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

K. Sugimura, J. Abramowitz, Y. Tsukamoto, Wadi N. Suki

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

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 languageEnglish (US)
Pages (from-to)F192-F200
JournalAmerican Journal of Physiology - Renal Fluid and Electrolyte Physiology
Volume263
Issue number2 32-2
DOIs
StatePublished - 1992

Keywords

  • Basolateral membranes
  • Calcium pump
  • Calcium-dependent adenosinetriphosphatase
  • Distal tubule
  • Proximal tubule

ASJC Scopus subject areas

  • Physiology

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