Novel ATP-dependent calcium transport component from rat liver plasma membranes. The transporter and the previously reported (Ca2+-Mg2+)-ATPase are different proteins.
An ATP-dependent calcium transport component from rat liver plasma membranes was solubilized by cholate and reconstituted into egg lecithin vesicles by a cholate dialysis procedure. The uptake of Ca2+ into the reconstituted vesicles was ATP-dependent and the trapped Ca2+ could be released by A23187. Nucleotides, including ADP, UTP, GTP, CTP, GDP, AMP, and adenyl-5'-yl beta, gamma-imidophosphate, and p-nitrophenylphosphate did not substitute for ATP. The concentration of ATP required for half-maximal stimulation of Ca2+ uptake into the reconstituted vesicles was 6.2 microM. Magnesium was required for calcium uptake. Inhibitors of mitochondrial calcium-sequestering activities, i.e. oligomycin, sodium azide, ruthenium red, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and valinomycin did not affect the uptake of Ca2+ into the vesicles. In addition, strophanthidin and p-chloromercuribenzoate did not affect the transport. Calcium transport, however, was inhibited by vanadate in a concentration-dependent fashion with a K0.5 of 10 microM. A calcium-stimulated, vanadate-inhibitable phosphoprotein was demonstrated in the reconstituted vesicles with an apparent molecular weight of 118,000 +/- 1,300. These properties of Ca2+ transport by vesicles reconstituted from liver plasma membranes suggest that this ATP-dependent Ca2+ transport component is different from the high affinity (Ca2+-Mg2+)-ATPase found in the same membrane preparation (Lotersztajn, S., Hanoune, J. and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215; Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020). When the entire reconstituted vesicle population was treated with ATP and 45Ca in a buffer containing oxalate, the vesicles with Ca2+ transport activity could be separated from other vesicles by centrifugation in a density gradient and the ATP-dependent Ca2+ transport component was purified approximately 9-fold. This indicates that transport-specific fractionation may be used to isolate the ATP-dependent Ca2+ transport component from liver plasma membrane.
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001386:Azides / Q000494:pharmacology; D001693:Biological Transport, Active; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000032:analysis*; D008274:Magnesium / Q000378:metabolism; D008297:Male; D008985:Monensin / Q000494:pharmacology; D010743:Phospholipids / Q000032:analysis; D051381:Rats; D012430:Ruthenium Red / Q000494:pharmacology; D019810:Sodium Azide; D013327:Strophanthidin / Q000494:pharmacology; D014634:Valinomycin / Q000494:pharmacology; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D001386:Azides; D002352:Carrier Proteins; D010743:Phospholipids; D014639:Vanadium; D012430:Ruthenium Red; D014634:Valinomycin; D014638:Vanadates; D013327:Strophanthidin; D000255:Adenosine Triphosphate; D008985:Monensin; D019810:Sodium Azide; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium