Calcium-independent myosin light chain kinase of smooth muscle. Preparation by limited chymotryptic digestion of the calcium ion dependent enzyme, purification, and characterization.
Calcium-independent myosin light chain kinase of smooth muscle. Preparation by limited chymotryptic digestion of the calcium ion dependent enzyme, purification, and characterization.
复制标题
平滑肌的钙非依赖性肌球蛋白轻链激酶。
DOI：
10.1021/bi00537a034
复制
发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Hartshorne,DJ
中科院分区：
文献类型：
--
作者：
Walsh,MP;Dabrowska,R;Hinkins,S;Hartshorne,DJ
Michael P.沃尔什、* Renata Dabrowska、* Susan Hinkins和大卫J.哈茨霍恩摘要：对从平滑肌（火鸡砂囊）中部分纯化的钙调蛋白依赖性肌球蛋白轻链激酶进行有限的α-糜蛋白酶消化，产生了一种不依赖于钙的酶。消化产生的Ca 2 +-非依赖性激酶需要的酶与Ca 2 +-钙调蛋白复合物，当消化进行脱辅基酶，即，在没有Ca 2+，依赖于Ca 2+的激酶活性被保留。通过离子交换色谱法纯化了Ca 2+非依赖性激酶，并显示其表观分子量约为80 000。以肌球蛋白轻链为底物，酶的比活力在Ca ~（2+）存在时为6.5±0.2 μ mol·min ~（-1）·mg ~（-1），在Ca ~（2+）缺乏时为8.3±0.3 μ mol·min ~（-1）·mg ~（-1）。不依赖Ca ~（2+）的酶也能磷酸化纯化的肌球蛋白和火鸡肌球蛋白粗品的20000-道尔顿轻链。Ca 2+非依赖性激酶对Mg 2 +-ATP的Km（54 μ M）与天然Ca 2+依赖性酶的Km（68 μ M）没有显著差异。这些观察结果表明在用α-胰凝乳蛋白酶消化后活性位点的完整性得以维持。这表明，在有限的蛋白水解后的激酶的钙离子敏感性的损失是由于从80 000-道尔顿片段的钙调素结合位点的损失。胰凝乳蛋白酶水解也去除了环AMP依赖性蛋白激酶磷酸化的两个位点。调节平滑肌肌动球蛋白的最广泛接受的理论涉及肌球蛋白的20000-道尔顿轻链的磷酸化（Adelstein & Eisenberg，1980;哈茨霍恩& Siemankowski，1981）。从各种实验方法中已经积累了支持磷酸化理论的证据。其中包括：腺苷5 '-0-（3-硫代三磷酸）（ATP 7S），1，其产生稳定的硫代磷酸化状态，并导致肌球蛋白和带皮平滑肌纤维中Ca 2+敏感性的丧失;肌球蛋白的磷酸化与几种体外肌球蛋白系统的ATP酶活性或各种平滑肌纤维制备物中的张力发展之间的正相关;最后，使用钙调素拮抗剂，如吩噻嗪类，它们抑制肌球蛋白磷酸化和肌球蛋白的ATP酶活性以及肌条的张力发展[参考文献见哈茨霍恩& Mrwa（1982）]。
Michael P. Walsh,* Renata Dabrowska,* Susan Hinkins, and David J. Hartshorne abstract: Limited a-chymotryptic digestion of Ca2+-, calmodulin-dependent myosin light chainkinase partially purified from smooth muscle (turkey gizzard) yielded a Ca2+-independent form of the enzyme. Digestion to yield the Ca2+-independent kinase required the enzyme complexed with Ca2+-calmodulin; when digestion was performed on the apoenzyme, ie, in the absence of Ca2+, the dependence of kinase activity on Ca2+ was retained. The Ca2+-independent kinase was purified by ion-exchange chromatography and shown to have an apparent molecular weight of~ 80 000. The specific activity of the freshly prepared enzyme was 6.5±0.2 umol of P; incorporated min'1 mg'1 in thepresence of Ca2+ and 8.3±0.3/imol min'1 mg'1 in the absence of Ca2+, using the isolated light chains of gizzard myosin as the substrate. The Ca2+-independent enzyme also phosphorylated the 20 000-dalton light chains of purifiedmyosin and crude actomyosin from turkey gizzard. The Km of the Ca2+-independent kinase for Mg2+-ATP (54 jtM) was not significantly different from that of the native, Ca2+-dependent enzyme (68 uM). These observations indicate maintenance of the integrity of the active site after digestion with a-chymotrypsin. It is suggested that the loss of Ca2+ sensitivity of the kinase after limited proteolysis is due to loss of the calmodulin-binding site from the 80 000-dalton fragment. The two sites of phosphorylation by the cyclic AMP dependent protein kinase were also removed by the chymotryptic hydrolysis. e most widely accepted theory for the regulation of smooth muscle actomyosin involves the phosphorylation of the 20 000-dalton light chains of myosin (Adelstein & Eisenberg, 1980; Hartshorne & Siemankowski, 1981). Evidence has been accumulated in support of the phosphorylation theory from a variety of experimental approaches. These include the following: the use of adenosine 5'-0-(3-thiotriphosphate)(ATP7S), 1 which generates a stable thiophosphorylated state and a resultant loss of Ca2+ sensitivity in gizzard actomyosin and in skinned smooth muscle fibers; a positive correlation between the phosphorylation of myosin and either the ATPase activities of several in vitro actomyosin systems or tension development in various smooth muscle fiber preparations; and, finally, the use of calmodulin antagonistssuch as the pheno-thiazines, which inhibit both myosin phosphorylation and ATPase activity of gizzardactomyosin and tension develop-ment in muscle strips [for bibliography, see Hartshorne & Mrwa (1982)].