Functional properties of a sarcoplasmic reticulum Ca(2+)-ATPase with an altered Ca(2+)-binding mechanism.
Treatment of sarcoplasmic reticulum vesicles with diethylpyrocarbonate in the presence of a large excess of reagent, at pH 6.2 and at room temperature, reveals both a fast- and a slow-reacting population of protein residues. The loss of the Ca(2+)-ATPase activity is mainly associated with the fast-reacting population being partially sensitive to hydroxylamine. There is also an effect on the Ca(2+)-binding mechanism. Shorter derivatization times (5 min) produce a loss of the positive cooperativity of Ca2+ binding. When the treatment was prolonged for 30 min there was an additional decrease in the overall Ca2+ affinity. Curve-fitting procedures applied to the non-cooperative binding isotherms provide the equilibrium constants for the two Ca2+ sites, although they cannot discriminate between interacting and independent site mechanisms. Prestationary kinetics assays show 2 Ca2+:1 ATP ratios, at any extent of Ca2+ saturation, indicating that the Ca2+ sites are not independent. The Ca2+ dissociation profile after derivatization shows a decrease in the dissociation constant for the release of the second Ca2+, which is consistent with interacting sites. Isotopic exchange experiments show fast and slow components of equal amplitude even at subsaturating Ca2+ concentrations, which is incompatible with independent binding sites. The experimental data suggest a modification of the equilibrium binding constants making them more similar, but keeping the interacting character. The structural position of the external (cytoplasmic) and the internal (lumenal) Ca2+ sites remains unaltered in the absence of positive cooperativity.
Martinez-Azorin
F
F
Departamento de Bioquimica y Biologia Molecular, Edificio de Veterinaria, Universidad de Murcia, Spain.
Soler
F
F
Gomez-Fernandez
J C
JC
Fernandez-Belda
F
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D007700:Kinetics; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D004047:Diethyl Pyrocarbonate; D002118:Calcium