Biochemical characterization of an electrogenic vacuolar proton pump in purified chicken osteoclast plasma membrane vesicles

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Abstract
A well-characterized chicken osteoclast plasma membrane vesicle preparation manifested Mg2+-dependent ATP hydrolyzing activity of 0.213 μmol inorganic phosphate released per mg protein per minute (n = 7). The Mg2+ dependence showed a high-affinity component with a Kmg of 1.293 μM and Vmax of 0.063 μmol Pi per mg protein per minute, and a low-affinity component with a KMg of 297.6 μM and a Vmax of 0.232 μmol Pi per mg protein per minute. The Mg2+-ATPase activity was inhibited by N, N1-dicyclohexylcarbodiimide (DCCD, 0.2 mM, 50.7%), N-ethylmaleimide (0.5 mM, 34.6%), nolinium bromide (1 mM, 29.9%), 4,4′-di-isothiocyano-2,2′-stilbene sulfonic acid (DIDS, 1 mM, 45.1%), and p-chloromercuribenzoic acid (PCMB, 0.1 mM, 33.8%). Sodium orthovanadate (Na3VO4) at 1 μM had no effect but caused 29.5% inhibition at 1 mM. Na+ could substitute for K+ without loss of activity, NO3 caused 19.5% inhibition when substituted for CI−, and acetate replacement of CI− resulted in 36.4% stimulation of Mg2+-ATPase. ATP, GTP, ITP, CTP, and ADP were all hydrolyzed effectively. DCCD (0.2 mM), NEM (0.5 mM), nolinium bromide (1 mM), and DIDS (50 μM) almost completely abolished proton transport as measured spectrofluorometrically by acridine orange quenching. Na3VO4 (1 mM) had no effect, and duramycin (80 μg/ml) inhibited transport 52.7%. K+ replacement of Na+ caused a 79.2% increase in initial proton transport rate. NO3- and acetate substitution of CI− resulted in a 46.1 and 55.7% decrease in transport, respectively. ATP supports transport far more effectively than the other nucleotides tested. ADP was ineffective. Experiments using the potassium ionophore, valinomycin, indicated that the proton pump functions electrogenically, with CI− most likely cotransported by an anion transporter. The proton pump also seems to have at least one anion-sensitive site, elucidated by experiments in the presence of NO3- and CI−.
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Funding Information
  • NIH (DE-04345)
  • NASA (NAGW-1196)