Hayashi H, Yamamoto K, Yonekawa H, Morisawa M
Institute of Molecular Biology, Faculty of Science, Nagoya University, Japan.
J Biol Chem. 1987 Dec 5;262(34):16692-8.
Initiation of flagellar motility in spermatozoa of the rainbow trout, Salmo gairdneri, is closely related to phosphorylation of a protein of molecular mass 15 kDa (Morisawa, M., and Hayashi, H. (1986) Biomed. Res. 6, 181-184). We have been able to solubilize the protein and its kinase and then construct an assay system in vitro for the phosphorylation of the 15-kDa protein. In vitro, the protein was phosphorylated in a cAMP-dependent manner. The phosphorylation absolutely required the presence of Mg2+ ions. at millimolar concentrations, but not of Ca2+ ions. The amino acid residue which was phosphorylated in the 15-kDa protein was tyrosine. The 15-kDa protein was purified to near homogeneity by affinity chromatography on a column of adenosine nucleotides conjugated to Eupergit and ion-exchange chromatography on DEAE-cellulose. The effects of synthetic inhibitors of protein kinase on the phosphorylation of the 15-kDa protein were also studied.
