Katoh T, Morita F
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Japan.
Eur J Biochem. 1995 Oct 1;233(1):123-31. doi: 10.1111/j.1432-1033.1995.123_1.x.
The two heads of porcine aorta smooth muscle myosin can be cross-linked by a disulfide bridge between the two 17-kDa essential light chains with 5,5'-dithiobis(2-nitrobenzoic acid) [Katoh, T., Tanahashi, K., Hasegawa, Y. & Morita, F. (1995) Eur. J. Biochem. 227, 459-465]. When the cross-linked myosin sample was visualized by rotary shadowing, the two heads of myosin molecules appeared predominantly to adhere to each other. The cross-linking of dephosphorylated myosin in the presence of ATP was greatly inhibited by a decrease in the concentration of NaCl from 0.4 M to 0.15 M, suggesting that the cross-linking of the two heads was suppressed in 10S myosin. However, the fraction of dephosphorylated myosin in a filamentous state at 0.1 M NaCl in the presence of 1 mM ATP was increased from 33% to 83% by the cross-linking. The cross-linking of the two heads might inhibit the formation of the 10S conformation, leading to the increase in the fraction of filamentous myosin. The filaments of the cross-linked myosin sample were visualized by electron microscopy and appeared morphologically similar to those of uncross-linked myosin. The ATPase activity of the cross-linked dephosphorylated myosin sample was more than three times as high as that of an uncross-linked control. The increase in the activity may be related to the increase in the fraction of filamentous myosin caused by the cross-linking. The ATPase activity of dephosphorylated myosin in the presence of actin was increased more than twofold by the cross-linking, but the activity of phosphorylated myosin was affected only slightly. The degree of phosphorylation-dependent regulation of actin-activated ATPase activity decreased with an increase in the degree of cross-linking and was extrapolated to zero at 100% cross-linking. Superprecipitation of acto-cross-linked dephosphorylated myosin was activated, while that of acto-cross-linked phosphorylated myosin was inhibited only slightly. These results suggest that the freedom of each head in myosin molecules may be required to keep the ATPase activity and superprecipitation of acto-dephosphorylated myosin low but not for keeping these activity levels high in acto-phosphorylated myosin.
猪主动脉平滑肌肌球蛋白的两个头部可通过两个17 kDa必需轻链之间的二硫键与5,5'-二硫代双（2-硝基苯甲酸）交联[加藤，T.，田桥，K.，长谷川，Y. & 森田，F.（1995年）《欧洲生物化学杂志》227，459 - 465]。当通过旋转阴影法观察交联的肌球蛋白样品时，肌球蛋白分子的两个头部主要表现为相互粘附。在ATP存在下，去磷酸化肌球蛋白的交联在NaCl浓度从0.4 M降至0.15 M时受到极大抑制，这表明在10S肌球蛋白中两个头部的交联受到抑制。然而，在1 mM ATP存在下，0.1 M NaCl中丝状状态的去磷酸化肌球蛋白比例通过交联从33%增加到了83%。两个头部的交联可能会抑制10S构象的形成，从而导致丝状肌球蛋白比例增加。通过电子显微镜观察交联的肌球蛋白样品的细丝，其形态与未交联的肌球蛋白细丝相似。交联的去磷酸化肌球蛋白样品的ATP酶活性比未交联对照高3倍以上。活性的增加可能与交联导致的丝状肌球蛋白比例增加有关。在肌动蛋白存在下，去磷酸化肌球蛋白的ATP酶活性通过交联增加了两倍多，但磷酸化肌球蛋白的活性仅受到轻微影响。肌动蛋白激活的ATP酶活性的磷酸化依赖性调节程度随着交联程度的增加而降低，在100%交联时外推至零。肌动蛋白交联的去磷酸化肌球蛋白的超沉淀被激活，而肌动蛋白交联的磷酸化肌球蛋白的超沉淀仅受到轻微抑制。这些结果表明，肌球蛋白分子中每个头部的自由度可能是保持肌动蛋白去磷酸化肌球蛋白的ATP酶活性和超沉淀较低所必需的，但对于保持肌动蛋白磷酸化肌球蛋白的这些活性水平较高则不是必需的。
