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Unification and reconstruction of myosin phosphorylation theory on contractile response of smooth muscle and nonmuscle cells.

平滑肌和非肌肉细胞收缩反应的肌球蛋白磷酸化理论的统一和重建。

基本信息

批准号：
01044066
负责人：
HIDAKA Hiroyoshi
金额：
$ 4.67万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

Myosin phosphorylation-dephosphorylation is the primary Ca^2-mediated regulatory process in smooth muscle. However, recent physiological studies showed that the tension in intact smooth muscle fiber is maintained in spite of the dephosphorylation of myosin, and have suggested that other control mechanisms may exist which modulate the contractile state of the muscle. Can contraction be regulated by protein kinase (s) other than myosin light chain kinase (MLCK), and by Ca2^2-binding proteins other than calmodulin? In this international scientific research program, we have attempted to unify and reconstruct the myosin phosphorylation theory on contractile response of smooth muscle and non-muscle cells, and obtained the following results, according to the schedule.1) We prepared monoclonal antibodies directed against chicken gizzard MLCK. One of the monoclonal antibody, MM-7 inhibited the kinase activity and the superprecipitation of bovine aortic smooth muscle actomyosin. We also demonstr … More ated the existence of it least 4 subspecies of MLCK in chicken tissues and the heterogeneity of tissue- and species-specific isozyme forms.2) Caldesmon, an actin and calmodulin binding protein, was phosphorylated by PK-C and calmodulin-dependent protein kinase.3) The calmodulin-dependent caldesmon kinase was an isozyme of the brain-rich calmodulin-dependent protein kinase II (CaM KII).4) CaM KII phosphorylated purified myosin light chain at same sites, as MLCK did. Our original CaM KII specific inhibitor, KN-62 inhibited the various agonist-induced contraction in rabbit common carotid arterial strips. CaM KII may be involved in smooth muscle contraction.5) We detected and purified three new Ca^<2+> binding proteins, using our original compounds affinity chromatography. One was calcyclin and the others were novel Ca^<2+>-binding proteins (tentatively designated calgizzarin and calvasculin). The presence of these Ca^<2+>binding proteins in smooth muscle cells show that novel intracellular Ca^<2+> messenger system (s) may exist. Less

肌球蛋白磷酸化-去磷酸化是平滑肌中Ca^2介导的主要调节过程。然而，最近的生理学研究表明，尽管肌球蛋白去磷酸化，但完整平滑肌纤维中的张力仍得以维持，并表明可能存在其他调节肌肉收缩状态的控制机制。收缩是否受肌球蛋白轻链激酶（MLCK）以外的蛋白激酶和钙调蛋白以外的钙结合蛋白的调节？本研究在国际科研计划中，尝试统一和重建肌球蛋白磷酸化对平滑肌和非肌细胞收缩反应的理论，并按计划取得了以下成果：1）制备了抗鸡砂囊MLCK的单克隆抗体。单克隆抗体MM-7可抑制牛主动脉平滑肌肌动球蛋白的激酶活性和过沉淀。我们还展示了 ...更多信息证明MLCK在鸡组织中至少存在4个亚种，且具有组织特异性和种特异性同工酶的异质性。2）Caldesmon，一种肌动蛋白和钙调素结合蛋白，3）钙调素依赖性钙调素激酶是脑富含钙调素依赖性蛋白激酶II（CaM KII）的同工酶。CaM KII在与MLCK相同的位点磷酸化纯化的肌球蛋白轻链。我们最初的CaM KII特异性抑制剂KN-62抑制各种激动剂诱导的兔颈总动脉条的收缩。CaM KII可能参与平滑肌收缩。5）我们用我们的化合物亲和层析法检测并纯化了三个新的Ca^<2+>结合蛋白。其中一种是钙周期蛋白，另外两种是新的钙离子结合蛋白（暂命名为钙离子结合蛋白和钙血管蛋白）。平滑肌细胞中这些Ca^2+结合蛋白的存在表明可能存在新的细胞内Ca^2+信使系统。少