Purification and characterization of a Ca2+/Mg2+ ecto-ATPase from rat heart sarcolemma.
The Ca2+/Mg2+ ATPase of the rat heart sarcolemmal particles was solubilized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in non-denaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240,000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12-0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca2+ (Ka = 0.4 mM) and Mg2+ (Ka = 0.2 mM) as well as by other cations in the order Ca2+ greater than Mg2+ greater than Mn2+ greater than Sr2+ greater than Ba2+ greater than Ni2+ greater than Cu2+. The ATPase activity in the presence of Ca2+ was markedly inhibited by Mg2+, Mn2+, Ni2+ and Cu2+ whereas the monovalent cations such as Na+ and K+ were without effect. The enzyme did not exhibit Ca2+ stimulated Mg2+ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca2+ or Mg2+ ATPase activity was 8.5-9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca2+ metabolism in the myocardium is discussed.
Tuana
B S
BS
Division of Cardiovascular Sciences, St. Boniface General Hospital, Winnipeg, Canada.
Dhalla
N S
NS
D000251:Adenosine Triphosphatases / Q000032:analysis / Q000302:isolation & purification*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002412:Cations; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D012508:Sarcolemma / Q000201:enzymology*; D012685:Sepharose; D012995:Solubility; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D002147:Calmodulin; D002412:Cations; D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; D012685:Sepharose; D014357:Trypsin; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C027201:ectoATPase; D000252:Calcium-Transporting ATPases