Ikebe M
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106.
Biochem Biophys Res Commun. 1990 Apr 30;168(2):714-20. doi: 10.1016/0006-291x(90)92380-i.
The functions associated with the inhibitory region and calmodulin binding region of smooth muscle myosin light chain kinase (MLCK) were studied using various synthetic peptide analogs. Peptides 480-501 and 483-498 strongly inhibited 61 kDa Ca2+/calmodulin-independent MLCK activity with Ki of 25 nM. Peptides 493-512 and 493-504 were considerably less effective as inhibitor of the Ca2+/calmodulin-independent MLCK and Kiapp. were 2 and 3 microM, respectively. Inhibition of Ca2+/calmodulin-independent MLCK by the peptides 480-501 and 483-498 were competitive with ATP and 20,000 dalton smooth muscle myosin light chain. The inhibition of native MLCK by peptide 493-512 was explained by the calmodulin depletion model in which the peptide binds to free calmodulin and prevents it from activating MLCK. On the other hand, the inhibition of native MLCK by the peptides 480-501 and 483-498 was explained by the binding of these peptides to the MLCK-calmodulin complex. The present study suggests that the inhibitory region of MLCK directly binds to MLCK active site and competes with both ATP and 20,000 dalton light chain so as to inhibit the enzyme.
利用各种合成肽类似物研究了平滑肌肌球蛋白轻链激酶（MLCK）的抑制区域和钙调蛋白结合区域相关的功能。肽480 - 501和483 - 498强烈抑制61 kDa的不依赖钙调蛋白的MLCK活性，其抑制常数（Ki）为25 nM。肽493 - 512和493 - 504作为不依赖钙调蛋白的MLCK抑制剂的效果要差得多，其表观抑制常数（Kiapp.）分别为2 μM和3 μM。肽480 - 501和483 - 498对不依赖钙调蛋白的MLCK的抑制作用与ATP和20,000道尔顿的平滑肌肌球蛋白轻链存在竞争性。肽493 - 512对天然MLCK的抑制作用可通过钙调蛋白耗竭模型来解释，即该肽与游离钙调蛋白结合并阻止其激活MLCK。另一方面，肽480 - 501和483 - 498对天然MLCK的抑制作用可通过这些肽与MLCK - 钙调蛋白复合物的结合来解释。本研究表明，MLCK的抑制区域直接与MLCK活性位点结合，并与ATP和20,000道尔顿的轻链竞争，从而抑制该酶。
