Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes.
The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification* / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D014268:Trifluoperazine; D004533:Egtazic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium