title: Comparison between calcium transport and adenosine triphosphatase activity in membrane vesicles derived from rabbit kidney proximal tubules.
abstract: Characteristics of Ca2+ uptake were studied in a vesicular preparation of proximal tubule plasma membranes from rabbit kidney and compared with the properties of both membrane-bound and solubilized Ca2+-ATPase activities. Calcium uptake required both ATP and MgCl2 and revealed two kinetic components with respect to Ca2+ concentration requirements, one with a high affinity for Ca2+ (1.8 microM), operative in the range of cytosolic Ca2+ activity, and one with a low affinity for Ca2+ (250 microM) which may become active only at abnormally high cytosolic Ca2+ concentrations. The high- and low-affinity components were stimulated to similar extents by phosphate, and required similar concentrations of ATP (0.6 mM) for half-maximal activity. The amount of membrane-bound phosphoenzyme formed from ATP in the presence of Ca2+ was the same regardless of whether only one or both sites were saturated, suggesting that occupancy of the second Ca2+ binding site accelerates the enzyme turnover. Inhibition of Ca2+ transport by Na+ was reversed by the addition of ouabain or an ATP-regenerating system, indicating that this inhibitory effect of Na+ on Ca2+ uptake may be due to the accumulation of ADP in the medium as a result of Na+ pump activity. Low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and valinomycin (2.5 and 1 microM, respectively) were without effect on Ca2+ uptake in the presence of phosphate, whereas higher concentrations of the ionophores (200 and 100 microM, respectively) reduced uptake by 60% or more. The calmodulin antagonist 48/80 also reduced Ca2+ uptake with half-maximal effectiveness at 100 micrograms/ml. None of these drugs affected either ATPase activity or the EGTA-induced Ca2+ efflux from preloaded vesicles. The Ca2+ dependence of ATP hydrolysis by the membrane-bound enzyme preparation was similar to that observed for Ca2+ uptake by the vesicles. However, with solubilized enzyme, concentrations of Ca2+ similar to that found in the plasma reduced Ca2+-stimulated ATP hydrolysis to one-half of its maximal rate. This indicates that peritubular Ca2+ may play a role in the regulation of Ca2+ transport across the tubular epithelium. ATP could not be replaced by ITP as a substrate for Ca2+ uptake, and the (Ca2+ + Mg2+)ITPase activity of soluble enzyme was 25-fold lower than in the presence of ATP. This is an indication that the active Ca2+ pumping mechanism in proximal tubules is critically dependent on the nucleoside moiety of the substrate.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008566:Membranes / Q000201:enzymology; D017830:Octoxynol; D010042:Ouabain / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D011755:Pyrophosphatases / Q000378:metabolism; D011817:Rabbits; D014634:Valinomycin / Q000494:pharmacology; D003189:p-Methoxy-N-methylphenethylamine / Q000494:pharmacology; D000097630:Inosine Triphosphatase
chemical_list: D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D002118:Calcium; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D008274:Magnesium; D017830:Octoxynol; D010042:Ouabain; D010710:Phosphates; D011092:Polyethylene Glycols; D011755:Pyrophosphatases; D014634:Valinomycin; D003189:p-Methoxy-N-methylphenethylamine; D000097630:Inosine Triphosphatase