The binding of smooth muscle heavy meromyosin to actin in the presence of ATP. Effect of phosphorylation.
Phosphorylation of the 20,000-dalton light chains of smooth muscle heavy meromyosin (HMM) from turkey gizzards results in a large increase in the actin-activated MgATPase activity over that observed with unphosphorylated HMM. In an attempt to define which step in the kinetic cycle is affected by phosphorylation, we have measured the binding of both unphosphorylated and phosphorylated HMM to actin in the presence of ATP using sedimentation. There was only a 4-fold difference in the actin binding constants of unphosphorylated HMM (5.35 x 10(3) M-1) and fully phosphorylated HMM (2.35 x 10(4) M-1). In contrast, the maximum rate of the actin-activated MgATPase activity (Vmax) of phosphorylated HMM was 25 times greater than that for unphosphorylated HMM. These data rule out a mechanism whereby the unphosphorylated light chain of myosin regulates actin-myosin interaction by directly or indirectly blocking the binding of HMM to actin. This implies that some step in the kinetic cycle other than the binding of HMM to actin must be regulated. We have also measured the rate constant for ATP hydrolysis (the initial phosphate burst) under the same conditions and found that this step was very fast compared to the steady state ATPase rate and was unaffected by phosphorylation. This suggests that the step which is regulated by phosphorylation is either phosphate release or a step preceding phosphate release but following ATP hydrolysis.
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D014422:Turkeys
D000199:Actins; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase