Abstract. Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts were shown to have ATP-dependent H+-transport activity. This activity was N-ethylmaleimide-sensitive but resistant to oligomycin and orthovanadate, suggesting a vacuolar-type ATPase. Furthermore, immunological cross-reactivity of 60-and 70-kD osteoclast membrane antigens with Neurospora crassa vacuolar ATPase was observed when analyzed by immunoblotting. Same antibodies labeled only osteoclasts in chicken and rat bone in immunohistochemistry. Immunoelectronmicroscopy localized these antigens in apical membranes of rat osteoclasts and kidney intercalated cells of inner stripe of outer medulla. Pretreatmerit of animals with parathyroid hormone enhanced the immunoreaction in the apical membranes of osteoclasts. No immunoreaction was seen in osteoclasts when antibodies against gastric H+,K+-ATPase were used. These results suggest that osteoclast resorbs bone by secreting protons through vacuolar H+-ATPase.STEOCLASTS are multinucleated giant cells that are responsible for bone resorption. Like secreting epithelial cells, they are polarized when active in bone resorption, showing three distinct specialized cell membrane areas (for review see Vaes et al., 1988). In addition to the basolateral membrane, which is rich in Na+,K+-ATPase (Baron et al., 1986), resorbing osteoclast exhibits a clear zone that mediates the attachment of resorbing cells to the bone matrix. The cytoplasm in the vicinity of this clear zone contains specialized cytoskeletal structures (Holtrop et al., 1974;Lakkakorpi et al., 1989). The third specialized membrane area, the ruffled border, faces the actual bone resorption site on the bone surface. The resorption lacuna underneath the ruffled border membrane is acidic. This has been shown by acridine orange accumulation experiments (Anderson et al., 1986; Bar6n et al., 1985) and direct micropuncture measurements (Fallon, 1984). The acidic pH favors dissolution of the bone mineral. In addition, proteinases active at acid pH and capable of collagen degradation are present in osteoclasts (Vaes et al., 1988; Blair et al., 1986). Enzyme histochemistry suggests the presenc$ of ATPase activity in the plasma membrane of the osteoclast- (Akisaka et al., 1986). Baron et al. (1985) found at the ruffled:border of the osteoclast a 100-kD lysosomal membrane polypeptide that showed immunological similarity to gastric H+,K+-ATPase. Now we report that osteoclasts contain an ATP-dependent proton pump that is clearly different from the gastric proton pump and from the mitochondrial proton pump but shows considerable immunological similarities to the vacuolar type H+-ATPase of Neurospora crassa.
Materials and Methods
Preparation of Bone MicrosomesBone microsomes were prepared from medullary bone of regularly laying hens. Medullary bone from the tibia and femur was dissected out and immediately homogenized in a medium containing 5 mM Tris pH 7.4, 250 mM sucrose, 1 mM K2CO3, 1 mM DTT, and 1 mM EGTA in a glass-Teflun ...
