Trybus K M
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254-9110.
J Cell Biol. 1989 Dec;109(6 Pt 1):2887-94. doi: 10.1083/jcb.109.6.2887.
The enzymatic activity of filamentous dephosphorylated smooth muscle myosin has been difficult to determine because the polymer disassembles to the folded conformation in the presence of MgATP. Monoclonal antirod antibodies were used here to "fix" dephosphorylated myosin in the filamentous state. The steady-state actin-activated ATPase of phosphorylated filaments was 30-100-fold higher than that of antibody-stabilized dephosphorylated filaments, suggesting that phosphorylation can activate ATPase activity independent of changes in assembly. The degree of regulation may exceed 100-fold, because steady-state measurements slightly overestimate the rate of product release from dephosphorylated filaments. Single-turnover experiments in the absence of actin showed that although dephosphorylated folded myosin released products at the low rate of 0.0005 s-1 (Cross, R. A., K. E. Cross, A. Sobieszek. 1986. EMBO [Eur. Mol. Biol. Organ.] J. 5:2637-2641) the rate of product release from dephosphorylated filaments was only 3-12-fold higher, depending on the ionic strength. The addition of actin did not increase this rate to any appreciable extent. Dephosphorylated filaments and dephosphorylated heavy meromyosin (Sellers, J. R. 1985. J. Biol. Chem. 260:15815-15819) thus have similar low rates of phosphate release both in the presence and absence of actin. These results show that light chain phosphorylation alone, without invoking other mechanisms, is an effective switch for regulating the activity of smooth muscle myosin filaments.
丝状去磷酸化平滑肌肌球蛋白的酶活性一直难以测定,因为在MgATP存在下,聚合物会解聚为折叠构象。在此使用单克隆抗杆状抗体将去磷酸化肌球蛋白“固定”在丝状状态。磷酸化细丝的稳态肌动蛋白激活的ATP酶比抗体稳定的去磷酸化细丝高30 - 100倍,这表明磷酸化可以独立于组装变化激活ATP酶活性。调节程度可能超过100倍,因为稳态测量略微高估了去磷酸化细丝中产物释放的速率。在没有肌动蛋白的情况下进行的单周转实验表明,尽管去磷酸化的折叠肌球蛋白以0.0005 s-1的低速率释放产物(克罗斯,R.A.,K.E.克罗斯,A.索别谢克。1986年。《欧洲分子生物学组织杂志》5:2637 - 2641),但去磷酸化细丝中产物释放的速率仅高3 - 12倍,这取决于离子强度。添加肌动蛋白并没有将该速率提高到任何可观的程度。因此,无论有无肌动蛋白,去磷酸化细丝和去磷酸化重酶解肌球蛋白(塞勒斯,J.R. 1985年。《生物化学杂志》260:15815 - 15819)都有相似的低磷酸盐释放速率。这些结果表明,仅轻链磷酸化,无需借助其他机制,就是调节平滑肌肌球蛋白细丝活性的有效开关。