Interdependence of Ca2+ occlusion sites in the unphosphorylated sarcoplasmic reticulum Ca(2+)-ATPase complex with CrATP.
The beta, gamma-bidentate chromium(III) complex of ATP (CrATP) was used as a substrate analog to stabilize a form of the Ca(2+)-ATPase of the sarcoplasmic reticulum containing both of the bound calcium ions in an occluded state without enzyme phosphorylation. The kinetics of dissociation of Ca2+ from the occlusion sites in the CrATP-enzyme complex were consistent with the existence of two nonequivalent and interdependent Ca2+ occlusion sites, both in the membranous Ca(2+)-ATPase and in a detergent-solubilized monomeric Ca(2+)-ATPase preparation. The rate constant for release of the first calcium ion was k1 = 0.99 h-1, whereas the second calcium ion was released with a rate constant of k2 = 0.25 h-1 when the first site was empty and with a rate constant of k3 = 0.13 h-1 when the first site was occupied by Ca2+. Ca2+ binding at the first site occurred with a rate constant of k-1 = 0.96 microM-1 h-1 (apparent Kd = 1.0 microM). The Ca(2+)-occluded state was further stabilized by ADP, binding in exchange with ATP with an apparent Kd of 8.6 microM. Two kinetic classes of CrATP-binding sites were observed, each with a stoichiometry of 3-4 nmol/mg of protein; but only the fast phase of CrATP binding was associated with Ca2+ occlusion. Derivatization of the Ca(2+)-ATPase with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)carbodimide resulted in inactivation of phosphorylation of the enzyme from MgATP, whereas the ability to occlude Ca2+ in the presence of CrATP was retained, albeit with a reduced apparent affinity for Ca2+.
Vilsen
B
B
Danish Biomembrane Research Center, University of Aarhus.
Andersen
J P
JP
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002132:Calcium Radioisotopes; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002250:Carbon Radioisotopes; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D011817:Rabbits; D011865:Radioisotope Dilution Technique; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014357:Trypsin
D002132:Calcium Radioisotopes; D002250:Carbon Radioisotopes; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D002118:Calcium