Disulphide cross-linking of smooth-muscle and non-muscle caldesmon to the C-terminus of actin in reconstituted and native thin filaments.
Disulphide cross-linking of smooth-muscle and non-muscle caldesmon to the C-terminus of actin in reconstituted and native thin filaments.
复制标题
平滑肌和非肌肉钙结合蛋白与重构和天然细丝中肌动蛋白 C 末端的二硫键交联。
DOI：
10.1042/bj2940063
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Lehman,W
中科院分区：
文献类型：
--
作者：
Graceffa,P;Adam,LP;Lehman,W
据报道，鸡砂囊平滑肌caldesmon Cys-580可以与肌动蛋白c端笔尖末端残基（Cys-374）二硫交联，表明这些残基在蛋白质复合体中是接近的[Graceffa， P. and Jancso， A. (1991) J. Biol]。化学学报，2004,23(2):444 - 444。由于不能完全排除交联涉及肌动蛋白Cys-374以外的半胱氨酸残基的可能性，因此需要寻找更直接的证据来鉴定交联中涉及的半胱氨酸残基。我们在这里证明caldesmon不能与肌动蛋白二硫交联，肌动蛋白Cys-374被羧肽酶A消化去除，直接支持肌动蛋白Cys-374参与与caldesmon的交联。为了确定参与交联的caldesmon半胱氨酸残基，使用了来自猪胃肌的caldesmon，它显示在580位置附近含有一个半胱氨酸残基，而鸡胗caldesmon在153位置有一个额外的半胱氨酸残基。猪胃caldesmon也与肌动蛋白形成二硫交联，进一步支持了鸡胗caldesmon的Cys-580与肌动蛋白交联的最初结论。在鸡砂囊肌细丝中也观察到类似产率的二硫交联，说明肌动蛋白与caldesmon c端结构域的相互作用在原生和重组细丝中是相同的。来自兔肝的小得多的caldesmon非肌肉同型体可以类似地与肌动蛋白交联，这与肌肉和非肌肉caldesmon的c端结构域之间的序列相似性一致。caldesmon Cys-580与肌动蛋白Cys-374交联的能力表明，caldesmon的Cys-580区域和肌动蛋白的c端可能构成了肌动蛋白-caldesmon结合界面的一部分。
It was reported that chicken gizzard smooth-muscle caldesmon Cys-580 can be disulphide-cross-linked to the C-terminal pen-ultimate residue (Cys-374) of actin, indicating that these residues are close in the protein complex [Graceffa, P. and Jancso, A. (1991) J. Biol. Chem. 266, 20305-20310]. Since the possibility that the cross-link involves a cysteine residue other than actin Cys-374 was not absolutely excluded, more direct evidence was sought for the identify of the cysteine residues involved in the cross-link. We show here that caldesmon could not be disulphide-cross-linked to actin which had Cys-374 removed by carboxypeptidase A digestion, providing direct support for the participation of actin Cys-374 in the cross-link to caldesmon. In order to assign the caldesmon cysteine residue involved in the cross-link, use was made of caldesmon from porcine stomach muscle, which is shown to contain one cysteine residue close to, or at, position 580, in contrast with chicken gizzard caldesmon, which has an additional cysteine residue at position 153. The porcine stomach caldesmon also formed a disulphide-cross-link to actin, further supporting the original conclusion that Cys-580 of the chicken gizzard caldesmon had been cross-linked to actin. Disulphide-cross-linking with similar yield was also observed in native chicken gizzard muscle thin filaments, indicating that the interaction between actin and the C-terminal domain of caldesmon is the same in native and reconstituted thin filaments. The much smaller non-muscle isoform of caldesmon, from rabbit liver, could be similarly cross-linked to actin, consistent with the sequence similarity between the C-terminal domain of muscle and non-muscle caldesmon. The ability to cross-link caldesmon Cys-580 to actin Cys-374 suggests the possibility that the Cys-580 region of caldesmon and the C-terminus of actin form part of the actin-caldesmon binding interface.