| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1993: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1992: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
It was shown that polymerization of G-actin in the presence of salts is blocked by treatment of G-actin with m-maleimidebenzoic acid N-hydroxysuccinimide ester(MBS)]. MBS-actin retains the ability to bind(and make chemical crosslinks)to myosin head [Bettache, N., Bertrand, R., & Kassab, R.(1989)Proc.Natl.Acad.Sci.U.S.A.86, 6028-6032 ; Arata, T.(1991)J.Biochem.109, 335-340]. Here, the interaction between MBS-actin and myosin subfragment 1(S1)has been further studied by proteolytic susceptibility and chemical crosslinking. Two moles of MBS-actin monomers bound to 1 mole of myosin heads or S1. The first binding of MBS-actin to S1 strongly protected a 27kDa/50kDa junction of S1 heavy chain from trypsin digestion and also weakly protected a 50kDa/20kDa junction. The second binding protected a 50kDa/20kDa junction more strongly. From this proteolytic analysis, the dissociation constants for the first and second binding sites were determined to be 1-2 and 4-5 x 10^<-6>M, respectively. ATP weakened these bindings more than 10-fold. MBS-actin was crosslinked by MBS to S1 at the first binding site and the resulting complex was migrating at 180kDa on electrophoretic gels. In the presence of ATP or ADP, an 140 kDa complex was produced together with the 180 kDa complex.
The electron-microscopic study is now in progress to examine the monomeric MBS-G-actin-HMM complexes which have been produced by crosslinking in the absence and presence of ATP.Preliminary experiment showed that the location of actin binding site of the acto-HMM molecules produced in the absence of ATP is 14-15 nm away from the head-rod junction. In contrast, the acto-HMM molecules produced in teh presence of ATP showed that the location is about 13 nm from the junction, slightly more proximal to the neck region.
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