Cancellation of the cooperativity of Ca 2+ binding to sarcoplasmic reticulum Ca 2+ ‐ATPase by the non‐ionic detergent dodecylmaltoside

The perturbation of the kinetics of the sarcoplasmic reticulum (SR) membranous Ca 2+ ‐ATPase cycle by the non‐ionic detergent dodecylmaltoside (DM) has been shown to exhibit specific features which were not observed with the related detergents octa(ethylene glycol) monododecylether and Triton X‐100 [de Foresta, B., Henao, F. & Champeil, P. (1992) Eur. J. Biochem. 209 , 1023–1034]. This previous study has been completed here by a detailed analysis of the perturbation by DM of the interaction of Ca 2+ with membranous ATPase, both in its unphosphorylated and phosphorylated form. Equilibrium binding measurements, performed at pH 7.5 and 20°C, showed that only one 45 Ca 2+ was bound with high affinity to the ATPase in the presence of maximally perturbing concentrations of DM, as compared to two 45 Ca 2+ in the absence of detergent. This binding was also assessed by a small decrease in the tryptophan fluorescence intensity. Binding of a second Ca 2+ occurred only with a much lower affinity. In the presence of DM, the pCa dependence of the phosphorylation by [γ‐ 32 P]ATP of the ATPase shifted towards 50‐fold higher Ca 2+ concentrations than in its absence. Furthermore, DM completely inhibited the cooperativity of this dependence. This shift strongly suggests that the phosphorylation of DM‐perturbed ATPase requires the binding of this second, low‐affinity Ca 2+ . In order to assess this, samples of ATPase were intramolecularly cross‐linked with glutaraldehyde. This treatment stabilized the phosphorylated intermediate with occluded Ca 2+ [Ross, D. C., Davidson, G. A. & McIntosh, D. B. (1991) J. Biol. Chem. 266 , 4613–4621]. Both in the absence and presence of DM, the cross‐linked enzyme occluded close to two Ca 2+ /phosphorylated molecule. Finally, the pCa dependences of the ATPase hydrolytic activity, measured with two different high‐energy substrates, ATP or p ‐nitrophenylphosphate ( P Np P ), were also found to shift towards higher Ca 2+ concentrations in the presence of DM, which was again consistent with a normal coupling ratio, i.e. two bound Ca 2+ /substrate hydrolyzed. As compared to other detergents, the maltoside head group of DM might favor a stronger interaction with membranous ATPase, resulting in its high perturbing effect on Ca 2+ binding. The loss of cooperativity of Ca 2+ binding evidenced here makes DM a useful tool in the analysis of the sequence of events occurring during Ca 2+ binding.