Mezgueldi M, Derancourt J, Calas B, Kassab R, Fattoum A
Centre de Recherches de Biochimie Macromoléculaire du CNRS, INSERM U 249, Université de Montpellier I, France.
J Biol Chem. 1994 Apr 29;269(17):12824-32.
The precise location of the regulatory F-actin- and calmodulin-binding sites in the COOH-terminal sequence Trp659-Pro756 of gizzard caldesmon was investigated by subjecting the corresponding 10-kDa CNBr fragment, characterized earlier (Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238), to limited chymotryptic reactions conducted in the absence and presence of F-actin-tropomyosin. As a result, the F-actin-binding and actomyosin ATPase inhibitory activity was separated from the regulatory Ca(2+)-calmodulin-binding site. Seven chymotryptic peptides accounting for the entire primary structure of the CB10 fragment were isolated, and their complete amino acid sequences were established by combining NH2-terminal sequencing, mass spectrometry, and gel electrophoresis. Reversed-phase high performance liquid chromatography analyses of the binding of F-actin to these peptides revealed the 30-residue sequence Leu693-Trp722 as the unique crucial stretch for actin interaction and ATPase inhibition. This segment was also specifically protected by F-actin against proteolytic degradation. We further determined the functional properties of three synthetic peptides which successively cover the sequences Asn675-Lys695, Leu693-Trp722, and Arg711-Lys729. The first peptide segment specifically bound Ca(2+)-calmodulin as assessed by affinity chromatography and spectrofluorometry and should contain a potent novel calmodulin-binding subsite. The second immediately adjacent peptide inhibited the actomyosin ATPase in a tropomyosin-sensitive manner, as expected. In contrast, the third peptide displayed no detectable function. The results indicate that the overall sequence Asn675-Trp722 represents the essential regulatory unit of the COOH-terminal 10-kDa domain of caldesmon.
通过对先前已鉴定的（Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231 - 15238）相应10 kDa溴化氰片段进行有限的胰凝乳蛋白酶反应，研究了肌胃钙调蛋白COOH末端序列Trp659 - Pro756中调节性F - 肌动蛋白和钙调蛋白结合位点的精确位置，反应分别在不存在和存在F - 肌动蛋白 - 原肌球蛋白的情况下进行。结果，F - 肌动蛋白结合和肌动球蛋白ATP酶抑制活性与调节性Ca(2 + ) - 钙调蛋白结合位点分离。分离出了七个解释CB10片段完整一级结构的胰凝乳蛋白酶肽段，并通过结合氨基末端测序、质谱分析和凝胶电泳确定了它们完整的氨基酸序列。对这些肽段与F - 肌动蛋白结合的反相高效液相色谱分析表明，30个残基的序列Leu693 - Trp722是肌动蛋白相互作用和ATP酶抑制的唯一关键区域。该片段也受到F - 肌动蛋白的特异性保护而免受蛋白水解降解。我们进一步确定了三个合成肽段的功能特性，它们依次覆盖序列Asn675 - Lys695、Leu693 - Trp722和Arg711 - Lys729。通过亲和色谱和荧光光谱法评估，第一个肽段特异性结合Ca(2 + ) - 钙调蛋白，并且应该包含一个有效的新型钙调蛋白结合亚位点。如预期的那样，第二个紧邻的肽段以原肌球蛋白敏感的方式抑制肌动球蛋白ATP酶。相比之下，第三个肽段未显示出可检测的功能。结果表明，整个序列Asn675 - Trp722代表钙调蛋白COOH末端10 kDa结构域的基本调节单元。
