| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Earlier kinetic studies have revealed that the ATP hydrolysis of myosin is a multi-transition process with many intermediates. From crystallographic comparisons of myosin heads with different nucleotide analogues, Fisher et al. have proposed that in one of these transitions, M・ATP to M・ADP・P,a rotation around Ile466-Ala467-Gly468 (of chicken smooth muscle myosin) occurs and that the rotation allows formation of a salt-bridge between Glu470 and Arg247 in the narrow 50 kDa cleft [Biochemistry 34,8960-8972(1995)]. Here, we made mutants of smooth muscle heavy meromyosin (HMM ; N-terminal truncated myosin fragment) to test whether both rotational movement and salt-bridge formation really occur in the normal ATP hydrolysis or not Smooth muscle 11MM mutants were expressed by using a baculovirus-cultured insect cell system. All mutant HMMs expressed here decorated actinfilaments, forming the characteristic arrowheads and were dissociated from actin by the addition of ATP.Therefore, their mutat
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ional effects on functions are neither global nor gross. A mutant G468A exhibited no ATPase activity, no initial phosphate burst, and no Trp fluorescence enhancement upon adding ATP.A mutant E470A also exhibited no ATPase activity and no phosphate burst, but it showed the Trp fluorescence enhancement corresponding to MATP^*. To test whether this failure of hydrolysis is due to the loss of a proton acceptor or to the loss of an essential salt-bridge suggested by Fisher et al., we made a double-mutant HMM (E470R/R247E) and the constituent single-mutant HMMs (E470R and R247E). The double-mutant HMM, like wild-type HMM, had ATPase activity, a phosphate burst, and a high-level Trp fluorescence enhancement corresponding toM・ADP P^<**>, whereas the two single-mutant HMMs had no ATPase activity, no phosphate burst, and no fluorescence enhancement This result suggests that whether in the normal or in the inverted direction, an intact salt-bridge is necessary for ATP hydrolysis. Thus, we propose that both the rotation at Gly468 and the salt-bridge formation really occur in the ATP hydrolytic process. Less
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