Characterization of ATP-dependent proton transport in medullary bone-derived microsomes.

Proton transport in microsomal vesicles derived from medullary bone of laying hens was observed to be inhibited in a dose-dependent manner with fusidic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diatzole (NBD-Cl), duramycin and dicyclohexylcarbodiimide (DCCD). The IC50 values were 570 microM, 4.5 microM, 10 micrograms/ml and 32 microM for fusidic acid, NBD-Cl, duramycin and DCCD, respectively. 14C-DCCD labeled a single protein band of 15-17 kDa from bone-derived microsomes in SDS-electrophoresis. A protein of this size is a proton-conducting subunit of the vacuolar ATPases. Further, the proton transport was found to be electrogenic, thus it generates the membrane potential across the vesicle membrane. The generation of membrane potential was inhibited using 100 nM bafilomycin A1, which in low concentrations is a specific inhibitor of vacuolar ATPases. The presence of Cl- was essential for maximal proton transport activity. These results confirm the electrogenicity and extend the characterization of the osteoclastic H(+)-ATPase.