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ATP analogue binding to the A subunit induces conformational changes in the E subunit that involves a disulfide bond formation in plant VâATPase
- Kawamura, Yukio, Arakawa, Keita, Maeshima, Masayoshi, Yoshida, Shizuo
- European journal of biochemistry 2001 v.268 no.10 pp. 2801-2809
- H-transporting ATP synthase, adenosine, adenosine triphosphate, cysteine, disulfide bonds, lighting, oxidation, polyacrylamide gel electrophoresis, polypeptides, reducing agents, ultraviolet radiation
- Vacuolar H+âATPase (VâATPase) consists of a catalytic head, a stalk part and a membrane domain. We indirectly investigated the interaction between the Aâsubunit (catalytic head) and the Eâsubunit (stalk part) using an ATP analogue, adenosine 5â²â[Î²,Î³âimino]triphosphate (AMPâPNP), which holds the enzyme in the substrateâbinding state. AMPâPNP treatment caused a mobility shift of the Eâsubunit with a faster migration in SDS/polyacrylamide gel electrophoresis without a reductant, while ATP treatment did not. A mobility shift of the Eâsubunit has been detected in several plants. As polypeptides with intramolecular disulfide bonds migrate faster than those without disulfide bonds, the mobility shift may be due to the formation of an intramolecular disulfide bond by two cysteine residues conserved among several plant species. The mobility shift may be involved in the binding of AMPâPNP to the ATPâbinding site, which exists in the A and B subunits, as it was inhibited by the addition of ATP. Pretreatment with 2â²â3â²âOâ(4âbenzoylbenzoyl)âATP (BzâATP), which modifies the ATPâbinding site of the Bâsubunit under UV illumination, did not inhibit the mobility shift of the Eâsubunit caused by AMPâPNP treatment. The response of VâATPase following the AMPâPNP binding may cause a conformational change in the Eâsubunit into a form that is susceptible to oxidation of cysteine residues. This is the first demonstration of interaction between the A and E subunits in the substrateâbinding state of a plant VâATPase.