Characterization of antibodies to smooth muscle myosin kinase and their use in localizing myosin kinase in nonmuscle cells.
Characterization of antibodies to smooth muscle myosin kinase and their use in localizing myosin kinase in nonmuscle cells.
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平滑肌肌球蛋白激酶抗体的表征及其在非肌肉细胞中定位肌球蛋白激酶中的应用。
DOI：
10.1073/pnas.78.8.4738
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发表时间：
1981
影响因子：
11.1
通讯作者：
Burridge,K
中科院分区：
文献类型：
--
作者：
deLanerolle,P;Adelstein,RS;Feramisco,JR;Burridge,K
从火鸡肌胃平滑肌中纯化肌球蛋白轻链激酶，在兔中开发抗体，并通过肌球蛋白轻链激酶-Sepharose 4 B柱上的亲和层析纯化。纯化的抗体与纯化的平滑肌肌球蛋白轻链激酶交叉反应，但不与各种收缩或细胞骨架蛋白交叉反应。抗体抑制平滑肌肌球蛋白轻链激酶的催化活性，并且激酶活性与测定中存在的抗体量之间存在反比关系。肌球蛋白激酶活性的半数最大抑制发生在抗体/肌球蛋白激酶摩尔比为10：1时。用亲和纯化的平滑肌肌球蛋白激酶抗体研究肌球蛋白激酶在多种非肌细胞中的定位。免疫荧光研究表明，肌球蛋白轻链激酶是本地化的微丝束（应力纤维）在培养的成纤维细胞。当在染色细胞之前将抗体与纯化的平滑肌肌球蛋白轻链激酶一起孵育时，应力纤维染色模式被消除，而当在染色之前将抗体与肌动蛋白、肌球蛋白、α-辅肌动蛋白或原肌球蛋白一起孵育时，染色模式不受影响。此外，应力纤维染色模式是周期性的沙鼠纤维瘤细胞和实验表明，肌球蛋白轻链激酶似乎具有相同的周期性分布的肌球蛋白，但相对于α-辅肌动蛋白的反周期分布。这些数据表明，肌球蛋白轻链激酶和它的底物，肌球蛋白，是在密切接近，并与肌球蛋白轻链激酶调节肌动蛋白-肌球蛋白在非肌肉细胞的相互作用的假设是一致的。
Antibodies to myosin light chain kinase, purified from turkey gizzard smooth muscle, were developed in rabbits and purified by affinity chromatography on a myosin light chain kinase-Sepharose 4B column. The purified antibodies crossreact with purified smooth muscle myosin light chain kinase but not with a variety of contractile or cytoskeletal proteins. The antibodies inhibit the catalytic activity of smooth muscle myosin light chain kinase and there is an inverse relationship between the kinase activity and the amount of antibody present in an assay. Half-maximal inhibition of myosin kinase activity occurs at an antibody/myosin kinase molar ratio of 10:1. The affinity-purified antibodies to smooth muscle myosin kinase were used to study the location of myosin kinase in a variety of nonmuscle cells. Immunofluorescence studies indicate that myosin light chain kinase is localized on microfilament bundles (stress fibers) in cultured fibroblasts. The stress fiber staining pattern is abolished when the antibodies are incubated with purified smooth muscle myosin light chain kinase prior to staining cells, while the staining pattern is unaffected when the antibodies are incubated with actin, myosin, alpha-actinin, or tropomyosin prior to staining. Moreover, the stress fiber staining pattern is periodic in well-spread gerbil fibroma cells and experiments have demonstrated that myosin light chain kinase appears to have the same periodic distribution as myosin but an antiperiodic distribution relative to alpha-actinin. These data indicate that myosin light chain kinase and its substrate, myosin, are in close proximity and are consistent with the hypothesis that myosin light chain kinase regulates actin-myosin interactions in nonmuscle cells.