Effects of phospholipids on binding of calcium to calcium-magnesium ATPase

Accumulation of Ca2+ by the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum has been measured in reconstituted, sealed vesicles as a function of lipid composition. Measurements were performed in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to eliminate any effects of H+ transport; in the presence of FCCP, addition of valinomycin had no effect on the level or rate of accumulation of Ca2+ showing that, in the presence of FCCP, no electrical potential built up across the membrane. Levels of accumulation were low when the phospholipid was dioleoylphosphatidylcholine (DOPC), even though DOPC supports high ATPase activity. Inclusion of 10 mol% anionic phospholipid [dioleoylphosphatidic acid (DOPA) or dioleoylphosphatidylserine (DOPS)] led to higher levels of accumulation of Ca2+, 10 mol% being the optimum concentration. Cardiolipin or phosphatidylinositol 4-phosphate were more effective than DOPA or DOPS in increasing accumulation of Ca2+. Effects of anionic phospholipids were seen in the presence of an ATP-regenerating system to remove ADP, and in the presence of phosphate within the reconstituted vesicles to precipitate calcium phosphate. Rates of passive leak of Ca2+ from the reconstituted vesicles were slow. The Ca2+-accumulation process was simulated assuming either simple passive leak of Ca2+ from the vesicles or assuming slippage on the ATPase, a process in which the phosphorylated intermediate of the ATPase releases bound Ca2+ on the cytoplasmic rather than the lumenal side of the membrane. The experimental data fitted to a slippage model, with anionic phospholipids decreasing the rate of slippage.

Section: Figure 6 Simulations Of Ca 2 + Accumulationsupporting

confidence: 60%

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

Dalton

Pilot

Mall

et al. 1999

“…(1979) operative manner, in a channel-like structure (Inesi et al, 1990). However, after reconstitution of the (Ca2+-Mg2+)-ATPase into bilayers of (C14:1)PC, we found that only a single Ca2+ ion bound to the ATPase (Michelangeli et al, 1990b) and that in this form the ATPase could still be phosphorylated by MgATP, although at a slower rate than normal (Michelangeli et al, 1991 …”

Section: Introductionmentioning

confidence: 80%

“…In previous papers we have studied the effects of hydrophobic inhibitors such as nonylphenol (Michelangeli et al, 1990a) and abnormal phospholipids such as dimyristoleoylphosphatidylcholine [(C 14: I)PC] (Michelangeli et al, 1990b(Michelangeli et al, , 1991 on the activity of the (Ca2+-Mg2+)-ATPase purified from skeletalmuscle sarcoplasmic reticulum. We have shown that these effects can be understood in terms of the model for ATPase activity shown in simplified form in Scheme 1, based on that of de Meis & Vianna (1979).…”

Section: Introductionmentioning

confidence: 99%

Mechanism of inhibition of the calcium pump of sarcoplasmic reticulum by thapsigargin

Wictome

Henderson

Lee

et al. 1992

The steady-state ATPase activity of sarcoplasmic-reticulum (Ca2+-Mg2+)-ATPase is inhibited by thapsigargin at a molar ratio of 1:1, with a dissociation constant for thapsigargin estimated to be in the sub-nanomolar range. In the presence of thapsigargin, only a single Ca2+ ion binds to the ATPase. Similarly, addition of thapsigargin to the ATPase incubated in the presence of Ca2+ results in the release of one of the two originally bound Ca2+ ions. As monitored by the fluorescence of nitrobenzo-2-oxa-1,3-diazole-labelled ATPase, thapsigargin appears to shift the transition between El and E2 conformations towards E2. Addition of thapsigargin prevents phosphorylation of the ATPase by Pi and results in a very low steady-state level of phosphorylation of the ATPase by ATP, as observed previously for nonylphenol.

“…As we know that there are two Ca 2+ binding sites on SERCA and that a number of potential Ca 2+ entry pathway sites have been proposed by several groups [36][37][38][39][40] as to the exact route for Ca 2+ to enter and bind, it would be tempting to speculate that each Ca 2+ ion enters their respective binding sites via a distinct pathway, located on adjacent sides of the transmembrane helix bundle. One piece of evidence which may support this view is, when the SERCA is reconstituted with short chain length phospholipids, it reduces the stochiometry for Ca 2+ binding from 2 to 1 without significantly affecting its affinity or the phosphorylation level [41]. Thus this indicates that the two Ca 2+ binding events can, under some circumstances, be made distinct.…”

Section: Discussionmentioning

confidence: 98%

The widely utilized brominated flame retardant tetrabromobisphenol A (TBBPA) is a potent inhibitor of the SERCA Ca2+ pump

Ogunbayo

Michelangeli

2007

TBBPA (tetrabromobisphenol A) is currently the most widely used type of BFR (brominated flame retardant) employed to reduce the combustibility of a large variety of electronic and other manufactured products. Recent studies have indicated that BFRs, including TBBPA, are bio-accumulating within animal and humans. BFRs including TBBPA have also been shown to be cytotoxic and potentially endocrine-disrupting to a variety of cells in culture. Furthermore, TBBPA has specifically been shown to cause disruption of Ca 2+ homoeostasis within cells, which may be the underlying cause of its cytotoxicity. In this study, we have demonstrated that TBBPA is a potent non-isoform-specific inhibitor of the SERCA (sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase) (apparent K i 0.46-2.3 µM), thus we propose that TBBPA inhibition of SERCA contributes in some degree to Ca 2+ signalling disruption. TBBPA binds directly to the SERCA without the need to partition into the phospholipid bilayer.From activity results and Ca 2+ -induced conformational results, it appears that the major effect of TBBPA is to decrease the SERCA affinity for Ca 2+ (increasing the K d from approx. 1 µM to 30 µM in the presence of 10 µM TBBPA). Low concentrations of TBBPA can quench the tryptophan fluorescence of the SERCA and this quenching can be reversed by BHQ [2,5-di-(t-butyl)-1,4-hydroquinone] and 4-n-nonylphenol, but not thapsigargin, indicating that TBBPA and BHQ may be binding to similar regions in the SERCA.