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 ether (C12E9) and was purified 13-fold by glycerol gradient centrifugation. The isolated pump appears by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a subunit composition similar to that of the clathrin-coated vesicle proton pump, in that subunits of apparent 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 cross-react with specific antisera generated against the homologous subunits of the clathrin-coated vesicle proton pump. The isolated osteoclast H(+)-ATPase was reconstituted into liposomes prepared from purified lipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol) by a cholate-dilution, freeze-thaw method. Proton transport catalyzed by the reconstituted pump was inhibited by bafilomycin A1 (10 nM) and N-ethylmaleimide (1 mM) but was insensitive to vanadate. We propose that osteoclast-mediated bone resorption is effected by a vacuolar-type proton pump with functional and structural similarities to that isolated from clathrin-coated vesicles.
Mattsson
J P
JP
Department of Cell Biology, Astra Hässle AB, Mölndal, Sweden.
Schlesinger
P H
PH
Keeling
D J
DJ
Teitelbaum
S L
SL
Stone
D K
DK
Xie
X S
XS
D000818:Animals; D001862:Bone Resorption / Q000209:etiology; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D005260:Female; D008970:Molecular Weight; D010010:Osteoclasts / Q000201:enzymology* / Q000648:ultrastructure; D006180:Proton-Translocating ATPases / Q000302:isolation & purification*; D014617:Vacuoles / Q000201:enzymology*
D006180:Proton-Translocating ATPases