ACANTHAMOEBA-CASTELLANII CAPPING PROTEIN - PROPERTIES, MECHANISM OF ACTION, IMMUNOLOGICAL CROSS-REACTIVITY, AND LOCALIZATION
ACANTHAMOEBA-CASTELLANII CAPPING PROTEIN - PROPERTIES, MECHANISM OF ACTION, IMMUNOLOGICAL CROSS-REACTIVITY, AND LOCALIZATION
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DOI：
10.1083/jcb.99.1.217
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发表时间：
1984-01-01
影响因子：
7.8
通讯作者：
POLLARD, TD
中科院分区：
文献类型：
--
作者：
COOPER, JA;BLUM, JD;POLLARD, TD
报告了 A.castellanii 帽蛋白的物理和免疫特性、作用机制和细胞内定位的进一步表征。根据斯托克斯半径（凝胶过滤色谱法为 3.8 nm）和沉降系数（蔗糖梯度速度沉降为 4.8 S）的测量计算出的天然分子量为 74,000 道尔顿。亚基分子量分别为 31,000 和 28,000 道尔顿，因此天然分子是异二聚体。这两个亚基之间不发生免疫交叉反应，也不与来自棘阿米巴或其他几种生物体的任何其他蛋白质发生免疫交叉反应。在作用机制的研究中，Isenberg 发现加帽蛋白阻止了肌动蛋白丝带刺末端的聚合并与肌动蛋白丝一起沉积。通过凝胶过滤测定证实加帽蛋白与肌动蛋白丝结合。加帽蛋白降低了肌动蛋白丝的长度分布和高剪切粘度。加帽蛋白不会使肌动蛋白丝成束或交联。低浓度的加帽蛋白将Mg2+和EGTA[乙二醇-双(β-氨基乙基醚)N,N,N'',N''-四乙酸]中肌肉和阿米巴肌动蛋白聚合的临界浓度从0.1-0.6μM增加。增加加帽蛋白的量并没有进一步增加临界浓度。在 Ca2+ 中，加帽蛋白不会改变肌肉肌动蛋白的临界浓度，但会增加阿米巴肌动蛋白的临界浓度。 Ca2+ 对加帽蛋白降低肌动蛋白丝的低或高剪切粘度的能力没有影响。通过间接荧光抗体染色，加帽蛋白定位于细胞皮质，这是一个富含肌动蛋白丝的区域。在匀浆的亚细胞分级分离过程中，.apprx。 1/3 的细胞加帽蛋白与粗膜部分呈带状。另外 2/3 的细胞加帽蛋白是可溶的，其斯托克斯半径等于纯化蛋白的斯托克斯半径。细胞中加帽蛋白与肌动蛋白的摩尔比为1:150。
Further characterization of the physical and immunologic properties, mechanism of action and intracellular localization of A. castellanii capping protein is reported. The native MW calculated from measurements of Stokes'' radius (3.8 nm by gel filtration chromatography) and sedimentation coefficient (4.8 S by sucrose gradient velocity sedimentation) was 74,000 daltons. The subunit MW were 31,000 and 28,000 daltons, so the native molecule is a heterodimer. The 2 subunits did not immunologically cross-react with each other, or with any other proteins from Acanthamoeba or several other organisms. In studies of the mechanism of action, Isenberg found that capping protein blocked polymerization from the barbed end of actin filaments and sedimented with actin filaments. It was confirmed that capping protein binds to actin filaments with a gel filtration assay. Capping protein decreased the length distribution and high shear viscosity of actin filaments. Capping protein did not bundle or cross-link actin filaments. Low concentrations of capping protein increased the critical concentration for muscle and ameba actin polymerization from 0.1-0.6 .mu.M in Mg2+ and EGTA [ethyleneglycol-bis(.beta.-aminoethyl ether) N,N,N'',N''-tetraacetic acid]. Increasing amounts of capping protein did not increase the critical concentration further. In Ca2+ capping protein did not change the critical concentration for muscle actin, but did increase the critical concentration for ameba actin. Ca2+ had no effect on the ability of capping protein to decrease the low or high shear viscosity of actin filaments. By indirect fluorescent antibody staining, capping protein was localized to the cell cortex, an area rich in actin filaments. During subcellular fractionation of homogenates, .apprx. 1/3 of cellular capping protein banded with a crude membrane fraction. The other 2/3 of cellular capping protein was soluble, with a Stokes'' radius equal to that of the purified protein. The molar ratio of capping protein to actin in the cell was 1:150.