J. Biochem, 1988, Vol. 103, No. 2 336-341
© 1988 Japanese Biochemical Society
research-article |
Reaction of Two Heads of Gizzard Myosin with ATP1
Department of Biology,Osaka University Toyonaka, Osaka 560
The reaction intermediates formed by the two heads of smooth muscle myosin were studied. The amount of myosin-phosphate-ADP complex, MADPp, formed was measured from the P1-burst size over a wide range of ATP concentrations. At low concentrations of ATP, the P1-burst size was 0.5 mol/mol myosin head, and the apparent Kc value was about 0.15 µM. However, at high ATP concentrations, the P1 burst size increased from 0.5 to 0.75 mol/mol myosin head with an observed Kd, value of 15 µM. The binding of nucleotides to gizzard myosin during the ATPase reaction was directly measured by a centrifugation method. Myosin bound 0.5 mol of nucleotides (ATP and ADP) with high affinity (Kd 
1 µM) and 0.35 mol of nucleotides with low affinity (Kd =24 µM) for ATP. These results indicate that gizzard myosin has two kinds of nucleotide binding sites, one of which forms MADPp with high affinity for ATP while the other forms MADPp and MATP with low affinity for ATP. We studied the correlation between the formation of MADPp and the dissociation of actomyosin. The amount of P1-burst size was not affected by the existence of F-actin, and when 0.5 mol of ATP per mol of myosin head was added to actomyosin (1 mg/ml F-actin, 5, µM myosin at 0°C) most (93%) of the added ATP was hydrolyzed in the P1-burst phase. All gizzard actomyosin dissociated when 1 mol of ATP per mol myosin head was added to actomyosin. However, the extent of actomyosin dissociation at 0.5 ml of ATP per mol myosin head depended on the experimental conditions. In 0.35 mg/ml F-actin and at 0°C, where myosin binds weakly to F-actin, more than, half of the myosin molecule dissociated from F-actin. Under conditions where myosin binds tightly to F-actin (1 mg/ml F-actin and at 20°C), only less than 1/4 of myosin dissociated from F-actin. These results indicate that smooth muscle myosin forms MADPp with a high affinity for ATP in one of the two heads of the myosin molecule and that the formation of MADPp in the high affinity site results in a remarkable weakening of the binding of the other head to F-actin.
1 This work was supported by grants from the Ministry of Education, Science and Culture of Japan, the Ministry of Health and Welfare of Japan, and the Muscular Dystrophy Association, Inc., U.S.A.