THE EFFECT OF CROSS-LINKING OF THE 2 HEADS OF PORCINE AORTA SMOOTH-MUSCLE MYOSIN ON ITS CONFORMATION AND ENZYMATIC-ACTIVITY

The two heads of porcine aorta smooth muscle myosin can be cross-linke d by a disfulfide bridge between the two 17-kDa essential light chains with 5,5'-dithiobis(2-nitrobenzoic acid) [Katoh, T., Tanahashi, K, Ha segawa, Y. & Morita, F. (1995) Eur. J. Biochem. 227, 459-465]. When th e cross-linked myosin sample was visualized by rotary shadowing, the t wo 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 hea ds was suppressed in 10S myosin. However, the fraction of dephosphoryl ated 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-l inking of the two heads might inhibit the formation of the 10S conform ation, leading to the increase in the fraction of filamentous myosin. The filaments of the cross-linked myosin sample were visualized by ele ctron microscopy and appeared morphologically similar to those of uncr oss-linked myosin. The ATPase activity of the cross-linked control. Th e increase in the activity may be related to the increase in the fract ion of filamentous myosin caused by the cross-linking. The ATPase acti vity of dephosphorylated myosin in the presence of actin was increased more than twofold by the cross-linking, but the activity of phosphory lated myosin was affected only slightly. The degree of phosphorylation -dependent regulation of actin-activated ATPase activity decreased wit h an increase in the degree of cross-linking and was extrapolated to z ero at 100% cross-linking. Superprecipitation of acto-cross-linked dep hosphorylated myosin was activated, while that of acto-cross-linked ph osphorylated 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-dephosphorylat ed myosin low but not for keeping these activity levels high in acto-p hosphorylated myosin.