Reconstitution and partial purification of calcium transport activity from rat kidney cortex.

An ATP-dependent Ca2+ uptake system from rat renal cortical basolateral membranes was solubilized with Triton X-100 and reconstituted into liposomes with lecithin. In the presence of Mg2+, Ca2+ uptake in the reconstituted vesicles was time and ATP dependent and was inhibited by vanadate. Ca2+ uptake in basolateral membrane vesicles depleted of endogenous calmodulin was enhanced by exogenous calmodulin and depressed by R-24571. This sensitivity to calmodulin and R-24571 was lost upon reconstitution in the presence and absence of leupeptin. Vesicles containing Ca2+ uptake activity were separated by gradient centrifugation after Ca2+ was taken up and accumulated as calcium phosphate in the vesicles. This resulted in Ca2+ uptake activity that was enriched 25 times. However, Ca(2+)-dependent adenosinetriphosphatase (ATPase) activity was not enriched significantly. This Ca(2+)-ATPase had two kinetic forms for Ca2+: one was a high-affinity low-capacity form; the other had a low affinity and high capacity. The Ca(2+)-ATPase activity also had two kinetic forms for ATP. All kinetic forms were inhibited by Mg2+. Vanadate, calmodulin, and R-24571 had no effects on Ca(2+)-ATPase activity. A protein doublet of Ca(2+)-dependent hydroxylamine-sensitive phosphorylated intermediates was demonstrated at 125 and 136 kDa in the purified vesicles. This doublet was not altered by addition of leupeptin throughout the purification.