| Research Abstract |
(i) We succeeded in the large-scale purification of Enterococcus hirae V-ATPase achieving column chromatographies ; about 30 mg of purified enzyme comprising nine subunits, A, B, C, D, E, F, G, I, K, was obtained from 20 liter culture. SDS-PAGE revealed that seven subunits, A, B, C, D, E, F and G, are releasable as the V1 subunit form thew V0V1 complex by incubation with EDTA ; the V0 portion consists of I and K subunits.
(ii) E.hirae V-ATPase catalyzes translocation of Na+ or Li+ coupled with ATP hydrolysis. It is suggested that the glutamic acid residue (Glu139) of NtpK proteolipid subunit of this multisubunit enzyme is the binding site of these ions for translocation. We established a complementation system from the ntpK gene with its deletion mutant, and found that the ATPase activity disappeared upon replacement of Glu139 to aspartic acid. The side-chain length of this acidic residue of NtpK is thus important for this ATPase reaction.
(iii) We investigated the ion binding step to E.hirae Na+-translocating ATPase. The kinetics of Na+ binding to purified V-ATPase suggested 6 Na+ bound/enzyme molecule, with a single high affinity (Kd (Na+) =15 microM.The number of cation binding sites is consisitent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. This suggest that the Na+ binding sites of the Na+-ATPase are readily accessible from the aqueous phase.
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