Characterization of a Ca2+-ATPase in osteoclast plasma membrane

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Abstract
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 Ca2+-ATPase had a high-affinity (KCa = 0.22 μM; Kmax = 0.16 μmol/mg per min) and a low-affinity (KCa = 148 μM; Vmax = 0.37 μtmol/mg per min) component. Calmodulin (0.12 μM) had no effect on Ca2+-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 Ca2+ and Mg2+ chelating agent, high-affinity Ca2+-ATPase activity was abolished, indicating that trace Mg2+ was essential for activity. The Ca2+-ATPase substrate curve using ATP showed a high-affinity (Km = 12.3 μM; Kmax= 0.022 μmol/mg per min) and a low-affinity (Km = 43.8 μM; Vmax = 0.278 μmol/mg per min) component. These results demonstrate that osteoclasts have a plasma membrane Ca2+-ATPase with characteristics similar to the enzyme responsible for active calcium extrusion in other cells.
Funding Information
  • NIH (DE-04345)
  • NASA (NAGW-1196)