Komatsu H, Emoto Y, Tawada K
Department of Molecular Biology, Graduate School of Medical Science, Faculty of Science, Kyushu University, Fukuoka, Japan.
J Biol Chem. 1993 Apr 15;268(11):7799-808.
A molecule of myosin subfragment 1 (S1) from skeletal muscle has one reactive lysine residue (RLR) that is rapidly and stoichiometrically modified by trinitrobenzene sulfonate (TNBS). Previous studies on the RLR modification with TNBS in the presence of inorganic pyrophosphate (PPi) by others suggested the non-identical nature of the two myosin heads which is still controversial. To resolve this issue, we studied the effects of PPi and ADP on the trinitrophenylation reaction over a wide range of ligands more systematically, by using S1 containing the alkali 1 light chain. We herein show that MgPPi or MgADP reduces the maximum number of trinitrophenylated RLRs down to about 0.5 mol/mol of S1 and that this half-stoichiometric modification of RLRs is not the result of heterogeneity in the primary structure of S1. Instead, our results suggest the existence of two almost equimolar, different conformations of S1.PPi and S1.ADP. Furthermore, we show evidence that the two different conformations are in a slow equilibrium in the presence of TNBS. We also studied the effects of the stoichiometric and half-stoichiometric RLR modification on the EDTA- and Mg-ATPase activities of S1 and found no evidence for the functional heterogeneity of the myosin active site.
来自骨骼肌的肌球蛋白亚片段1（S1）分子有一个反应性赖氨酸残基（RLR），它会被三硝基苯磺酸（TNBS）快速且化学计量地修饰。此前其他人关于在无机焦磷酸（PPi）存在下用TNBS对RLR进行修饰的研究表明，两个肌球蛋白头部具有不同性质，但这一观点仍存在争议。为解决这个问题，我们通过使用含有碱1轻链的S1，更系统地研究了在广泛的配体范围内PPi和ADP对三硝基苯化反应的影响。我们在此表明，MgPPi或MgADP可将三硝基苯化RLR的最大数量降低至约0.5摩尔/摩尔的S1，并且RLR的这种半化学计量修饰并非S1一级结构异质性的结果。相反，我们的结果表明存在两种几乎等摩尔的、不同构象的S1·PPi和S1·ADP。此外，我们有证据表明，在TNBS存在下，这两种不同构象处于缓慢平衡状态。我们还研究了化学计量和半化学计量的RLR修饰对S1的EDTA和Mg - ATP酶活性的影响，未发现肌球蛋白活性位点功能异质性的证据。
