Preparation and properties of smooth muscle myosin from horse esophagus.

Abstract: Myosin was prepared from smooth muscle of horse esophagus in good yield (about 15 ° mg/Ioo g tissue) and was designated myosin S. Its properties were compared with those of myosin A from skeletal muscle. The ratio of the absorption of myosin S at 280 nm to that at 26o nm was about 1.8, and the amount of contaminating phosphorus was only o.91 g/io 5 g of myosin S, indicating that the latter is free of nucleic acid. The purity of this protein was examined by ultracentrifugation, gel filtration in the presence of 0.5 M KC1 and 6 M urea and chromatography on DEAE-cellulose columns. These experiments all indicated that myosin S was homogeneous, like highly purified rabbit skeletal myosin A. Amino acid analyses showed differences in the composition of smooth and skeletal myosins. Myosin S contained the same amount of sulfhydryl groups per lO 5 g of protein as horse and rabbit skeletal myosin A (about 8 moles/Io 5 g of protein). But it contained more asparatic acid or asparagine, more leucine and less lysine, glycine and proline. Ca~+-ATPase of myosin S in the presence of 0.5 M KC1 and Mg2+-ATPase in the presence of 0.05 M KC1 at 37 ° were very similar to those of skeletal myosin A. On the other hand, EDTA-ATPase and Ca~+-ATPase in the presence of 0.05 M KC1 were much lower than those of skeletal myosin A. Lowering the temperature from 37 to 25 °, the degree of decrease of the ATPase activities was much larger in myosin S than in skeletal myosin A. The reaction of N-ethylmaleimide with myosin S caused inhibition of the EDTA-ATPase but did not affect the Ca2+-ATPase activity. This behaviour was different from that of skeletal myosin A which exhibited an inhibition of EDTA-ATPase and an activation of Ca2+-ATPase during the course of the reaction of sulfhydryl groups of myosin with N-ethylmaleimide. These facts suggest that the structure of the active site of myosin S ATPase differs significantly from that of skeletal myosin A. These differences appear to influence the interaction of myosin with F-actin, so that the rate of superprecipitation found in an actomyosin reconstituted from myosin S and F-actin was only one fortieth of that found with skeletal myosin A. The difference in the speed of contraction of skeletal and smooth muscles is generally considered to be due to the differences in the processes taking place in the excitable membrane and in the arrangement of contractile filaments 1, Recently, several investigators have reported on myosin extracted from smooth muscle tissues: NEEDHAM AND WILLIAMS 2 and COHEN et al. 3 on uterus myosin, HAMOIR 4 and RC'EGG 32 on carotid muscle myosin, named tonomyosin, and B.X, RANY et al. 5 o11 chicken gizzard myosin. These myosins differ from myosin A (EC 3.6.1.3) of rabbit skeletal muscle in some respects, i.e. the dependence of their ATPase activity on KC1 concentration and the degree of the activation of Mg2+-ATPase induced by F-actin. These facts suggest that differences in the physiological properties of the two types of muscles might also be due to differences in the properties of myosin molecules. To study this problem, highly purified myosin had to be obtained from smooth muscle tissues, which contain more lipids and nucleic acids than skeletal muscle tissues. The present investigation describes the purity, the amino acid composition and the enzymatic properties of highly purified myosin (myosin S) obtained from horse esophagus.