Characterization of a Ca 2+ ‐ATPase in osteoclast plasma membrane

The plasma membrane fraction of chicken osteoclasts was purified utilizing 20% continuous Percoll gradients. Biochemical marker enzyme analysis (ouabain‐sensitive Na + , K + ‐ATPase and 5′‐nucleotidase) indicated that plasma membrane enrichment was 11.87‐fold and 7.25‐fold, respectively, and contamination with mitochondria, endoplasmic reticulum, and lysosomes was low as determined by succinic dehydrogenase, NADH dehydrogenase, and N ‐acetylglucosaminidase activities, respectively. SDS latency of Na + K + ‐ATPase and 5′‐nucleotidase activities of the isolated plasma membranes revealed that 43‐50% of vesicles were sealed, with 10‐16% in the inside‐out orientation, depending on the membrane fraction used. Electron microscopy confirmed the vesicular nature of the plasma membrane fraction. The plasma membrane Ca 2+ ‐ATPase had a high‐affinity (K Ca = 0.22 μM; K max = 0.16 μmol/mg per min) and a low‐affinity ( K Ca = 148 μM; V max = 0.37 μtmol/mg per min) component. Calmodulin (0.12 μM) had no effect on Ca 2+ ‐ATPase activity. However, trifluoperazine (0.1 mM), a calmodulin antagonist, strongly inhibited especially the high‐affinity component of the enzyme. Vanadate and lanthanum also caused inhibition. In the presence of CDTA, a potent Ca 2+ and Mg 2+ chelating agent, high‐affinity Ca 2+ ‐ATPase activity was abolished, indicating that trace Mg 2+ was essential for activity. The Ca 2+ ‐ATPase substrate curve using ATP showed a high‐affinity ( K m = 12.3 μM; K max = 0.022 μmol/mg per min) and a low‐affinity ( K m = 43.8 μM; V max = 0.278 μmol/mg per min) component. These results demonstrate that osteoclasts have a plasma membrane Ca 2+ ‐ATPase with characteristics similar to the enzyme responsible for active calcium extrusion in other cells.