The effect of cross-linking of the two heads of porcine aorta smooth muscle myosin on its conformation and enzymic activity.

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.