Reaction of Two Heads of Gizzard Myosin with ATP

Abstract

The reaction intermediates formed by the two heads of smooth muscle myosin were studied. The amount of myosin-phosphate-ADP complex, M^ADP_P, 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 K_d, 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 K_d 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 (K_d_??_1 μM) and 0.35 mol of nucleotides with low affinity (K_d=24 μM) for ATP. These results indicate that gizzard myosin has two kinds of nucleotide binding sites, one of which forms M^ADP_P with high affinity for ATP while the other forms M^ADP_P and MATP with low affinity for ATP. We studied the correlation between the formation of M^ADP_P 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 (1mg/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 0.5ml of ATP per mol myosin head depended on the experimental conditions. In 0.35mg/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 (1mg/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 M^ADP_P with a high affinity for ATP in one of the two heads of the myosin molecule and that the formation of M^ADP_P in the high affinity site results in a remarkable weakening of the binding of the other head to F-actin.