Vacuolar H+-ATPase activity and expression in mouse bone marrow cultures

We examined vacuolar H+-ATPase (V-ATPase) structure, enzymatic properties, and protein and mRNA expression from mouse marrow cultured in the presence or absence of 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3), which stimulates formation of bone-resorptive osteoclasts, V-ATPases from osteoclast-contain ing cultures were similar in ion and inhibitor sensitivities to the enzyme from kidney-derived sources. Immunopurified V-ATPase from 1,25(OH)(2)D-3-st imulated cultures exhibited 20-fold greater ATPase activity than the enzyme from unstimulated cultures, which do not contain osteoclasts, In contrast, 1,25(OH)(2)D-3-treated cultures contained only 2-fold more assembled V-ATP ase, as determined by immunoprecipitation, Quantitative reverse transcripti on-polymerase chain reaction (RT-PCR) and immunoblot analysis similarly sho wed similar to 2-fold increases of V-ATPase mRNA and protein levels in 1,25 (OH)(2)D-3-treated cultures, The bulk of the relative difference in V-ATPas e activity between the two cultures was due to a 10-fold difference in enzy me specific activity. Quantitative RT-PCR also revealed that expression lev els of V-ATPase mRNAs reflected the stoichiometry of enzyme subunits in the assembled complex. These data indicate that in mouse bone marrow cultures, V-ATPase expression is controlled at the level of mRNA, and that increases in subunit expression and assembly cannot account for the 20-fold differen ce in enzyme activity in osteoclast-containing cultures. Therefore, osteocl ast V-ATPase activity may be regulated by subtle alterations in enzyme stru cture or associated factors.