|Budget Amount *help
¥6,400,000 (Direct Cost : ¥6,400,000)
Fiscal Year 1992 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1991 : ¥4,500,000 (Direct Cost : ¥4,500,000)
Three type ATPase couple ATP hydrolysis(synthesis) and H^+ translocation : F-ATPase, synthesizing ATP and found in mitochondria, chloroplasts and bacterial membranes; V-ATPase, hydrolyzing ATP and forming electrochemical proton gradient in endomembranes such as lysosomes and synaptic vesicles; P-ATPase, forming phosphoenzyme intermediate such as gastric H^+/K^+ ATPase and yeast H^+ ATPase. We are studying these three type ATPase from the comparative aspects, and focussed mainly catalytic site and its coupling with H^+ translocation. Especially F-ATPase and V-ATPase were studied in the research. Major results are summarized as following.
(1) We showed Lys-155 and Thr-156 of F-ATPasse are shown to be catalytic residues participating substrate-binding steps : mutant enzymes (Lys-155* Ala, Thr, Ser, or Arg, Thr-156 Ala, Cys, Asp or Ser) had no ATPase and ATP synthase activities.
(2) Analysis of mutants and their surpressers indicated that Gly-172, Ser-174, Glu-192 and Gly-149 of the beta subunit are near or in the catalytic site of F-ATPase. These residues together with Lys-155 and Thr-156 are conserved in V-ATPase, suggesting they are also in the catalytic site of V-ATPase.
(3) The gamma subunit of F-ATPase was suggested to be regulating coupling between catalysis and H^+ transport by genetic studies. Met-23* Lys mutant of the gamma subunit had wild-type ATPase activity but no H^+ transport or ATP synthesis. Introducing the second mutation (eg. Gln-269* Arg) restored H^+ transport or ATP synthesis. This line of studies indicated that interaction of amino and carboxyl terminal regions are important for the coupling.
(4) Catalytic subunit and proton pathway of V-ATPase were cloned and formed similar to the corresponding subunits of F-ATPase.
(5) We found that synaptic vesicles contain large amount of V-ATPase : about 20% of the visicle membrane protein. This enzyme was shown to drive neurotransmitter transport Grant-in-Aid for Scientific Research(B)