Characterization of the purified rat heart plasma membrane Ca2+/Mg2+ ATPase.
The purified Ca2+/Mg2+ ATPase from rat heart plasma membrane was activated by Ca2+ and Mg2+ with Ka values of 1.47 mM and 2.51 mM, respectively; other divalent cations also activated the enzyme but to a lesser extent. Divalent cations like Cu2+, Zn2+, Ni2+, Cd2+ were potent inhibitors of the enzyme activity in the presence of Ca2+ or Mg2+ whereas Na+, K+ or HCO3- did not affect the Ca2+/Mg2+ ATPase activity; the pH optima was 8.5. The enzyme hydrolyzed ATP with a Km of 0.34 mM for Ca2+ ATPase and 0.48 mM for Mg2+ ATPase; various nucleoside triphosphate such as ITP, CTP, GTP, and UTP were also hydrolyzed. Phospholipase A and C as well as neuraminidase decreased the Ca2+/Mg2+ ATPase activity whereas phospholipase D was ineffective. The purified Ca2+/Mg2+ ATPase was found to bind ATP-r-35S with two affinities; the KD values were 50.9 +/- 0.8 and 1160 +/- 198 nM and the Bmax values were 8.71 +/- 0.16 and 145 +/- 9.7 nmol/mg protein for high and low affinity sites, respectively. Treatment of the enzyme preparation with phospholipases and neuraminidase did not affect the ATP-r-35S binding. Ca2+ was also found to bind with Ca2+/Mg2+ ATPase with a KD of 0.384 mM and a Bmax of 1.85 mumol/mg protein; Ni2+, Mn2+, Zn2+ at 1 mM concentrations inhibited the Ca2+ binding but Mg2+ and verapamil were without effect. Phospholipase A and neuraminidase decreased the Ca2+ binding by 20-30%; this indicated that Ca2+ binding with the purified enzyme may be partly due to the phospholipids and sialic acid residues associated with the enzyme. These results show that the purified Ca2+/Mg2+ ATPase is a Ca2+ binding glycoprotein having two binding sites for ATP. Furthermore, this study suggests that phospholipids associated with purified Ca2+/Mg2+ ATPase are required for maximal activity.
Zhao
D
D
Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Canada.
Elimban
V
V
Dhalla
N S
NS
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D006868:Hydrolysis; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D009439:Neuraminidase / Q000494:pharmacology; D010740:Phospholipases / Q000494:pharmacology; D010743:Phospholipids / Q000502:physiology; D011134:Polysaccharides / Q000502:physiology; D051381:Rats
D002413:Cations, Divalent; D010743:Phospholipids; D011134:Polysaccharides; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D010740:Phospholipases; D009439:Neuraminidase; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium