Purification and composition of Ca2+/Mg2+ ATPase from rat heart plasma membrane

The Ca²⁺/Mg²⁺ ATPase, which is activated by millimolar concentrations of Ca²⁺ or Mg²⁺, was solubilized from rat heart plasma membrane by employing lysophosphatidylcholine, CHAPS, Nal, EDTA and Tris-HCI at pH 7.4. The enzyme was purified by sucrose density gradient, Affi-Gel Blue column and Sepharose 6B column chromatography. The purified enzyme was seen as a single peptide band in the sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of about 90,000. The apparent molecular weight of the holoenzyme as determined under non-dissociating conditions by gel filtration on Sepharose 6B column was about 180,000 indicating two subunits. The enzyme was insensitive to ouabain, verapamil, vanadate, oligomycin, N,N-dicyclohexylcarbodiimide and NaN₃, but was markedly inhibited by 20 µM gramacidin S and 50 µM trifluoperazine. Analysis of the purified Ca²⁺/Mg²⁺ ATPase revealed the presence of 17 amino acids where leucine, glutamic acid and aspartic acid were the major components and histidine, cysteine and methionine were the minor components. The purified enzyme was associated with 19.7 µmol phospholipid/mg protein which was 60 times higher than the phospholipid content in plasma membrane. The cholesterol content in the purified enzyme preparation was 0.75 µmol/mg protein and this represented an 8-fold enrichment over plasma membrane. The glycoprotein nature of the enzyme was evident from the positive periodic acid-Schiff staining of the purified Cau2+/Mg^ATPase in the sodium dodecyl sulfate polyacrylamide gel. The polysaccharide content of the enzyme was enriched 8-fold over plasma membrane; neurominidase treatment decreased the polysaccharide content. Concanavalin A prevented the ATP-dependent inactivation of the purified Ca²⁺/Mg²⁺ ATPase and was found to bind to the purified enzyme with a K_D of 576 nM and Bmax of 4.52 nmol/mg protein. The results indicate that Ca²⁺/Mg²⁺ ATPase is a glycoprotein and contains a large amount of lipids.