Characterization of cardiac sarcoplasmic reticulum ATP-ADP phosphate exchange and phosphorylation of the calcium transport adenosine triphosphatase.
1. The terminal phosphate of (gamma-32P)ATP is rapidly incorporated into cardiac sarcoplasmic reticulum membranes (0.7--1.3 mumol/g protein) in the presence of calcium and magnesium. Cardiac sarcoplasmic reticulum membranes catalize an ATP-ADP phosphate exchange in the presence of calcium and magnesium. 2. Half-maximum activation of the phosphoprotein formation and ATP-ADP phosphate exchange is reached at an ionized calcium concentration of about 0.3 muM. The Hill coefficients are 1.3. 3. Transphosphorylation and ATP-ADP phosphate exchange require magnesium and are maximally activated at magnesium concentrations close to or equal to the ATP concentration. 4. The phosphoprotein level is reduced to about 45% at an ADP/ATP ratio of 0.1. The rate of calcium-dependent ATP splitting declines, whilst the rate of the calcium-dependent ATP-ADP phosphate exchange increases when the ADP/ATP ratio is varied from 0.1 to 1. The sum of both, the rate of ATP splitting and the rate of ADP-ATP phosphate exchange remains constant. 5. Phosphoprotein formation and ATP-ADP phosphate exchange are not affected by azide, dinitrophenol, dicyclohexyl carbodiimide and oubain, whilst both activities are reduced by blockade of -SH groups localized on the outside of the sarcoplasmic reticulum membrane. 6. The isolated phosphoprotein is acid stable. The trichloroacetic acid denatured 32P-labelled membrane complex is dephosphorylated by hydroxylamine, which might indicate that the phosphorylated protein is an acyl-phosphate. 7. Polyacrylamide gel elctrophoresis (performed with phenol/acetic acid/water) of phosphorylated sarcoplasmic reticulum fractions demonstrates that the 32P-incorporation occurs into a protein of about 100000 molecular weight. 8. It is suggested that the phosphoprotein represents a phosphorylated intermediate of the calcium-dependent ATPase which formation occurs as an early step in the reaction sequence of calcium translocation by cardiac sarcoplasmic reticulum similar as in skeletal muscle.
D000244:Adenosine Diphosphate / Q000378:metabolism* / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004140:Dinitrophenols / Q000494:pharmacology; D004285:Dogs; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001386:Azides; D004140:Dinitrophenols; D004024:Dicyclohexylcarbodiimide; D010042:Ouabain; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008274:Magnesium; D005033:Ethylmaleimide; D002118:Calcium