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J. Biol. Chem., Vol. 282, Issue 5, 2947-2955, February 2, 2007

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A Sea Urchin Sperm Flagellar Adenylate Kinase with Triplicated Catalytic Domains^*

Masashi Kinukawa¹², Mamoru Nomura¹, and Victor D. Vacquier

From the Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92093-0202 and Shimoda Marine Research Center, University of Tsukuba, 5-10-1 Shimoda, Shizuoka 415-0025, Japan

The mitochondrion of sea urchin sperm is located at the base of the sperm head, and the flagellum extends from the mitochondrion for 40 µm. 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 µM. In 1 mM ADP, flagella reactivate motility in 5 min; 1 µM 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 K_m for Sp-AK is 0.32 mM and the V_max is 2.80 µM ATP formed/min/mg of protein. When assayed at pH 7.6, the Sp-CK K_m is 0.25 mM and the V_max 5.25. At the measured in vivo concentrations of ADP of 114 µM, at pH 7.6, the axonemal Sp-AK could contribute 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.

Received for publication, August 21, 2006 , and in revised form, October 23, 2006.

^* This work was supported by National Institutes of Health Grant HD12986. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental movies S1–S4.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 858-534-2146; Fax: 858-534-7313; E-mail: makinukawa{at}ucsd.edu.

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				M. Kinukawa and V. D. Vacquier Recombinant Sea Urchin Flagellar Adenylate Kinase J. Biochem., October 1, 2007; 142(4): 501 - 506. [Abstract] [Full Text] [PDF]