Regulatory and essential light-chain interactions in scallop myosin. II. Photochemical cross-linking of regulatory and essential light-chains by heterobifunctional reagents.
Regulatory and essential light-chain interactions in scallop myosin. II. Photochemical cross-linking of regulatory and essential light-chains by heterobifunctional reagents.
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扇贝肌球蛋白的调节和重要轻链相互作用。
DOI：
10.1016/0022-2836(82)90464-8
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发表时间：
1982
影响因子：
5.6
通讯作者：
Szent-Györgyi,AG
中科院分区：
文献类型：
--
作者：
Wallimann,T;Hardwicke,PM;Szent-Györgyi,AG
分离的扇贝调节轻链的4 - 6个赖氨酸残基与N-羟基琥珀酰亚胺基-4-叠氮苯甲酸酯缀合;分离的MercenariaR-LC的单个巯基和分离的兔R-LC的两个巯基被对叠氮苯酰溴取代。通过这些异双功能光敏交联剂修饰的R-LC容易与扇贝肌原纤维结合，其中R-LC已经通过在35 °C下用EDTA处理而从扇贝肌原纤维中去除。修饰的扇贝和雇佣兵R-LC恢复了肌动蛋白激活的Mg-ATPase的钙敏感性。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳硝酸纤维素印迹的间接抗体染色，使用特异性抗扇贝R-LC和抗扇贝SH-LC抗体，然后分别用异硫氰酸荧光素标记或125 I标记的山羊抗兔免疫球蛋白G，观察和定量修饰的R-LC和必需轻链之间在光照下形成的复合物。在光解后形成的较高分子量复合物（Mr> 17，000）的条带中存在SH-LC被认为是取代的R-LC和扇贝SH-LC之间交联的证据。约1/3的扇贝SH-LC与p-APA-取代的MercenariaR-LC交联，当光照产生一条由一个R-LC和一个SH-LC组成的单一新带。没有形成更高分子量的复合物。用多取代的扇贝和兔R-LC，超过一半的扇贝SH-LC交联;约三分之一与修饰的R-LC形成一对一的复合物，并且显著量与肌球蛋白重链可变区R-LC交联。分子量约为53，000和66，000的复合物也与高度修饰的扇贝和兔R-LC少量形成，可能由三条和四条交联轻链组成。在12 °C下用EDTA处理的肌原纤维中证实了扇贝肌球蛋白分子的两个R-LC的一些交联，所述肌原纤维含有1摩尔完整的扇贝R-LC和1摩尔p-APA修饰的雇佣兵或兔R-LC。R-LCs与肌动蛋白或原肌球蛋白没有显著交联。光解不影响肌动蛋白激活的Mg-ATPase活性，但在无钙条件下通过激活降低钙敏感性。这些结果表明，扇贝肌球蛋白的R-LC和SH-LC非常接近（相距不超过6至8 μ m），并且至少有一半的长度重叠。R-LC和SH-LC的交联会干扰调节，这与SH-LC的调节作用一致。
From four to six lysine residues of isolated scallop regulatory light-chains were conjugated withN-hydroxysuccinimidyl-4-azidobenzoate; the single thiol group of isolatedMercenariaR-LC‡, and the two thiol groups of isolated rabbit R-LCs were substituted withp-azidophenacylbromide. R-LCs modified by these heterobifunctional photosensitive cross-linkers combined readily with scallop myofibrils from which R-LCs had been removed by treatment with EDTA at 35 °C. The modified scallop andMercenariaR-LCs restored the calcium sensitivity of the actin-activated Mg-ATPase. The complexes formed upon illumination between modified R-LCs and essential light-chains were visualized and quantitated by indirect antibody staining of electrophoretic nitrocellulose blots of sodium dodecyl sulfate/polyacrylamide gels using specific anti-scallop R-LC and anti-scallop SH-LC antibodies followed by fluorescein isothiocyanate-labeled or125I-labeled goat anti-rabbit immunoglobulin G, respectively. The presence of SH-LC in bands of higher molecular weight complexes (Mr> 17,000) formed upon photolysis was taken as evidence for cross-linking between the substituted R-LCs and scallop SH-LCs. About one-third of the scallop SH-LC cross-linked withp-APA-substitutedMercenariaR-LC, when illumination generated a single new band atMr35,000 consisting of one R-LC and one SH-LC. Higher molecular weight complexes were not formed. With the multi-substituted scallop and rabbit R-LCs, more than half of the scallop SH-LC was cross-linked; about one-third formed a one to one complex with the modified R-LC, and a significant amount was cross-linked to the myosin heavy-chainviathe R-LC. Complexes of molecular weights of approximately 53,000 and 66,000 were also formed in small amounts with highly modified scallop and rabbit R-LCs, probably consisting of three and four cross-linked light-chains. Some cross-linking of the two R-LCs of the scallop myosin molecule was demonstrated with myofibrils treated with EDTA at 12 °C and containing one mole of intact scallop R-LC and one mole ofp-APA-modifiedMercenariaor rabbit R-LC. R-LCs did not significantly cross-link with actin or tropomyosin. Photolysis did not inactivate the actin-activated Mg-ATPase activity, but reduced calcium sensitivity by activation in the absence of calcium. These results show that R-LCs and SH-LCs of scallop myosin are in close proximity (no more than 6 to 8 Å apart) and overlap for at least half of their lengths. Cross-linking of R-LCs and SH-LCs interferes with regulation, consistent with a regulatory role for the SH-LCs.
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