Characterization of the (Ca2+-Mg2+)ATPase purified by calmodulin-affinity chromatography from bovine aortic smooth muscle.
A calmodulin inhibitor, trifluoperazine, suppresses ATP-dependent Ca2+ uptake into microsomes prepared from bovine aortic smooth muscle. From this microsomal preparation which we expected to contain calmodulin-dependent Ca2+-transport ATPase [EC 3.6.1.3], we purified (Ca2+-Mg2+)ATPase by calmodulin affinity chromatography. The protein peak eluted by EDTA had calmodulin-dependent (Ca2+-Mg2+)ATPase activity. The major band (135,000 daltons) obtained after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) accounted for about 80% of the total protein eluted. This major band was phosphorylated by [gamma-32P]ATP in a Ca2+-dependent manner. All the 32P incorporated into the major band was released by hydroxylaminolysis. The ATPase reconstituted in soybean phospholipid liposomes showed ATP, calmodulin-dependent Ca2+ uptake. The affinity of the ATPase for Ca2+, Km, was 7 microM and the maximum ATPase activity was 1.4 mumol/mg/min. These values were changed to 0.17 microM and 3.5 mumol/mg/min, respectively by the addition of calmodulin. The activity of the purified (Ca2+-Mg2+)ATPase was inhibited by orthovanadate, and the concentration required for half-maximal inhibition was about 1.8 microM which is close to that of plasma membrane ATPases. Judging from the effect of orthovanadate and the molecular weight, the purified (Ca2+-Mg2+)ATPase was considered to have originated from the plasma membrane not from the sarcoplasmic reticulum.
D000818:Animals; D001011:Aorta / Q000201:enzymology; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002417:Cattle; D002846:Chromatography, Affinity; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D014268:Trifluoperazine / Q000494:pharmacology; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D002147:Calmodulin; D008081:Liposomes; D014639:Vanadium; D014268:Trifluoperazine; D014638:Vanadates; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium