Xie L, Schoenberg M
Laboratory of Physical Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland 20892, USA.
Biochemistry. 1998 Jun 2;37(22):8048-53. doi: 10.1021/bi980319k.
Myosin subfragment-1 (S1) was labeled with NPM in the presence of ATP or with pPDM in the presence of ADP at 0 degreesC, conditions which favor linking of maleimide groups to both Cys-707 (SH1) and Cys-697 (SH2). Unmodified S1 was removed by sedimentation with a small amount of F-actin, and the modified protein in the supernatant thoroughly dialyzed. The myosin high-salt EDTA and calcium ATPase activities of the isolated modified S1 were close to zero, suggesting nearly complete modification of SH1 and SH2. The binding of control and these modified myosins to actin was measured at 100 mM ionic strength using a co-sedimentation assay. In the presence of high MgATP concentration, control and NPM- and pPDM-reacted S1 all bind weakly to actin, with binding constants K3 of 4.9, 2.2, and 1.9 x 10(4) M-1, respectively. In the absence of MgATP, the binding constant K2 of pPDM-reacted S1 remains weak, 4.6 x 10(4) M-1,while that of NPM-reacted and control S1 becomes strong, 4.7 and 31 x 10(6) M-1, respectively. The binding constant for ATP to acto-NPM-reacted-S1 is approximately 2 x 10(4) M-1. Our data suggest that the binding of NPM-S1 to F-actin, in contrast to that of pPDM-S1, is ATP sensitive and can be quite strong at very low ATP concentration. They also suggest that while simple alkylation of SH1 and SH2 may be sufficient to inhibit myosin's ability to hydrolyze ATP, actual covalent linkage of SH1 and SH2 may be necessary to inhibit the weakly to strongly binding conformational change.
在0℃条件下，肌球蛋白亚片段-1（S1）在ATP存在时用NPM标记，或在ADP存在时用pPDM标记，这些条件有利于马来酰亚胺基团与Cys-707（SH1）和Cys-697（SH2）连接。用少量F-肌动蛋白沉降去除未修饰的S1，上清液中的修饰蛋白进行充分透析。分离得到的修饰S1的肌球蛋白高盐EDTA和钙ATP酶活性接近零，表明SH1和SH2几乎完全被修饰。使用共沉降分析法在100 mM离子强度下测量对照和这些修饰肌球蛋白与肌动蛋白的结合。在高MgATP浓度存在时，对照以及NPM和pPDM反应的S1与肌动蛋白的结合都很弱，结合常数K3分别为4.9、2.2和1.9×10⁴ M⁻¹。在没有MgATP时，pPDM反应的S1的结合常数K2仍然较弱，为4.6×10⁴ M⁻¹，而NPM反应的S1和对照S1的结合常数变强，分别为4.7和31×10⁶ M⁻¹。ATP与肌动蛋白-NPM反应的S1的结合常数约为2×10⁴ M⁻¹。我们的数据表明，与pPDM-S1相比，NPM-S1与F-肌动蛋白的结合对ATP敏感，并且在非常低的ATP浓度下可能相当强。数据还表明，虽然SH1和SH2的简单烷基化可能足以抑制肌球蛋白水解ATP的能力，但SH1和SH2的实际共价连接可能是抑制从弱结合到强结合构象变化所必需的。
