ISOLATION AND RECONSTITUTION OF A VACUOLAR-TYPE PROTON PUMP OF OSTEOCLAST MEMBRANES

A vacuolar-type proton-translocating ATPase was extracted from ruffled membranes of chicken osteoclasts with 1% polyoxyethylene 9-lauryl eth er (C(12)E(9)) and was purified 13-fold by glycerol gradient centrifug ation. The isolated pump appears by sodium dodecyl sulfate-polyacrylam ide gel electrophoresis to have a subunit composition similar to that of the clathrin-coated vesicle proton pump, in that subunits of appare nt molecular masses of 116, 71, 57, 40, 39, 33, and 17 kDa are present in the osteoclast pump preparation. In addition, the 116-, 71-, 57-, and 40-kDa components were shown to crossreact with specific antisera generated against the ho mologous subunits of the clathrin coated vesi cle proton pump. The isolated osteoclast H+-ATPase was reconstituted i nto liposomes prepared from purified lipids (phosphatidylcholine, phos phatidylethanolamine, phosphatidylserine, and cholesterol) by a cholat e-dilution, freeze thaw method. Proton transport catalyzed by the reco nstituted pump was inhibited by bafilomycin A(1) (10 nM) and N-ethyhna leimide (1 mM) but was insensitive to vanadate. We propose that osteoc last-mediated bone resorption is effected by a vacuolar-type proton pu mp with functional and structural similarities to that isolated from c lathrin-coated vesicles.