Purification of a protein kinase phosphorylating myosin regulatory light chain-a (RLC-a) from smooth muscle of scallop, Patinopecten yessoensis.

A protein kinase activity phosphorylating regulatory light chain-a (RLC-a) of scallop smooth muscle myosin was found to be present in scallop smooth muscle homogenate. The kinase was purified to homogeneity and named RLC-a myosin kinase (aMK). aMK was extracted from the muscle homogenate with a low salt solution and was purified by successive DE-32 ion exchange chromatography, gel filtration on Ultrogel AcA 44, and affinity chromatography on Sepharose 4B-6-aminohexyl-1-pyrophosphate. The molecular weight of aMK was estimated to be 40-kDa from the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 35-kDa from the elution volume on Sephadex G-150 gel filtration. The phosphorylation site of RLC-a by aMK was determined to be Ser residue(s). Only RLC-a was phosphorylated; the other regulatory light chain, RLC-b, was not. The phosphorylatable Ser of RLC-a is, therefore, considered to be Ser-11, which is located in the N-terminal region having a different amino acid sequence from that of RLC-b. RLC-a was phosphorylated by aMK 3 times faster in the free state than in the bound state to myosin. aMK does not require calmodulin and is rather inhibited by CaCl2.