ATP-dependent calcium transport and its correlation with Ca2+ -ATPase activity in basolateral plasma membranes of rat...

Isolated basolateral plasma membrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca2+ -dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate beta-gamma-(CH2)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent Km of 0.2 microM Ca2+ and a Vmax of 5.3 nmol Ca2+/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducible after a wash with a 5 mM EGTA. The kinetic parameters of Ca2+ -ATPase are: Km = 0.25 microM Ca2+ and Vmax = 19.2 nmol Pi/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethicin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the Vmax of ATP-dependent calcium uptake amounts to 25 nmol Ca2+/min per mg protein and of the Ca2+ -ATPase to 23 nmol Pi/min per mg protein. The close correlation between kinetic parameters of Ca2+ -ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca2+ -pump located in duodenal basolateral plasma membranes.