Kargacin G J, Ikebe M, Fay F S
Department of Physiology, University of Massachusetts Medical School, Worcester 01655.
Am J Physiol. 1990 Aug;259(2 Pt 1):C315-24. doi: 10.1152/ajpcell.1990.259.2.C315.
To examine the importance of myosin light chain kinase (MLCK) in the initiation of contraction in smooth muscle, we used a constitutively active form of MLCK (IMLCK) and two specific peptide inhibitors of MLCK to study the activation of skinned single smooth muscle cells. Although unregulated by Ca-calmodulin, IMLCK, in vitro, was found to have biochemical properties like those of MLCK. Upon photolysis of caged ATP, IMLCK caused Ca-free shortening of skinned cells similar in time course and extent to that induced by Ca2+. Two peptide probes, RS-20 and SM-1, patterned after the Ca-calmodulin binding site and a pseudosubstrate inhibitory site, respectively, of the native MLCK molecule, were shown to specifically inhibit MLCK in in vitro experiments. Both peptides dose dependently inhibited Ca-induced shortening of skinned single cells. These results indicate that MLCK plays an essential role in the activation process in the smooth muscle cell in that activation of this enzyme is both necessary and sufficient for the initiation of contraction.
为了研究肌球蛋白轻链激酶（MLCK）在平滑肌收缩起始中的重要性，我们使用了一种组成型活性形式的MLCK（IMLCK）和两种MLCK特异性肽抑制剂来研究去表皮单个平滑肌细胞的激活情况。尽管不受钙调蛋白调节，但体外实验发现IMLCK具有与MLCK相似的生化特性。在笼锁ATP光解后，IMLCK导致去表皮细胞在无钙情况下缩短，其时间进程和程度与Ca2+诱导的相似。两种肽探针RS - 20和SM - 1，分别根据天然MLCK分子的钙调蛋白结合位点和假底物抑制位点设计，在体外实验中显示能特异性抑制MLCK。两种肽均剂量依赖性地抑制Ca诱导的去表皮单细胞缩短。这些结果表明，MLCK在平滑肌细胞的激活过程中起重要作用，因为该酶的激活对于收缩的起始既是必要的也是充分的。
