A high-affinity, calmodulin-sensitive (Ca 2+ + Mg 2+)-ATPase and associated calcium-transport pump in the Ehrlich ascites tumor cell plasma membrane

Abstract

A unique cytoplast preparation from Ehrlich ascites tumor cells ( G. V. Henius, P. C. Laris, and J. D. Woodburn (1979) Exp. Cell. Res. 121, 337–345 ), highly enriched in plasma membranes, was employed to characterize the high-affinity plasma membrane calcium-extrusion pump and its associated adenosine triphosphatase (ATPase). An ATP-dependent calcium-transport system which had a high affinity for free calcium ( K 0.5 = 0.040 ± 0.005 μM) was identified. Two different calcium-stimulated ATPase activities were detected. One had a low ( K 0.5 = 136 ± 10 μM) and the other a high ( K 0.5 = 0.103 ± 0.077 μM) affinity for free calcium. The high-affinity enzyme appeared to represent the ubiquitous high-affinity plasma membrane (Ca 2+ + Mg 2+)-ATPase (calcium-stimulated, magnesium-dependent ATPase) seen in normal cells. Both calcium transport and the (Ca 2+ + Mg 2+)-ATPase were significantly stimulated by the calcium-dependent regulatory protein calmodulin, especially when endogenous activator was removed by treatment with the calcium chelator ethylene glycol bis(β-aminoethyl ether) N, N′-tetraacetic acid. Other similarities between calcium transport and the (Ca 2+ + Mg 2+)-ATPase included an insensitivity to ouabain (0.5 m m), lack of activation by potassium (20 m m) and a requirement for magnesium. These similar properties suggested that the (Ca 2+ + Mg 2+)-ATPase represents the enzymatic basis of the high-affinity calcium pump. The calcium pump/enzyme system was inhibited by orthovanadate at comparatively high concentrations (calcium transport: K 0.5 − 100 μM; (Ca 2+ + Mg 2+)-ATPase: K 0.5 > 100 μM). Upon Hill analysis, the tumor cell (Ca 2+ + Mg 2+)-ATPase failed to exhibit cooperative activation by calcium which is characteristic of the analogous enzyme in the plasma membrane of normal cells.