Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in calcium-ATPase/lipid interaction

Abstract

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Preservation of the native structure and function of Ca2+-ATPase from sarcoplasmic reticulum: Solubilization and reconstitution by new short-chain phospholipid detergent 1,2-diheptanoyl-sn-phosphatidylcholine

“…In the detergent-solubilized and sucrose-gradient-purified preparations, the DHPC enzyme retains its ability to undergo the Ca# + -induced conformational change, as well as remaining close to the maximal Ca# + -to-ATP coupling ratio, whereas the C "# E ) preparation has lost these native properties. This is consistent with the suggestion of de Foresta et al [21] that the C "# E ) displaces some lipids from the protein-lipid interface. The retention of these native properties by the DHPC preparation is consistent with a mechanism of solubilization of Ca# + -ATPase by DHPC in which the detergent either does not interact directly with the protein or, because it is itself a phospholipid, can replace these lipids without affecting the protein conformation and activity.…”

“…Because it is a natural phospholipid it has no particular interactions with the membrane protein interface, and is probably preferentially excluded from this domain by the chain length incompatibility. This is in contrast with the probable mechanism of solubilization of C "# E ) , which has been shown to displace lipids from the interfacial region of Ca# + -ATPase [21]. Our results in comparing the Ca# + -ATPase prepared with the use of solubilization and reconstitution by C "# E ) and by DHPC support this mechanism of solubilization for DHPC.…”

“…The reason for this difference in the properties is probably related to the mechanism of solubilization of the two detergents : in the DHPC preparation it is likely that the immediate surroundings of the protein might retain more endogenous lipids than the protein treated with C "# E ) , even though both preparations have approximately the same lipid-to-protein ratio. As discussed by de Foresta et al [21], C "# E ) interacts directly with the protein, replacing lipids in contact with the protein ; it is possible that the sucrose-gradient-purified C "# E ) -solubilized Ca# + -ATPase retains significant C "# E ) in the interfacial region of the protein.…”

“…Ionic detergents such as deoxycholate [3] and cholate [4] and nonionic detergents such as Triton X-100 [8], C "# E * [7] and poly(oxyethylene)8-lauryl ether (C "# E ) ) [9,21] have been employed for the solubilization and purification of Ca# + -ATPase from SR. These procedures have produced varied results in terms of active enzyme yield, specific activity and formation of phosphorylated enzyme.…”

Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

“…This was confirmed by addition of low concentrations (0n8 mg\ml) of the detergent C "# E ) to the reconstituted vesicles to make them leaky to ATP. C "# E ) at 0n8 mg\ml had no significant effect on the activity of the purified Ca# + -ATPase [26], and also had no significant effect on the ATPase activity of SR vesicles measured in the presence of the Ca# + ionophore A23187 ( Table 1). Addition of C "# E ) to the reconstituted vesicles led to about a doubling of ATPase activity, showing that in the reconstituted vesicles the distribution of ATPase molecules across the membrane is close to random, with about 50 % having a ' right-side-out ' orientation.…”