Myristoylation of flagellar creatine kinase in the sperm phosphocreatine shuttle is linked to its membrane association properties.
TCK, the flagellar creatine kinase (ATP:creatine N-phosphotransferase) of sperm from the sea urchin Strongylocentrotus purpuratus is a membrane-associated lipophilic protein involved in energy transport. The cDNA derived protein sequence contains a consensus site sufficient for the covalent attachment of myristate. To examine whether TCK was myristoylated, mouse fibroblast Swiss 3T3 and baby hamster kidney cell lines were transfected with a cDNA encoding the entire TCK protein linked to a metallothionein promotor. TCK expression was induced by zinc and paralleled by incorporation of [3H]myristic acid derived label into the protein. 3H Label incorporated into TCK was resistant to hydroxylamine treatment. The 3H-labeled material released from TCK by acid methanolysis eluted from a C18 reverse phase high pressure liquid chromatography column at the positions of myristic acid and methylmyristate. Thus, TCK expressed in transfected mammalian cell lines contains authentic myristic acid, covalently attached through amide linkage. [3H]Myristoyl TCK comigrated on two-dimensional gels with the purified lipophilic isoform TCK II from sea urchins. Furthermore, like TCK II, [3H]myristoyl TCK associated with phospholipid liposomes, suggesting that myristoylation may mediate the observed membrane association of TCK. Myristoylation of sea urchin sperm flagellar creatine kinase may play a role in confining this enzyme to the flagellum during spermatogenesis.
Quest
A F
AF
Department of Biochemistry, University of Washington, Seattle 98195.
Chadwick
J K
JK
Wothe
D D
DD
McIlhinney
R A
RA
Shapiro
B M
BM
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003402:Creatine Kinase / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D004247:DNA; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism; D015870:Gene Expression; D008081:Liposomes; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D010725:Phosphocreatine / Q000378:metabolism*; D010957:Plasmids; D012617:Sea Urchins; D013094:Spermatozoa / Q000201:enzymology*; D014162:Transfection
D005227:Fatty Acids; D008081:Liposomes; D009227:Myristic Acids; D010725:Phosphocreatine; D019814:Myristic Acid; D004247:DNA; D003402:Creatine Kinase