Biphasic kinetics of sarcoplasmic reticulum Ca2+-ATPase and the detergent-solubilized monomer.
The mechanism of ATP hydrolysis was studied at 0 degrees C and pH 7.5 using purified leaky vesicles of sarcoplasmic reticulum Ca2+-ATPase and enzyme solubilized in monomeric form with high concentrations of octaethylene glycol monododecyl ether (C12E8). The enzyme reaction of membranous Ca2+-ATPase was characterized by an initial burst in the hydrolysis of ATP and modulated by millimolar concentrations of ATP. For detergent-solubilized Ca2+-ATPase no burst and moderate low affinity modulation was observed, but the reaction was activated both at low (phosphorylating) and intermediate (K0.5 = 0.06 mM) ATP concentrations. A study of the partial reactions indicated that the effects of detergent and ATP were attributable to activation of the E1P----E2P transition which was rate-limiting. E32P dephosphorylation of membranous Ca2+-ATPase and the detergent-solubilized monomer comprised both a slow and a rapid component. The inhibitory effect of high Ca2+ was correlated with the development of a dominant contribution of slow phase dephosphorylation and with ATP-induced extra binding of Ca2+ binding which presumably takes place at the phosphorylation site (ECaP). Ca2+ was bound with lesser affinity to detergent-solubilized Ca2+-ATPase but with qualitatively the same characteristics as to membranous ECaP. Either Ca2+ or Mg2+ was required for dephosphorylation, also after detergent solubilization. It is concluded that ATP hydrolysis occurs by the same steps for membranous and monomeric Ca2+-ATPase and involves formation of either EMgP or ECaP as reaction intermediates, leading to biphasic kinetics, which, therefore, cannot be taken as evidence of an oligomeric function of the enzyme.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012995:Solubility; D013501:Surface-Active Agents / Q000494:pharmacology*; D013696:Temperature
D003902:Detergents; D013501:Surface-Active Agents; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium