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.
Sugimura
K
K
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.
Abramowitz
J
J
Tsukamoto
Y
Y
Suki
W N
WN
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000493:pharmacokinetics / Q000502:physiology; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology; D005591:Chemical Fractionation; D007672:Kidney Cortex / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D014638:Vanadates / Q000494:pharmacology
D002147:Calmodulin; D014638:Vanadates; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium