Characterization of the single Ca2+-binding site on the Ca2+-ATPase reconstituted with short- or long-chain phosphatidylcholines

Starling, A P; Khan, Y M; East, J. Malcolm; Lee, A G

doi:10.1042/bj3040569

Cited by 14 publications

(13 citation statements)

References 23 publications

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“…For the ATPase in di(C ") : " )PC, removal of Ca# + from the Ca# + -bound ATPase results in a decrease in fluorescence intensity. For the ATPase in di(C "% : " )PC, removal of Ca# + results in an increase in fluorescence intensity in the presence of Mg# + , but no change in fluorescence intensity in the absence of Mg# + [40]. In mixtures of di(C "% : " )PC and either di(C ") : " )PS or di(C ") : " )PA, the fluorescence response to the removal of Ca# + gradually decreases in magnitude with an increase in the content of di(C "% : " )PC (results not shown), consistent with the gradual increase in activity to the maximum observed in Figure 6.…”

Section: Reconstitution With Mixtures Of Di(c 14 : 1 )Pc and Anionic mentioning

confidence: 97%

“…Rapid kinetic and equilibrium binding experiments have shown that there is no one unique step in the reaction pathway of the Ca# + -ATPase that is sensitive to lipid structure, but rather that a number of different steps can be affected, with different changes in lipid structure affecting different steps. The Ca# + -binding sites on the ATPase are sensitive to the thickness of the phospholipid bilayer, reconstitution into bilayers of phosphatidylcholines containing short (C "% ) or long (C #% ) fatty acyl chains resulting in a change in the stoichiometry of Ca# + -binding to the ATPase [5,40]. However, changing the phase of the phospholipid from liquid crystalline bilayer to gel phase [32] or to hexagonal H II phase [33] has little effect on the Ca# + -binding sites.…”

Section: Mechanism Of Inhibitionmentioning

confidence: 99%

“…One of the changes in the ATPase induced by short-chain lipid is a change in the stoichiometry of Ca# + binding to one Ca# + ion bound per ATPase molecule ; this change can be reversed by addition of 50 mol-% di(C ") : " )PC [40]. It is not possible to test directly whether the anionic lipids have this same effect, because of extensive non-specific binding of Ca# + .…”

Section: Effects Of Phospholipid Chain Length and Headgroup Structurementioning

confidence: 99%

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Interaction of phosphatidic acid and phosphatidylserine with the Ca2+-ATPase of sarcoplasmic reticulum and the mechanism of inhibition

Dalton

East

Mall

et al. 1998

Biochemical Journal

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The sarcoplasmic reticulum of skeletal muscle contains anionic phospholipids as well as the zwitterionic phosphatidylcholine and phosphatidylethanolamine. Here we study the effects of anionic phospholipids on the activity of the Ca2+-ATPase purified from the membrane. Reconstitution of the Ca2+-ATPase into dioleoylphosphatidylserine [di(C18:1)PS] or dioleoylphosphatidic acid [di(C18:1)PA] leads to a decrease in ATPase activity. Measurements of the quenching of the tryptophan fluorescence of the ATPase by brominated phospholipids give a relative binding constant for the anionic lipids compared with dioleoylphosphatidylcholine close to 1 and suggest that phosphatidic acid only binds to the ATPase at the bulk lipid sites around the ATPase. Addition of di(C18:1)PS or di(C18:1)PA to the ATPase in the short-chain dimyristoleoylphosphatidylcholine [di(C14:1)PC] reverse the effects of the short-chain lipid on ATPase activity and on Ca2+ binding, as revealed by the response of tryptophan fluorescence intensity to Ca2+ binding. It is concluded that the lipid headgroup and lipid fatty acyl chains have separate effects on the function of the ATPase. The anionic phospholipids have no significant effect on Ca2+ binding to the ATPase; the level of Ca2+ binding to the ATPase, the affinity of binding and the rate of dissociation of Ca2+ are unchanged by reconstitution into di(C18:1)PA. The major effect of the anionic lipids is a reduction in the maximal level of binding of MgATP. This is attributed to the formation of oligomers of the Ca2+-ATPase, in which only one molecule of the ATPase can bind MgATP dimers in di(C18:1)PS and trimers or tetramers in di(C18:1)PA. The rates of phosphorylation and dephosphorylation for the proportion of the ATPase still able to bind ATP are unaffected by reconstitution. Larger changes were observed in the level of phosphorylation of the ATPase by Pi, which became very low in the anionic phospholipids. The fluorescence response to Mg2+ for the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin was also changed in di(C18:1)PS and di(C18:1)PA, so that effects of Mg2+ became comparable with those seen on phosphorylation for the unreconstituted ATPase. The anionic phospholipids could induce a conformational change in the ATPase on binding Mg2+ equivalent to that normally induced by phosphorylation or by binding inhibitors such as thapsigargin.

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Section: Reconstitution With Mixtures Of Di(c 14 : 1 )Pc and Anionic mentioning

confidence: 97%

Section: Mechanism Of Inhibitionmentioning

confidence: 99%

Section: Effects Of Phospholipid Chain Length and Headgroup Structurementioning

confidence: 99%

See 1 more Smart Citation

Interaction of phosphatidic acid and phosphatidylserine with the Ca2+-ATPase of sarcoplasmic reticulum and the mechanism of inhibition

Dalton

East

Mall

et al. 1998

Biochemical Journal

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“…Besides, replacement of endogenous SR phospholipids by di-C16:0PC completely inhibits the Ca 2+ -ATP-ase activity at 4°C (gel phase), but not at 29°C (liquid-crystalline phase) [31]. Activities in phosphatidylethanolamines under conditions where the phos- phatidylethanolamine is in the hexagonal HII phase are also low [32]. Thus a liquid crystalline bilayer of the appropriate thickness with zwitterionic headgroups is required to stimulate Ca 2+ ATPase activity.…”

Section: Ca 2+ Atpasementioning

confidence: 99%

Bilayer Hydrophobic Thickness and Integral Membrane Protein Function

Cybulski

Mendoza²

2011

CPPS

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The influence of the lipid environment on the function of membrane proteins is increasingly recognized as crucial. Nevertheless, the molecular mechanisms underlying protein-lipid interactions remain obscure. Membrane lipid composition has a regulatory effect on membrane protein activity, and for a number of membrane proteins a clear correlation was found between protein activity and properties of the membrane bilayer such as fluidity. Membrane thickness is an important property of a lipid bilayer. It is expected that hydrophobic thickness match the hydrophobic thickness of transmembrane segments of integral membrane proteins. Any mismatch between the hydrophobic thicknesses of the lipid bilayer and the protein would lead to some modification in either the structure of the protein or the structure of the bilayer, or both. Consequent rearrangements may result in changes in protein activity. Here we review the behavior of several transmembrane proteins whose activity is altered by hydrophobic core thickness.

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“…4 corresponds to the Ca2+-bound, E 1 conformation of the ATPase. It has been shown that the E1 conformation of the ATPase is favored by di(C14:1)PC, whereas di(C 18:1)PC favors the other major conformation of the ATPase, E2 (Starling et al, 1994). Thus, it is possible that conformational changes within the transmembrane region of the Ca2+-ATPase lead to changes in the interhelical loops and thus to changes in the effective hydrophobic thickness of the ATPase (Lee and East, 2001).…”

mentioning

confidence: 99%