Effect of metal bound to the substrate site on calcium release from the phosphoenzyme intermediate of sarcoplasmic reticulum ATPase.
The ADP-sensitive phosphoenzyme intermediate (E1P) of sarcoplasmic reticulum ATPase was formed using CaATP as a substrate and release of its bound calcium was investigated. Our previous study (Shigekawa, M., Wakabayashi, S., and Nakamura, H. (1983) J. Biol. Chem. 258, 14157-14161) indicated that 1 mol of E1P has 3 mol of high affinity binding sites for Ca2+, of which two are transport sites for Ca2+, whereas the remainder is presumably the nucleotide binding site. The calcium bound to the substrate site was readily replaced by other divalent cations or lanthanide ions. These ions induced a pronounced change in the rate of Ca2+ release from the transport sites on the same phosphoenzyme. In E1P, whose substrate site had been deprived of a metal, Ca2+ release from the transport sites was fast. It was similar to the Ca2+ release from the normal E1P, whose substrate site was occupied by either Ca2+ or Mg2+, in that it was accelerated by increasing pH or high ATP and inhibited markedly by the treatment of the enzyme with phospholipase C. Release of Ca2+ from E1P, whose substrate site had been rendered metal-free, increased the fluorescence intensity of the enzyme-bound 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, which decayed rapidly upon addition of Mg2+ plus K+. This result suggests that the active site of the resultant divalent cation-free phosphoenzyme has a conformation similar to that for the normal ADP-insensitive phosphoenzyme, E2P. These data suggest that the basic mechanism for Ca2+ release from the phosphoenzyme may be similar whether or not the phosphoenzyme has bound metal at its substrate site, although this metal affects the rate of Ca2+ release extensively.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007811:Lanthanum / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008670:Metals / Q000494:pharmacology*; D010710:Phosphates / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D010738:Type C Phospholipases / Q000378:metabolism
D002413:Cations, Divalent; D008670:Metals; D010710:Phosphates; D015636:Magnesium Chloride; D004533:Egtazic Acid; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D007811:Lanthanum; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; D000251:Adenosine Triphosphatases; D008274:Magnesium; D011188:Potassium; D002118:Calcium