Characterization of proton transport in bone-derived membrane vesicles.
ATP-dependent proton transport in membrane vesicles prepared from the medullary bone of egg-laying hens, a source rich in osteoclasts, was characterized. Proton transport was abolished by bafilomycin A1 (10 nM) and N-ethylmalemide (50 microM), but not by oligomycin (15 micrograms/ml), vanadate (100 microM) or SCH 28080 (100 microM), thereby differentiating this H(+)-ATPase from the F1F0- and phosphorylated-type of ATPases. Preincubation of the membrane vesicles at 0 degrees C for 1 h in the presence of KCl (0.3 M) and Mg-ATP (5 mM) resulted in almost complete loss of H(+)-transport activity (cold-inactivation). Preventing the formation of a membrane potential by voltage clamp (Kin+ = Kout+ + valinomycin) increased both the rate of H(+)-transport and the equilibrium delta pH, suggesting an electronic proton transport mechanism. Thus, the H(+)-ATPase in this bone-derived membrane vesicle preparation shows the characteristics of a vacuolar H(+)-ATPase in its inhibitor- and cold-sensitivity and its electrogenic mechanism. The anion sensitivity of the H(+)-ATPase was investigated by varying the intra- and/or extra-vesicular salt composition. The H(+)-ATPase had no absolute requirement for any specific anion, but membrane permeable anions were found to stimulate proton transport activity, presumably by acting as charge compensators for the electrogenic hydrogen ion transport. However, some anions, such as sulfate, acetate and nitrate were directly inhibitory to the ATPase. The results are in agreement with the recently proposed mechanism of osteoclast acidification: a vacuolar H(+)-ATPase working in parallel with a Cl(-)-channel resulting in electroneutral HCl secretion.
Mattsson
J P
JP
Department of Cell Biology, Astra Hässle AB, Mölndal Sweden.
Lorentzon
P
P
Wallmark
B
B
Keeling
D J
DJ
D000818:Animals; D000838:Anions; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism*; D002413:Cations, Divalent; D002645:Chickens; D003080:Cold Temperature; D005033:Ethylmaleimide / Q000494:pharmacology; D018942:Macrolides; D008566:Membranes / Q000378:metabolism; D009840:Oligomycins / Q000494:pharmacology; D010010:Osteoclasts / Q000378:metabolism; D011506:Proteins / Q000032:analysis; D017494:Proton Pumps; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D000838:Anions; D000900:Anti-Bacterial Agents; D002413:Cations, Divalent; D018942:Macrolides; D009840:Oligomycins; D011506:Proteins; D017494:Proton Pumps; D014638:Vanadates; C040929:bafilomycin A1; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide