Membrane association of flagellar creatine kinase in the sperm phosphocreatine shuttle.
The flagellar creatine kinase (TCK) of Strongylocentrotus purpuratus sperm is both a principal component of sperm tail membrane preparations and a cytosolic enzyme. An improved purification scheme identified three pools of TCK, termed TCK I, TCK II, and TCK III. TCK I and II were essentially homogeneous protein preparations, while TCK III was heavily contaminated with other flagellar proteins, predominantly guanylate cyclase, and alpha- and beta-tubulin. The three TCK species are roughly present in a 1:10:1 ratio as assessed by activity measurements. TCK I and II are similar proteins as shown by two-dimensional gel electrophoresis, partial proteolytic fragmentation, and cellulose polyacetate electrophoresis and have the same pH-dependent specific activity. However, they are functionally distinct with respect to their capacity to associate with lipids. TCK II associated readily with phospholipid liposomes and detergent micelles, while TCK I did not. Association of TCK II was as a protein monomer with an apparent Kd of approximately 1-2 mM at a 10(4):1 lipid or detergent to protein ratio. Whereas the Kd estimates were pH independent, the rate of association increased 2-3-fold between pH 6.5 and 8. The data are consistent with membrane-association of TCK II being a two-step process, involving a pH-dependent, intramolecular, TCK-specific step and a charge-facilitated, but pH-independent, membrane association step. Membrane association of TCK may, together with microtubule association (Tombes, R.M., Farr, A., and Shapiro, B.M. (1988) Exp. Cell Res. 178, 307-317) represent a mechanism required for specific accumulation of the enzyme within the flagellum.
Department of Biochemistry, University of Washington, Seattle 98195.
A F
AF
Quest
B M
BM
Shapiro
D000818:Animals; D002853:Chromatography, Liquid; D003402:Creatine Kinase / Q000378:metabolism*; D003902:Detergents; D004588:Electrophoresis, Cellulose Acetate; D004591:Electrophoresis, Polyacrylamide Gel; D006162:Guanylate Cyclase / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D008823:Micelles; D010725:Phosphocreatine / Q000378:metabolism*; D012617:Sea Urchins / Q000201:enzymology*; D013082:Sperm Tail / Q000201:enzymology*; D014404:Tubulin / Q000378:metabolism
D003902:Detergents; D007527:Isoenzymes; D008823:Micelles; D014404:Tubulin; D010725:Phosphocreatine; D003402:Creatine Kinase; D006162:Guanylate Cyclase