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

肌球蛋白的磷酸化-去磷酸化是钙离子介导的平滑肌细胞的主要调节过程。然而，最近的生理学研究表明，尽管肌球蛋白去磷酸化，完整的平滑肌纤维中的张力仍保持不变，并提示可能存在其他调节肌肉收缩状态的控制机制。肌球蛋白轻链激酶(MLCK)以外的蛋白激酶(S)和钙调素(CaM)以外的钙离子结合蛋白(CaM)能否调节收缩？在这项国际科研计划中，我们试图统一和重建肌球蛋白磷酸化理论对平滑肌和非肌肉细胞收缩反应的影响，并取得了以下结果：1)制备了针对鸡胆MLCK的单抗。单抗MM-7抑制了牛主动脉平滑肌肌动球蛋白的激酶活性和超沉淀。我们还演示了…2)肌动蛋白和钙调蛋白结合蛋白Caldesmon被PK-C和钙调蛋白依赖的蛋白激酶磷酸化。3)CaM依赖的Caldesmon是富含脑的钙调蛋白依赖蛋白激酶II(CaM KII)的同工酶。4)与MLCK一样，CaM KII在相同的位点磷酸化纯化的肌球蛋白轻链。我们独创的CaM KII特异性抑制剂KN-62可抑制不同激动剂诱导的兔颈总动脉条收缩。CaM KII可能参与了平滑肌收缩。5)我们利用我们最初的化合物亲和层析，检测并纯化了三个新的钙结合蛋白。一个是钙周期蛋白，另一个是新的钙结合蛋白(暂定名为Calgizzarin和Calculculin)。这些Ca~(2+)结合蛋白在平滑肌细胞中的存在，提示可能存在新的细胞内Ca~(2+)信使系统(S)。较少

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