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, Ca²⁺-dependent adenosinetriphosphatase (ATPase) activity was not enriched significantly. This Ca²⁺-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 Ca²⁺-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 Ca²⁺-ATPase activity. A protein doublet of Ca²⁺-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.