Vascular smooth muscle caldesmon.
Vascular smooth muscle caldesmon.
复制标题
血管平滑肌钙化蛋白。
DOI：
10.1016/s0021-9258(19)57507-x
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Michael P. Walshs
中科院分区：
文献类型：
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作者：
Timothy Clark;Philip;Ngai;Cindy Sutherland;Ute Groschel;Michael P. Walshs
钙调素，一个主要的肌动蛋白和钙调素结合蛋白，已被确定在不同的牛组织，包括平滑肌和横纹肌和各种非肌肉组织，变性聚丙烯酰胺凝胶电泳的组织匀浆和免疫印迹使用兔抗鸡砂囊钙调素。通过热处理组织匀浆、离子交换层析和固定化钙调蛋白柱上的亲和层析，从血管平滑肌（牛主动脉）中纯化钙调蛋白。分离的蛋白质具有许多与鸡砂囊钙调素相同的性质：免疫交叉反应性，与钙调素的相互作用依赖于Ca 2+，与F-肌动蛋白的相互作用不依赖于Ca 2+，肌动蛋白和钙调素之间竞争钙调素结合仅在Ca 2+存在下，和抑制肌动蛋白激活的平滑肌肌球蛋白的Mg 2 +-ATP酶活性而不影响肌球蛋白的磷酸化状态。主动脉钙调蛋白与肌动蛋白的最大结合发生在1摩尔钙调蛋白：9-10摩尔肌动蛋白，结合不受原肌球蛋白的影响。肌动蛋白激活的肌球蛋白Mg 2 +-ATP酶的半数最大抑制发生在约1摩尔钙调蛋白：12摩尔肌动蛋白。这种抑制作用也不受原肌球蛋白的影响。在没有肌动蛋白的情况下，Caldesmon对平滑肌肌球蛋白的Mg 2 +-ATP酶活性没有影响。牛主动脉和鸡砂囊在几个方面不同：先生（牛主动脉钙调素为149，000，鸡胗钙调素为141，000），消光系数（牛主动脉和鸡肌钙蛋白的E1%280nm分别为19.5和5.0），氨基酸组成，以及通过有限的胰凝乳蛋白酶和金黄色葡萄球菌V8蛋白酶消化获得的一维肽图。在竞争性酶联免疫吸附试验中，使用抗鸡砂囊钙调素，相对于鸡砂囊钙调素，需要174倍摩尔过量的牛主动脉钙调素的半数最大抑制。这些研究建立了广泛的组织和物种分布的钙调素，并表明，血管平滑肌钙调素表现出理化差异，但结构和功能的相似性，从鸡砂囊分离的钙调素。
Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard.