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

Abstract

A well-characterized chicken osteoclast plasma membrane vesicle preparation manifested Mg2(+)-dependent ATP hydrolyzing activity of 0.213 mumol inorganic phosphate released per mg protein per minute (n = 7). The Mg2+ dependence showed a high-affinity component with a KMg of 1.293 microM and Vmax of 0.063 mumol Pi per mg protein per minute, and a low-affinity component with a KMg of 297.6 microM and a Vmax of 0.232 mumol Pi per mg protein per minute. The Mg2(+)-ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (DCCD, 0.2 mM, 50.7%), N-ethylmaleimide (0.5 mM, 34.6%), nolinium bromide (1 mM, 29.9%), 4,4'-diisothiocyano-2,2'-stilbene sulfonic acid (DIDS, 1 mM, 45.1%), and p-chloromercuribenzoic acid (PCMB, 0.1 mM, 33.8%). Sodium orthovanadate (Na3 VO4) at 1 microM 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 Cl-, and acetate replacement of Cl- 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 microM) almost completely abolished proton transport as measured spectrofluorometrically by acridine orange quenching. Na3 VO4 (1 mM) had no effect, and duramycin (80 micrograms/ml) inhibited transport 52.7%. K+ replacement of Na+ caused a 79.2% increase in initial proton transport rate. NO3- and acetate substitution of Cl- 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 Cl- 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 Cl-.

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

“…(39.40) Although it is called vacuolar, this term may prove to be inaccurate. In addition to the kidney and bladder, the osteoclast may also be an exception, since it appears to manifest an electrogenic H' pump of the "vacuolar" type on its plasma membrane and, more specifically, on the ruffled border, as demonstrated by Akisaka and Gay,(4) Ghiselli et (41) and also in the present report.…”

Biological Calcification

“…A wide variety of contributions to the literature (Blair and Ghandur-Mnaymneh 1985;Vaes 1988;Blair et al 1989;Teti et al 1989;Bekker and Gay 1990;Marks and Popoff 1990;Sundquist et al 1990;Väänänen et al 1990Väänänen et al , 1996Väänänen et al , 2000Laitala and Väänänen 1993;Teti 1993;Baron 1995;Hall and Chambers 1996;Katsunuma 1997;Salo et al 1997;Schlesinger et al 1997;Rousselle and Heymann 2002) unanimously support the view that osteoclastic bone resorption occurs in an acidic environment and that in the earliest phase an osteoclast proton pump acidifies the compartment encompassed by the bone surface and the osteoclast brush border, so inducing decalcification of the bone matrix, while in the second phase osteoclast-synthesized metalloproteases digest the unmasked collagen fibrils (Vaes 1988;Delaissé et al 1993;Hill et al 1995;Holliday et al 1997). It is, in fact, accepted that bone resorption occurs in two phases, the first consisting of the decalcification of the bone matrix, the second of the extracellular digestion of its organic components.…”

Unique uptake and efflux systems of inorganic phosphate in osteoclast-like cells

“…An acidic pH is essential for solubilization of the alkaline salts of bone mineral hydroxyapatite ([Ca 3 (PO 4 ) 2 ] 3 Ca(OH) 2 ) as well as for digestion of the organic bone matrix by acid lysosomal enzymes secreted by osteoclasts (9,26). The primary cellular mechanism responsible for this acidification is active secretion of protons by vacuolar-type H ϩ -ATPase (V-ATPase), which is localized in the ruffled border of osteoclasts (2). The energy requirement to drive this process is high and is manifested by the presence of abundant mitochondria.…”

“…These cells are required not only for the development of the skeleton but also for mineral homeostasis and normal remodeling of bone in adult animals (33,38). Bone resorption depends on the ability of the osteoclast to generate an acid extracellular compartment between it and the bone surface (2). An acidic pH is essential for solubilization of the alkaline salts of bone mineral hydroxyapatite ([Ca 3 (PO 4 ) 2 ] 3 Ca(OH) 2 ) as well as for digestion of the organic bone matrix by acid lysosomal enzymes secreted by osteoclasts (9,26).…”