Kwon H, Hardwicke P M, Collins J H, Zhao X, Szent-Györgyi A G
Department of Biology, Brandeis University, Waltham, MA 02254.
J Muscle Res Cell Motil. 1994 Oct;15(5):555-62. doi: 10.1007/BF00121161.
Reaction of rabbit skeletal muscle F-actin with the lysine-directed photolabile cross-linker, N-5-azido-2-nitrobenzoyloxy succinimide was limited to Lysine-328 and Lysine-326, with Lysine-328 being labelled to a greater extent. Photolysis of the modified actin enhanced the actin-activated MgATPase activity of filamentous scallop myosin 3-4-fold more than unmodified actin, without affecting calcium sensitivity. Unphotolysed modified actin behaved as untreated actin, indicating that photolysis was essential for the effect. The actin-activated ATPase of filamentous rabbit myosin was similarly increased by photolysed N-5-azido-2-nitrobenzoyloxy succinimide-modified actin. After photolysis in either the monomeric (G-) or filamentous (F-) form, N-5-azido-2-nitrobenzoyloxy succinimide-modified actin moved as a monomeric (42 kDa) species on SDS gels, and depolymerized and polymerized readily, demonstrating that any cross-linking event produced by photolysis must be intramolecular. In contrast to the substantial increase in actin-activated ATPase activity observed when photolysed ANB-NOS-modified actin was added to filamentous myosin, the enhancement was not observed with the soluble HMM and S-1 fragments of myosin. Photolysed modified actin showed only poor movement on a rabbit HMM-coated surface in vitro motility assays. These results can be explained if the internally cross-linked G-actin subunits which comprise only a fraction of the actin population, either weaken the actin-actin contacts or have an increased affinity for myosin.
兔骨骼肌F-肌动蛋白与赖氨酸定向光不稳定交联剂N-5-叠氮基-2-硝基苯氧基琥珀酰亚胺的反应仅限于赖氨酸-328和赖氨酸-326，其中赖氨酸-328的标记程度更高。修饰后的肌动蛋白经光解后，其对丝状扇贝肌球蛋白的肌动蛋白激活的MgATP酶活性的增强幅度比未修饰的肌动蛋白高3至4倍，且不影响钙敏感性。未光解的修饰肌动蛋白表现得与未处理的肌动蛋白一样，这表明光解对于该效应至关重要。光解后的N-5-叠氮基-2-硝基苯氧基琥珀酰亚胺修饰的肌动蛋白同样能使丝状兔肌球蛋白的肌动蛋白激活的ATP酶活性增加。在以单体（G-）或丝状（F-）形式进行光解后，N-5-叠氮基-2-硝基苯氧基琥珀酰亚胺修饰的肌动蛋白在SDS凝胶上以单体（42 kDa）形式迁移，并且易于解聚和聚合，这表明光解产生的任何交联事件必定是分子内的。与将光解后的ANB-NOS修饰的肌动蛋白添加到丝状肌球蛋白时观察到的肌动蛋白激活的ATP酶活性大幅增加相反，在肌球蛋白的可溶性HMM和S-1片段中未观察到这种增强。在体外运动分析中，光解后的修饰肌动蛋白在兔HMM包被的表面上仅表现出微弱的运动。如果仅占肌动蛋白群体一部分的内部交联的G-肌动蛋白亚基要么削弱了肌动蛋白-肌动蛋白接触，要么对肌球蛋白具有更高的亲和力，那么这些结果就可以得到解释。
