INTERACTION OF HEAVY MEROMYOSIN WITH SUBSTRATE .2. RATE OF FORMATION OF ATP-INDUCED ULTRAVIOLET DIFFERENCE SPECTRUM OF HEAVY MEROMYOSIN MEASURED BY STOPPED-FLOW METHOD

1. 1. The rate of formation of the ultraviolet difference spectrum of heavy meromyosin, induced by ATP, was measured by the stopped-flow method. The initial velocity of formation was proportional to the initial concentration of ATP, indicating a second-order reaction. The second-order rate constant decreased with decrease of MgCl2 and with increase of KCl concentrations, but did not depend on the kind of divalent cation (Mg2+ or Ca2+) in the medium. 2. 2. The maximum difference absorbance Δ;Amax attained after addition of ATP was obtained as a function of initial concentration of ATP. A strong binding of ATP to heavy meromyosin was suggested in the presence of MgCl2. The minimum ATP concentration giving the maximum Δ;Amax was 2-fold the protein concentration. In the presence of CaCl2, the binding was not as strong. 3. 3. Decay of the difference spectrum was measured. In the presence of MgCl2, ks calculated using Chance's equation agreed well with the maximum velocity of the steady-state ATPase. In the presence of CaCl2, ks was 3 times larger than the maximum velocity. 4. 4. It is inferred that the difference spectrum accompanies only the Michaelis-Menten complex formed at one of the two sites in heavy meromyosin. © 1960.