Tokumitsu H, Hagiwara M, Onoda K, Hidaka H
Department of Pharmacology, Nagoya University School of Medicine, Aichi.
J Biochem. 1989 Sep;106(3):511-4. doi: 10.1093/oxfordjournals.jbchem.a122883.
We prepared monoclonal antibodies directed against chicken gizzard myosin light chain kinase (MLCK) and used them to study the contractile system of aortic smooth muscle. One monoclonal antibody, MM13, dose dependently inhibited actomyosin superprecipitation of bovine aortic smooth muscle, in accord with the suppression of 20 kDa myosin light chain phosphorylation by endogenous kinase. Immunoblotting analysis demonstrated that MM13 cross-reacted with the 150,000 Mr peptide of bovine aortic actomyosin preparation. The bovine aortic MLCK was purified approximately 2,400-fold to apparent homogeneity by three steps of column chromatography. The purified enzyme has a molecular weight of 150,000 and a slower mobility than chicken gizzard MLCK (130,000 Mr), as determined by SDS-polyacrylamide gel electrophoresis. MM13 also cross-reacted with purified bovine aortic MLCK and inhibited the kinase activity, in vitro. We interpret these findings to mean that binding of the anti-gizzard MLCK monoclonal antibody directly to aortic smooth muscle MLCK (150,000 Mr) decreases the phosphorylation of the 20 kDa myosin light chain, thus suppressing the aortic smooth muscle myosin-actin interaction.
我们制备了针对鸡肌胃肌球蛋白轻链激酶（MLCK）的单克隆抗体，并利用它们来研究主动脉平滑肌的收缩系统。一种单克隆抗体MM13能剂量依赖性地抑制牛主动脉平滑肌的肌动球蛋白超沉淀，这与内源性激酶对20 kDa肌球蛋白轻链磷酸化的抑制作用一致。免疫印迹分析表明，MM13与牛主动脉肌动球蛋白制剂的150,000 Mr肽发生交叉反应。通过三步柱层析将牛主动脉MLCK纯化至表观均一性，纯化倍数约为2400倍。经SDS-聚丙烯酰胺凝胶电泳测定，纯化后的酶分子量为150,000，迁移率比鸡肌胃MLCK（130,000 Mr）慢。MM13也与纯化的牛主动脉MLCK发生交叉反应，并在体外抑制其激酶活性。我们将这些发现解释为，抗肌胃MLCK单克隆抗体与主动脉平滑肌MLCK（150,000 Mr）的直接结合会降低20 kDa肌球蛋白轻链的磷酸化，从而抑制主动脉平滑肌肌动蛋白-肌球蛋白相互作用。
