To determine whether the nonidentical two-headed structure of myosin is common to various kinds of muscle, we compared the stoichiometry in the reactions of ATP with chicken gizzard myosin and rabbit skeletal muscle myosin. We also compared the properties of acto-gizzard myosin ATPase with those of skeletal muscle actomyosin ATPase, to clarify the roles of the burst head and the nonburst head in muscle contraction. The findings obtained are as follows.
1. The maximum amount of total nucleotides (ATP+ADP) bound to HMM during the ATPase reaction was 2 mol/mol for both smooth and skeletal muscles. However, the amount of ADP bound to HMM increased linearly with increase in the ATP concentration, reaching the maximum level of 1 and 1.35-1.60 mol/mol HMM at 1 and 2 mol of ATP per mol of HMM for skeletal and smooth muscles, respectively. Also in the case of skeletal muscle, the ATP binding to HMM was observed only after saturation of the ADP binding with the maximum value of 1 mol/mol HMM, while in the case of smooth muscle, the amount of ATP bound to HMM was proportional to that of ADP at all the ATP concentrations tested.
2. The maximum amounts of P bound to HMM were the same as those of ADP, being 1 and 1.60 mol/mol HMM, respectively, for skeletal and smooth muscles.
3. The amounts of ATP required to induce maximum enhancement of the fluorescence intensity of HMM in the steady state in the presence of sufficient amounts of PK and PEP were 1 and 2 mol/mol HMM for skeletal and smooth muscles, respectively.
4. The ATPase activity of smooth muscle myosin was inhibited by trinitrophenylation, and the inhibition was prevented by the addition of PP_i. The amount of lysine residues involved in the ATPase inhibition was 2-3 mol/mol myosin.
5. The extent of dissociation of skeletal muscle actomyosin increased linearly, while that of acto-gizzard myosin (hybrid actomyosin reconstituted from gizzard myosin and skeletal muscle F-actin) increased sigmoidally with increase in the ATP concentration. The amounts of ATP required for the complete dissociation were 1 and 2 mol/mol myosin for skeletal muscle actomyosin and acto-smooth muscle myosin, respectively.
6. The amounts of ATP required for the maximum Mg²⁺-ATPase activity were 1 and 2 mol/mol HMM for skeletal muscle acto-HMM and acto-smooth muscle thiophosphorylated HMM, respectively.
7. The Mg²⁺-ATPase reaction of skeletal muscle acto-HMM in the presence of TM and TN exhibits a marked substrate inhibition upon removal of Ca²⁺, and the extent of the inhibition is proportional to the amount of ATP bound to the nonburst head [Inoue, A. & Tonomura, Y. (1975) J. Biochem. 78, 83-92]. In contrast to the above report, the Mg²⁺-ATPase activity of acto-smooth muscle thiophosphorylated HMM measured in the presence of skeletal muscle TM and TN increased linearly both in the presence and absence of Ca²⁺ with increase in the ATP concentration, reaching the maximum level when 2 mol ATP/mol HMM was added. The activity decreased markedly upon removal of Ca²⁺, but exhibited no substrate inhibition.
The findings 1-4 clearly show that smooth muscle and skeletal muscle myosins have an identical and a nonidentical two-headed structure, respectively. The findings 5-7 also indicate that the burst head and the nonburst head are involved in the energy transduction and in the control by high concentrations of ATP in muscle contraction, respectively.