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Structure and function of Ca-sensitive myosins

钙敏感肌球蛋白的结构和功能

基本信息

批准号：
10044236
负责人：
KOHAMA Kazuhiro
金额：
$ 3.26万
依托单位：
Gunma University, School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10044236/
关键词：
calcium myosin scallop Physarum recombinant protein regulation

项目摘要

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.

钙敏感肌球蛋白可从扇贝和绒泡菌中提取。这两种肌凝蛋白都能以高亲和力结合Ca γ γ + γ γ γ。然而，钙结合的作用是非常明显的。Ca φ D12+ φ D1可作为扇贝肌凝蛋白与肌动蛋白相互作用的激活剂，但可作为绒泡菌肌动蛋白-肌凝蛋白相互作用的抑制剂。目前的工作旨在通过国际合作研究来分析这些钙开关的分子机制。首先，我们从绒泡菌cDNA文库中克隆了编码重链、钙结合轻链和磷酸化轻链的cDNA。然后，我们在大肠杆菌中表达CaLc和PLc。我们还同时表达了CaLc、PLc和含有CaLc和PLc结合位点的10 kDa重链短片段。通过柱层析纯化CaLc、PLc和CaLc、PLc和重链的复合物（调节结构域，RD），并在流动透析室进行钙结合测定。在CaLc中检测到钙结合活性，而在PLc中检测不到。当钙c掺入RD时，与单独钙c的钙结合活性相比，钙结合活性在较低的Ca γ γ - D12+ γ - D1浓度下观察到。CaLc在E26A、S124A和D126A位点发生突变。E26A突变影响钙结合活性，表明含有E26残基的EF手结构的重要性。