Perturbation of the Structure and Function of a Membranous Ca2+ ‐ATPase by Non‐solubilizing Concentrations of a Non‐ionic Detergent

Abstract

The effect of octa(ethyleneglycol)-monododecylether (C12E8) on Ca2+-ATPase membranes, prepared form sarcoplasmic reticulum (SR), is characterized. At low concentrations C12E8 is incorporated into the membrane (.ltoreq. 0.2 g/g protein), without any solubilization or appreciable morphological changes of freeze-fracture replica. Binding studies of C12E8 to ATPase membranes and SR lipid liposomes suggest that the major part of the detergent interacts with lipid. Solubilization of ATPase membranes occurs at a free concentration of C12E8 close to the critical micellar concentration (c.m.c); at low temperatures (2.degree. C) phospholipid is extracted somewhat more easily than ATPase. ESR spectra of appropriate spin labels, incorporated into ATPase membranes, show that C12E8 strongly increases the fluidity of the lipid phase and the rotational diffusion of ATPase in the membrane. The effect of C12E8 on the ESR spectra is indistinguishable from that produced by a rise in temperature. Incorporation of C12E8 alters the functional properties of Ca2+-ATPase in a characteristic way: Vmax is decreased and the modulatory effect of high ATP concentrations is reduced, in contrast to what occurs by a rise of temperature. The intrinsic fluorescence of protein is increased, especially in the absence of Ca2+, suggesting that C12E8 modified in particular the E* form (Ca2+-depleted conformation) of the enzyme. Stopped-flow data indicate that C12E8 strongly activates the E* to E transition, which may account for the effect of the detergent on ATP modulation during steady-state ATP hydrolysis. C12E8 perturbs ATPase turnover by direct interaction with the enzyme, rather than by an indirect effect exerted via a change in the lipid phase or protein aggregation.