Enzyme termini of a phosphocreatine shuttle. Purification and characterization of two creatine kinase isozymes from sea urchin sperm.
Two isozymes of creatine kinase have been purified from sperm of the sea urchin, Strongylocentrotus purpuratus. One isozyme was purified from the sperm flagellum, and the other from the head. Both require nonionic detergent for extraction from sperm. The flagellar isozyme is a monomeric species with an Mr of 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126,000 from sucrose density gradient and gel filtration analyses. Creatine kinase from sperm heads was localized to the mitochondrion by an antibody raised against mouse muscle creatine kinase. This purified mitochondrial isozyme is multimeric, with an Mr of 47,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but 240,000 for the native enzyme. Peptide mapping indicates that the two isozymes are not related. The following kinetic characteristics were observed for the purified flagellar and mitochondrial isozymes, respectively. In the direction of ATP formation, at pH 6.6 and 25 degrees C, specific activities were 235 and 180 units/mg; pH optima were 6.7 and 6.9 and Michaelis constants were 0.13 and 0.055 mM for ADP and 5.8 and 2.7 mM for phosphocreatine. In the direction of phosphocreatine formation, at pH 7.5 and 25 degrees C, specific activities were 29 and 47 units/mg; pH optima were 7.5 and 7.7 and Michaelis constants were 0.89 and 0.31 mM for ATP and 39 and 62 mM for creatine. These unique isozymes constitute the termini of the phosphocreatine shuttle of sea urchin sperm that is responsible for energy transport from the mitochondrion to the distal flagellum (Tombes, R. M., and Shapiro, B. M. (1985) Cell 41, 325-334; Tombes, R. M., Brokaw, C. J., and Shapiro, B. M. (1987) Biophys. J., 52, 75-86).
Tombes
R M
RM
Department of Biochemistry, University of Washington, Seattle 98195.
Shapiro
B M
BM
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003402:Creatine Kinase / Q000302:isolation & purification* / Q000378:metabolism; D005407:Flagella / Q000201:enzymology; D007527:Isoenzymes; D007700:Kinetics; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D010725:Phosphocreatine / Q000378:metabolism*; D012617:Sea Urchins; D013077:Sperm Head / Q000201:enzymology; D013094:Spermatozoa / Q000201:enzymology*
D000596:Amino Acids; D007527:Isoenzymes; D010725:Phosphocreatine; D003402:Creatine Kinase