DEMONSTRATION OF CALMODULIN-SENSITIVE CALCIUM TRANSLOCATION BY ISOLATED OSTEOCLAST PLASMA-MEMBRANE VESICLES

Plasma membrane vesicles were prepared from chicken osteoclasts, and active calcium transport was demonstrated in a spectrofluorimetric assay using the fluorescent calcium concentration indicator, fura-2. Transport activity was inhibited by quercetin (10-mu-M), sodium vanadate (10-mu-M), and the anticalmodulin agents, compound 48/80 (20 and 200-mu-g/ml) and calmidazolium (10 and 20-mu-M). The transport rate (V(max), 1.3 nmol/mg protein/min) was not altered in the presence of the protonophore, nigericin (1-mu-M), indicating that proton transport was not driving calcium transport. Release of accumulated calcium in the vesicles occurred with the addition of bromo-A23187 (5-mu-M) or ionomycin (5-mu-M). Increasing calcium transport occurred with increasing calcium concentration. Finally, the calmodulin content of the vesicles was demonstrated to be 54-134 U/mg protein. These results demonstrate that a calmodulin-sensitive, ATP-dependent calcium transporter is present in the osteoclast plasma membrane.