CANCELLATION OF THE COOPERATIVITY OF CA2-RETICULUM CA2+-ATPASE BY THENONIONIC DETERGENT DODECYLMALTOSIDE( BINDING TO SARCOPLASMIC)

The perturbation of the kinetics of the sarcoplasmic reticulum (SR) me mbranous Ca2+-ATPase cycle by the non-ionic detergent dodecylmaltoside (DM) has been shown to exhibit specific features which were not obser ved with the related detergents octa(ethylene glycol) monododecylether and Triton X-100 [de Foresta, B., Henao, E and Champeil, P. (1992) Eu r J. Biochem. 209, 1023-1034]. This previous study has been completed here by a detailed analysis of the perturbation by DM of the interacti on of Ca2+ with membranous ATPase, both in its unphosphorylated and ph osphorylated form. Equilibrium binding measurements, performed at pH 7 .5 and 20 degrees C, showed that only one Ca-45(2+) was bound with hig h affinity to the ATPase in the presence of maximally perturbing conce ntrations of DM, as compared to two Ca-45(2+) in the absence of deterg ent. This binding was also assessed by a small decrease in the tryptop han fluorescence intensity. Binding of a second Ca2+ occurred only wit h a much lower affinity. In the presence of DM, the pCa dependence of the phosphorylation by [gamma-P-32]ATP of the ATPase shifted towards 5 0-fold higher Ca2+ 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 req uires the binding of this second, low-affinity Ca2+. In order to asses s this, samples of ATPase were intramolecularly cross-linked with glut araldehyde. This treatment stabilized the phosphorylated intermediate with occluded Ca2+ [Ross, D. C., Davidson, G. A. and 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 Ca2+/phosphoryla ted molecule. Finally, the pCa dependences of the ATPase hydrolytic ac tivity, measured with two different high-energy substrates, ATP or p-n itrophenylphosphate (PNpP), were also found to shift towards higher Ca 2+ concentrations in the presence of DM, which was again consistent wi th a normal coupling ratio, i.e. two bound Ca2+/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 Ca2+ binding. The loss of cooperativity of C a2+ binding evidenced here makes DM a useful tool in the analysis of t he sequence of events occurring during Ca2+ binding.