title: [3H]Bafilomycin as a probe for the transmembrane proton channel of the osteoclast vacuolar H(+)-ATPase.
abstract: Bone resorption by the osteoclast is dependent on acidification of the bone surface by a vacuolar type H+-ATPase (V-ATPase) present in the ruffled membrane of the actively resorbing cell. V-ATPases are a highly conserved family of proton pumps consisting of two functional complexes: a cytoplasmic catalytic sector (VC) and a membrane bound proton channel (VB). Bafilomycin A1, a macrolide antibiotic, is a highly potent inhibitor of V-ATPases, and inhibits bone resorption in vitro in isolated rat calvariae. In order to investigate the binding of bafilomycin to the osteoclast V-ATPase, we used a tritiated bafilomycin which had been prepared by acetylating the 21-hydroxyl group of bafilomycin A1. Osteoclast ruffled membrane vesicles were prepared from purified chicken osteoclasts by differential centrifugation and proton transport in these vesicles was shown to be inhibited by [3H]bafilomycin (IC50 approximately 2 nM). Control membrane vesicles or membrane vesicles partially inhibited with [3H]bafilomycin were solubilized and separated by centrifugation on 15-30% glycerol gradients. V-ATPase activity and reconstitutable proton transport activity could be recovered in high density fractions of the gradient. However, the peak of [3H]bafilomycin radioactivity (>70% of total radioactivity in the gradient) was present in a single peak at lower density. Antibodies against subunits of VC (70, 56 and 40 kDa) reacted only in fractions containing the peak V-ATPase activity whereas an antibody to the 39 kDa subunit of VB reacted both with fractions containing the peak V-ATPase activity but also, and more strongly, in fractions containing the peak [3H]bafilomycin. The fractions in the control gradient corresponding to the peak of [3H]bafilomycin were reconstituted into liposomes and shown to mediate passive bafilomycin A1-inhibitable proton conductance. SDS-PAGE followed by autoradiography, indicated that the bafilomycin was not covalently bound to the V-ATPase or the proton channel. Quantification of VB by [3H]bafilomycin binding or by antibody staining suggested an excess of the free proton channel to that of the intact holoenzyme. A corresponding amount of free catalytic sector could not be found in any fraction throughout the isolation procedure of the V-ATPase from the initial homogenate. Thus, in conclusion, bafilomycin inhibits the V-ATPase by binding tightly but non-covalently to the proton channel region of the V-ATPase which appears to be present in excess over the intact holoenzyme in the osteoclast. The possible role of an excess of the proton channel subcomplex in the osteoclast is discussed.
authors.affiliation: Department of Cell Biology, Astra Hässle AB, Mölndal, Sweden. jan.mattsson@hassle.se.astra.com
authors.forename: J P
authors.initials: JP
authors.lastname: Mattsson
authors.forename: D J
authors.initials: DJ
authors.lastname: Keeling
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D015153:Blotting, Western; D001862:Bone Resorption; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002645:Chickens; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005260:Female; D005990:Glycerol; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D018942:Macrolides; D008564:Membrane Potentials; D010010:Osteoclasts / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D014316:Tritium; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D008081:Liposomes; D018942:Macrolides; D014316:Tritium; C040929:bafilomycin A1; D006180:Proton-Translocating ATPases; D005990:Glycerol