Structure and function of Ca-sensitive myosins

Calcium sensitive myosin can be obtained from scallop and Physarum. Both myosins can bindCa D12+ D1 with a high affinity. However，calcium binding is quite distinct. Ca D12+ D1 works as an activator for theinteractions of scallop myosin with actin，but does as an inhibitor for the actin-myosin interaction of Physarum. the present works are aimedto analyze the molecular mechanisms of these calcium switches by the international co-operativestudy.For the first step of the analysis, we cloned cDNAs coding heavy chain，calcium-binding light chain (CaLc) and phosphorylatable light chain from the cDNA library ofPhysarum. Then, we expressed CaLc and PLc in E. Coli. we also expressed CaLc, PLc，10 kDa short fragment of heavy chain containg the binding sites for CaLc and PLc at once. CaLc，PLc and the complex (Regulatory domain， RD) of CaLc, PLcheavy chain were purifed by the column chromatography and subjected to the calcium-binding assaywith a flow-dialysis chamber.Calcium-binding activity was detected in CaLc not in PLc. When CaLcwas incorporated into RD，the calcium-binding was observed in the lower Ca D12+ D1 concentration as compared withCaLc -binding activity of CaLc alone. CaLc was mutated at E26A, S124A，D126A. The E26A mutation affected calcium-binding activity，indicating the importance of the EF hand structure containing the E26 residue。

Calcium sensitive myosin can be obtained from scallop and Physarum. Both myosins can bind CaィイD12+ィエD1 with a high affinity. However, the effect of calcium binding is quite distinct. CaィイD12+ィエD1 works as an activator for the interactions of scallop myosin with actin, but does as an inhibitor for the actin-myosin interaction of Physarum. The present works are aimed to analyze the molecular mechanisms of these calcium switches by the international co-operative study.For the first step of the analysis, we cloned cDNAs coding heavy chain, calcium-binding light chain (CaLc) and phosphorylatable light chain from the cDNA library of Physarum. Then, we expressed CaLc and PLc in E. Coli. We also expressed CaLc, PLc, and 10 kDa short fragment of heavy chain containg the binding sites for CaLc and PLc at once. CaLc, PLc and the complex (Regulatory domain, RD) of CaLc, PLc, and heavy chain were purifed by the column chromatography and subjected to the calcium-binding assay with a flow-dialysis chamber.Calcium-binding activity was detected in CaLc not in PLc. When CaLc was incorporated into RD, the calcium-binding was observed in the lower CaィイD12+ィエD1 concentration as compared with calcium-binding activity of CaLc alone. CaLc was mutated at E26A, S124A, and D126A. The E26A mutation affected calcium-binding activity, indicating the importance of the EF hand structure containing the E26 residue.