title: Characterization of the functional coupling of bovine brain vacuolar-type H(+)-translocating ATPase. Effect of divalent cations, phospholipids, and subunit H (SFD).
abstract: 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.
authors.affiliation: Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8591, USA.
authors.forename: Bill P
authors.initials: BP
authors.lastname: Crider
authors.forename: Xiao-Song
authors.initials: XS
authors.lastname: Xie
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology*; D002417:Cattle / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007476:Ionophores / Q000494:pharmacology; D008081:Liposomes / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials; D010743:Phospholipids / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism; D021122:Protein Subunits / Q000494:pharmacology / Q000502:physiology*; D017494:Proton Pumps / Q000502:physiology; D011522:Protons; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000502:physiology; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D007476:Ionophores; D008081:Liposomes; D018942:Macrolides; D010743:Phospholipids; D021122:Protein Subunits; D017494:Proton Pumps; D011522:Protons; D014634:Valinomycin; C040929:bafilomycin A1; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D011188:Potassium; D002118:Calcium