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J. Biol. Chem., Vol. 278, Issue 45, 44281-44288, November 7, 2003

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Characterization of the Functional Coupling of Bovine Brain Vacuolar-type H⁺-translocating ATPase

EFFECT OF DIVALENT CATIONS, PHOSPHOLIPIDS, AND SUBUNIT H (SFD)^*

Bill P. Crider and Xiao-Song Xie

From the Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8591

Vacuolar-type H⁺-translocating ATPases (V-ATPases or V-pumps) are complex proteins containing multiple subunits and are organized into two functional domains: a peripheral catalytic sector V₁ and a membranous proton channel V₀. The functional coupling of ATP hydrolysis activity to proton transport in V-pumps requires a regulatory component known as subunit H (SFD) as has been shown both in vivo and in vitro (Ho, M. N., Hirata, R., Umemoto, N., Ohya, Y., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1993) J. Biol. Chem. 268, 18286–18292; Xie, X. S., Crider, B. P., Ma, Y. M., and Stone, D. K. (1994) J. Biol. Chem. 269, 25809–25815). Ca²⁺ is thought to uncouple V-pumps because it is found to support ATP hydrolysis but not proton transport, while Mg²⁺ supports both activities. The direct effect of phospholipids on the coupling of V-ATPases has not been reported, likely due to the fact that phospholipids are constituents of biological membranes. We now report that Ca²⁺-induced uncoupling of the bovine brain V-ATPase can be reversed by imposition of a favorable membrane potential. Furthermore we report a simple "membrane-free" assay system using the V₀ proton channel-specific inhibitor bafilomycin as a probe to detect the coupling of V-ATPase under certain conditions. With this system, we have characterized the functional effect of subunit H, divalent cations, and phospholipids on bovine brain V-ATPase and have found that each of these three factors plays a critical role in the functional coupling of the V-pump.

Received for publication, July 9, 2003 , and in revised form, August 18, 2003.

^* This work was supported by NIDDK, National Institutes of Health Grant 33627. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 214-648-7700; Fax: 214-648-7720; E-mail: xiao-song.xie{at}UTSouthwestern.edu.

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