Yuji Miyazawa

Preparation and properties of smooth muscle myosin from horse esophagus.

Abstract Myosin was prepared from smooth muscle of horse esophagus in good yield (about 150 mg/100 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 260 nm was about 1.8, and the amount of contaminating phosphorus was only 0.91 g/10 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 KCl 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 10 5 g of protein as horse and rabbit skeletal myosin A (about 8 moles/10 5 g of protein). But it contained more asparatic acid or asparagine, more leucine and less lysine, glycine and proline. Ca 2+ -ATPase of myosin S in the presence of 0.5 M KCl and Mg 2+ -ATPase in the presence of 0.05 M KCl at 37° were very similar to those of skeletal myosin A. On the other hand, EDTA-ATPase and Ca 2+ -ATPase in the presence of 0.05 M KCl 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 Ca 2+ -ATPase activity. This behaviour was different from that of skeletal myosin A which exhibited an inhibition of EDTA-ATPase and an activation of Ca 2+ -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.

Partial melting of the segment around pseudouridine in yeast 5S RNA

Pseudouridine in yeast 5S RNA was modified with 4-bromomethyl-7-methoxy-coumarin(BMC). Temperature dependence of fluorescence intensity was measured at various concentrations of Mg2+ and K+ cations. Hyperchromicity was also measured. At 100mM KCl and 10mM Mg2+, fluorescence intensity decreased with temperature as free BMC except a plateau at 45 degrees C. Withdrawal of Mg2+ from the buffer resulted in a large quenching at 20 degrees C and showed a gradual increase of fluorescence intensity with temperature, indicating a partial melting of the segment around pseudouridine. The temperature range agrees with the low melting temperature shown by hyperchromicity. In 10 mM KCl solution, the effects are more exaggerated.