Characterization of the purified rat heart plasma membrane Ca²⁺/Mg²⁺ ATPase

Abstract

The purified Ca²⁺/Mgu2+ 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 HC0 ³₋ 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 K_D 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 K_D of 0.384 mM and a Bmax of 1.85 µmol/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.