Tissue levels and purification by affinity chromatography of the calmodulin-stimulated Ca2+ -transport ATPase in pig antrum smooth muscle.
The Ca2+ -transport ATPase [Ca2+ + Mg2+)-ATPase) in a plasma membrane-rich fraction of porcine antrum (stomach) smooth muscle, is stimulated 2.9-times by calmodulin in the presence of 0.2 mg/ml saponin and reaches a value of 12.0 +/- 2.0 (4) mumol/100 mg protein (equivalent to 110 g wet tissue) per min at 37 degrees C and 10(-5) M [Ca2+]. Saponin was found to specifically potentiate the calmodulin-(Ca2+ + Mg2+)-ATPase interaction, even in the Triton X-100 solubilized enzyme. The conditions for purification of the (Ca2+ + Mg2+)-ATPase by affinity chromatography on a calmodulin-Sepharose 4B gel were optimized. The purified enzyme has a specific activity of 11.9 mumol/mg protein per min at 37 degrees C, 10(-5) M [Ca2+], 0.6 microM calmodulin, and shows a double polypeptide band at 140 and 150 kDa. The (Ca2+ + Mg2+)-ATPase can be incorporated in artificial liposomes that thereupon show an ATP-dependent Ca2+ uptake (Ca:ATP = 1.0). The magnitude of the calmodulin stimulation of the isolated enzyme depends on its phospholipid environment. When isolated in the presence of phosphatidylserine no calmodulin stimulation is observed. After reconstitution in phosphatidylcholine the calmodulin stimulation amounts to 4.05 +/- 0.63 (n = 12) times.
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology*; D011706:Pyloric Antrum / Q000201:enzymology*; D013552:Swine
D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases