Evolution of phosphagen kinase VII. Isolation of glycocyamine kinase from the polychaete Neanthes diversicolor and the cDNA-derived amino acid sequences of alpha and beta chains.

Glycocyamine kinase (GK) was isolated from the marine polychaete Neanthes diversicolor by gel filtration, DEAE-cellulose chromatography, butyl-Toyopearl hydrophobic chromatography, and chromatofocusing. The GK was eluted as a single peak on the latter three chromatographies, and the molecular mass for the native GK was estimated to be about 80 kDa. The SDS-PAGE showed that the isolated GK consists of two distinct subunits in equal proportion, alpha and beta chains, with molecular masses of 42.2 and 43.8 kDa, respectively. The present results suggest that the Neanthes GK has a heterodimeric structure. The cDNAs for alpha and beta chains of Neanthes GK were amplified by PCR and their cDNA-derived amino acid sequences were determined. The alpha and beta chains are composed of 374 and 390 amino acids, and the molecular masses were calculated to be 42,392 and 43,966 Da, respectively, in good agreement with the apparent masses on SDS PAGE. The beta chain has a characteristic N-terminal extension of 15 amino acids, and all of the sequence differences between alpha and beta chains were restricted in the N-terminal region of 50 residues. The overall sequence identity was 92%. The occurrence of heterodimeric nature in Neanthes GK is of great interest from the evolutionary point of view, because the heterodimeric structure is only known for creatine kinase MB-isozyme specific for mammalian heart muscle among phosphagen kinases.