 mM ATP时，Ap5A不会抑制运动性。Sp-AK的最适pH为7.7，这是精子完全有运动能力时的内部pH。Sp-CK的最适pH为6.7，这是精子无运动能力时的内部pH。在pH 7.6下测定的分离的、经去污剂通透处理的鞭毛中，Sp-AK的Km为0.32 mM，Vmax为每分钟每毫克蛋白质形成2.80 microM ATP。在pH 7.6下测定时，Sp-CK的Km为0.25 mM，Vmax为5.25。在体内测得的ADP浓度为114 microM时，在pH 7.6下，轴丝Sp-AK可贡献与去膜轴丝相关联的非线粒体ATP合成总量的约31%，Sp-CK为69%。因此，Sp-AK可对用于运动的ATP合成有显著贡献。或者，Sp-AK可在去除ADP方面发挥作用，ADP是动力蛋白ATP酶的有效抑制剂。
