Abstract
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.
Original language | English (US) |
---|---|
Pages (from-to) | F192-F200 |
Journal | American Journal of Physiology - Renal Fluid and Electrolyte Physiology |
Volume | 263 |
Issue number | 2 32-2 |
State | Published - Aug 1992 |
Keywords
- Basolateral membranes
- Calcium pump
- Calcium-dependent adenosinetriphosphatase
- Distal tubule
- Proximal tubule
ASJC Scopus subject areas
- Physiology