Thiophosphorylated HMM was prepared by incubating chicken gizzard HMM with a partially purified preparation of gizzard myosin light-chain kinase and ATP-γ-S. The ATPase reactions catalyzed by gizzard un-thiophosphorylated HMM (or gizzard un-thiophosphorylated myosin) and by acto-gizzard un-thiophosphorylated HMM (or acto-gizzard un-thiophosphorylated myosin) were compared with those catalyzed by gizzard thiophosphorylated HMM (or gizzard thiophosphorylated myosin) and by acto-gizzard thiophosphorylated HMM (or acto-gizzard thiophosphorylated myosin). The following results were obtained.
1. The second-order rate constants for the ATP-induced fluorescence enhancement and for the Pi burst of gizzard HMM (or gizzard myosin), and those for ATP-induced dissociation of acto-gizzard HMM (or acto-gizzard myosin) were practically unaffected by thiophosphorylation of the light-chain subunit of HMM (or myosin) either in 5mM KCI or in 0.5M KCl.
2. The ATPase activity (Δv0) of acto-gizzard HMM, the apparent first-order rate constant (vrecomb) for recombination of gizzard HMMADPP with F-actin, which occurred after hydrolysis of ATP, and the extent (1-α) of binding of F-actin with gizzard HMM during the ATPase reaction were measured in 5mM KCl. The actinactivated ATPase reaction, either with gizzard un-thiophosphorylated HMM or with gizzard thiophosphorylated HMM, was found to fit the following equation:
where Δv0 was obtained by extrapolating Δvo to an infinite concentration of F-actin. 3. The first-order rate constant (Κrecomb) for recombination of HMMADPP with F-actin at the infinite concentration of F-actin, was increased approximately 3 times by thiophosphorylation of the light-chain subunit. Δv0 was increased approximately 10 times and the apparent dissociation constant of acto-gizzard HMM in the presence of ATP was decreased to approximately one tenth by thiophosphorylation of the light-chain subunit. As a result, Κrecomb became much smaller than Δv0 in the KCl concentration range from 5 to 90mM. In other words, ATP hydrolysis via direct decomposition of acto-HMMADPP was the main pathway in a wide KCl concentration range.
4. The extent (α) of AMPPNP-induced dissociation of acto-gizzard HMM was practically unaffected by thiophosphorylation of the light-chain subunit, whereas, as described above, the extent (α) of dissociation of acto-gizzard HMM during the ATPase reaction was markedly decreased by thiophosphorylation of the light-chain subunit. On addition of F-actin to HMMADPP which was produced by preincubating gizzard thiophosphorylated HMM with ATP, the amount of Pi liberation linearly increased with reaction time, showing no lag phase in the early stage. On the basis of these results, we suggest that in the ATPase reaction catalyzed by acto-HMM, the reaction equilibrium, acto-HMMATP→←acto-HMMADPP, was shifted by thiophosphorylation of the light-chain subunit, and that the refractory state did not occur on the reaction of gizzard HMM with ATP.
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