Interactions of cholesterol hemisuccinate with phospholipids and calcium-magnesium ATPase

Abstract

Cholesterol hemisuccinate has been shown to equilibrate readily with liposomes and with the (Ca2+-Mg2+)-ATPase from sarcoplasmic reticulum and has been used to modify the sterol content of these membranes. Cholesterol hemisuccinate incorporates into dioleoylphosphatidylcholine (DOPC) up to a molar ratio of 3:1 sterol to DOPC. Effects on lipid order as detected by electron spin resonance and fluorescence polarization are comparable to those of cholesterol. Binding constants have been determined, and the uncharged form of the sterol binds more strongly than the anionic form. Binding to DOPC and to the lipid component of the ATPase system is comparable. From use of the fluorescence quenching properties of 1,2-bis(9,10- dibromooleoyl )phosphatidylcholine and dibromocholesterol hemisuccinate, two classes of binding sites on the ATPase have been deduced. At the lipid/protein interface, the binding constant for cholesterol hemisuccinate is considerably less than that for DOPC. At the second set of sites ( nonannular sites), binding occurs with Kd = 0.55 in molar ratio units. The effect of cholesterol hemisuccinate on the activity of the ATPase depends on the phospholipid present in the system: ATPase reconstituted with DOPC is inhibited whereas ATPase reconstituted with dimyristoleoylphosphatidylcholine is activated. We conclude that changes in membrane fluidity are not important in determining ATPase activity in these systems.

Transmembrane α helices

“…This is rather surprising, given the relatively rigid structure of the steroid ring of cholesterol and the molecularly rough surface of the peptide. In other studies, it has been shown that cholesterol binds relatively weakly at the lipid-protein interface of the ATPase (Simmonds et al, 1982(Simmonds et al, , 1984; comparison with the peptide studies reported here suggests that weak binding of cholesterol to the ATPase involves interactions in the lipid headgroup region rather than interactions between the sterol ring and the hydrophobic transmembrane helices.…”

Effects of phospholipid fatty acyl chain length on phosphorylation and dephosphorylation of the Ca2+-ATPase

“…The response to the binding of Mg2+ at the gating site on the ATPase is normal in di(C24:1)PC but no response is seen in di(C14: 1)PC, as detected by changes in the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin [12]. Lastly, we have shown that effects of di(C14:1)PC can be reversed by addition of a variety of hydrophobic molecules (including cholesterol) whereas effects of di(C24: 1)PC cannot be reversed in this way [11,[13][14][15][16][17][18]. We have shown that the effects of cholesterol follow from binding to sites on the ATPase other than those accessible to phosphatidylcholines [13,19].…”

Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers

“…Numerous studies have shown that cholesterol affects the behavior of membrane inserted helices and the function of many membrane-associated proteins directly or by means of its effect on the physical properties of the phospholipid bilayer, i.e., bilayer hydrophobic thickness and material moduli (35)(36)(37)(38)(39)(40)(41)(42). For example, cholesterol can stabilize ␣-helices in membrane proteins (43,44); it also affects the function of many membrane proteins (36,39,(45)(46)(47)(48) and can alter ion channel gating (49-51).…”