Johnson J D, Holroyde M J, Crouch T H, Solaro R J, Potter J D
J Biol Chem. 1981 Dec 10;256(23):12194-8.
The interaction of calmodulin with myosin light chain kinase produces an approximately 30% increase in myosin light chain kinase tryptophan fluorescence. This represents the first report of calmodulin-induced structural changes in a protein which it activates. We fund that the calmodulin-myosin light chain kinase interaction is: 1) dependent on [Ca2+] (half-maximal binding at pCa 6.2) and essentially independent of [Mg2+], 2) occurs before saturation of all four reported Ca2+-specific sites on calmodulin. 3) saturates with 1 mol of calmodulin bound per mol of kinase with an apparent affinity of approximately 2.0 X 10(7) M-1, 4) is specific for calmodulin over troponin-C, 5) is directly related to the activation of myosin light chain kinase for phosphorylation of myosin light chain. Fluorescence stopped flow studies of these calmodulin-induced fluorescence changes in myosin light chain kinase indicate that Ca2+ binding to calmodulin occurs very rapidly and is not rate-limiting while the calmodulin-induced fluorescence increase in myosin light chain kinase occurs as a biphasic process with rates of approximately 65 s-1 and 6 s-1. The fluorescence increase produced by calmodulin binding to myosin light chain kinase is completely reversed by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at a rate of approximately 2 s-1.
钙调蛋白与肌球蛋白轻链激酶相互作用，使肌球蛋白轻链激酶的色氨酸荧光增加约30%。这是关于钙调蛋白诱导其激活的蛋白质发生结构变化的首次报道。我们发现钙调蛋白与肌球蛋白轻链激酶的相互作用：1）依赖于[Ca2+]（在pCa 6.2时半最大结合）且基本不依赖于[Mg2+]；2）发生在钙调蛋白上所有四个已报道的Ca2+特异性位点饱和之前；3）每摩尔激酶结合1摩尔钙调蛋白时达到饱和，表观亲和力约为2.0×10(7) M-1；4）对钙调蛋白比对肌钙蛋白C具有特异性；5）与肌球蛋白轻链激酶对肌球蛋白轻链磷酸化的激活直接相关。对这些钙调蛋白诱导的肌球蛋白轻链激酶荧光变化进行的荧光停流研究表明，Ca2+与钙调蛋白的结合非常迅速且不是限速步骤，而钙调蛋白诱导的肌球蛋白轻链激酶荧光增加是一个双相过程，速率约为65 s-1和6 s-1。钙调蛋白与肌球蛋白轻链激酶结合产生的荧光增加可被乙二醇双（β-氨基乙醚）-N,N,N',N'-四乙酸以约2 s-1的速率完全逆转。
