Bartegi A, Fattoum A, Kassab R
Institut National de la Sante et de la Recherche Médicale U.249 Université de Montpellier I, France.
J Biol Chem. 1990 Feb 5;265(4):2231-7.
The cross-linking of the F-actin-caldesmon complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the presence of N-hydroxysuccinimide generated four major adducts which were identified on polyacrylamide gels. By cross-linking 3H-actin to 14C-caldesmon, these were found to represent 1:1 cross-linked complexes of actin and caldesmon displaying different electrophoretic mobilities. Tropomyosin did not noticeably affect the cross-linking process. The same four fluorescent species resulting from the cross-linking of caldesmon to F-actin labeled with N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide were subjected separately to partial cleavages with hydroxylamine or cyanogen bromide. These treatments yielded fluorescent 41- and 37-kDa fragments, respectively, from each cross-linked entity indicating unambiguously that caldesmon was cross-linked only to the NH2-terminal actin stretch of residues 1-12. This region is also known to serve for the carbodiimide-mediated cross-linking of the myosin subfragment-1 heavy chain (Sutoh, K. (1982) Biochemistry 21, 3654-3661). A covalent caldesmon-F-actin conjugate containing a protein molar ratio close to 1:19 was isolated following dissociation of uncross-linked caldesmon. It showed a low level of activation of the ATPase activity of skeletal myosin subfragment-1, and the binding of Ca2(+)-calmodulin to the derivative did not cause the reversal of the ATPase inhibition. In contrast, the reversible binding of caldesmon to F-actin cross-linked to myosin subfragment-1 did not inhibit the accelerated ATPase of the complex. The overall data point to the dual involvement of the actin's NH2 terminus in the inhibitory binding of caldesmon and in actomyosin interactions in the presence of ATP.
在N - 羟基琥珀酰亚胺存在下，用1 - 乙基 - 3 - [3 -（二甲基氨基）丙基]碳二亚胺使F - 肌动蛋白 - 钙调蛋白复合物交联，产生了四种主要加合物，这些加合物在聚丙烯酰胺凝胶上得到了鉴定。通过将³H - 肌动蛋白与¹⁴C - 钙调蛋白交联，发现它们代表肌动蛋白和钙调蛋白的1:1交联复合物，显示出不同的电泳迁移率。原肌球蛋白对交联过程没有明显影响。用N - [7 -（二甲基氨基）- 4 - 甲基 - 3 - 香豆素基]马来酰亚胺标记的钙调蛋白与F - 肌动蛋白交联产生的相同四种荧光物质，分别用羟胺或溴化氰进行部分切割。这些处理分别从每个交联实体产生了荧光的41 kDa和37 kDa片段，明确表明钙调蛋白仅与残基1 - 12的NH₂ - 末端肌动蛋白片段交联。该区域也已知用于碳二亚胺介导的肌球蛋白亚片段 - 1重链的交联（Sutoh，K.（1982年）《生物化学》21，3654 - 3661）。在未交联的钙调蛋白解离后，分离出一种蛋白质摩尔比接近1:19的共价钙调蛋白 - F - 肌动蛋白缀合物。它显示出骨骼肌肌球蛋白亚片段 - 1的ATP酶活性的低水平激活，并且Ca²⁺ - 钙调蛋白与衍生物的结合不会导致ATP酶抑制的逆转。相反，钙调蛋白与交联到肌球蛋白亚片段 - 1的F - 肌动蛋白的可逆结合不会抑制复合物加速的ATP酶活性。总体数据表明，肌动蛋白的NH₂末端在钙调蛋白的抑制性结合以及ATP存在下的肌动球蛋白相互作用中具有双重作用。
