Biomedical subjects

A P Starling

Publications and source records attributed to A P Starling.

At least 19 recordsLinked to original sources

PubMed1998

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

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.

Adenosine Triphosphate↗

PubMed1996

Stimulation of the Ca(2+)-ATPase of sarcoplasmic reticulum by disulfiram.

Disulfiram [bis(diethylthiocarbamoyl)disulphide] has been found to stimulate reversibly the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum. At pH 7.2, 2.1 mM ATP and 25 degrees C, ATPase activity was found to double on addition of 120 microM disulfiram. Stimulation fitted to binding of disulfiram at a single site with a Kd of 61 microM. Disulfiram had no effect on the Ca2+ affinity of the ATPase or on the rate of phosphorylation of the ATPase by ATP, but increased the rate of dissociation of Ca2+ from the phosphorylated ATPase (the transport step) and increased the rate of dephosphorylation of the phosphorylated ATPase. It also decreased the level of phosphorylation of the ATPase by Pi, consistent with a 7.5-fold decrease in the equilibrium constant of the phosphorylated to non-phosphorylated forms (E2PMg/E2PiMg) at 80 microM disulfiram. Disulfiram had no significant effect on the concentration of ATP resulting in stimulation of ATPase activity, suggesting that it does not bind to the empty nucleotide-binding site on the phosphorylated ATPase. Studies of the effects of mixtures of disulfiram and jasmone (another molecule that stimulates the ATPase) suggest that they bind to separate sites on the ATPase.

Alcohol Deterrents↗

PubMed1996

Effects of phosphatidylethanolamines on the activity of the Ca(2+)-ATPase of sarcoplasmic reticulum.

ATPase activities for the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum reconstituted into dioleoylphosphatidylethanolamine [di(C18:1)PE] are, at temperatures higher than 20 degrees C, lower than in dioleoylphosphatidylcholine [di(C18:1)PC], whereas in egg yolk phosphatidylethanolamine the activities are the same as in di(C18:1)PC up to 25 degrees C, suggesting that low ATPase activities occur when the phosphatidylethanol-amine species is in the hexagonal H11 phase. ATPase activities measured in mixtures of di(C18:1)PC and di(C18:1)PE do not change with changing di(C18:1)PE content up to 80%. It is concluded that curvature frustration in bilayers containing di(C18:1)PE has no effect on ATPase activity. The rates of phosphorylation and of Ca2+ transport are identical for the native ATPase and for the ATPase in di(C18:1)PE. Dephosphorylation of the phosphorylated ATPase in di(C18:1)PE at 25 degrees C is, however, slower than for the native ATPase, explaining the lower steady-state rate of ATP hydrolysis; in egg yolk phosphatidylethanolamine at 25 degrees C the rate of dephosphorylation is equal to that for the unreconstituted ATPase. Phosphorylation of the ATPase by P1 in the absence of Ca2+ is unaffected by reconstitution in di(C18:1)RE. The stoichiometry of Ca2+ binding to the ATPase is also unaltered. Studies of the effect of di(C18:1)PE on the fluorescence intensity of the ATPase labelled with 7-chloro-4-nitro-2,1,3-benzoxadiazole are consistent with an increase in the E1/E2 equilibrium constant, where E1 is the conformation of the ATPase with two high-affinity binding sites for Ca2+ exposed to the cytoplasm, and E2 is a conformation unable to bind cytoplasmic Ca2+. A slight increase in affinity for Ca2+ can be attributed to the observed increase in the E1/E2 equilibrium constant.

Calcium-Transporting ATPases↗

PubMed1996

Separate effects of long-chain phosphatidylcholines on dephosphorylation of the Ca(2+)-ATPase and on Ca2+ binding.

The steady-state activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) is low when reconstituted into bilayers of the long-chain phosphatidylcholines dierucyl phosphatidylcholine [di(C22:1)PC] or dinervonyl phosphatidylcholine [di(C24:1)PC]. In di(C24:1)PC the ATPase binds a single Ca2+ ion, whereas in di(C22:1)PC it binds two, as in the native SR [Starling, East and Lee (1993) Biochemistry 32, 1593-1600]. In di(C22:1)PC, rates of phosphorylation of the ATPase by ATP and the rate of ATP-induced Ca2+ dissociation are slightly lower than in the native ATPase. However, a much more marked decrease is observed in di(C22:1)PC in the rate of dephosphorylation of the phosphorylated ATPase, which explains the low steady-state ATPase activity. The level of phosphorylation of the ATPase by Pi was little affected by reconstitution in di(C22:1)PC, suggesting that the rate of phosphorylation by Pi is also decreased. The very similar effects of di(C22:1)PC and di(C24:1)PC (Starling, East and Lee (1995) Biochem. J. 310, 875-879) on phosphorylation and dephosphorylation suggest that changes in these steps and the change in Ca2+ binding stoichiometry observed in di(C24:1)PC represent independent changes on the ATPase.

Adenosine Triphosphate↗

PubMed1996

An investigation of the mechanism of inhibition of the Ca(2+)-ATPase by phospholamban.

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum has been reconstituted with peptides corresponding to the hydrophobic domain of phospholamban (PLB) with or without the three Cys residues replaced by Ala, and with PLB with the three Cys residues replaced by Ala [PLBcys-(1-52)]. Reconstitution with the hydrophobic domain of PLB[PLB(25-52)] was found to decrease the apparent affinity of the ATPase for Ca2+ with no effect on the maximal rate of ATP hydrolysis observed at saturating concentrations of Ca2+. Reconstitution with PLBCys-(1-52) decreased both the apparent affinity for Ca2+ and the maximal activity; the effect on maximal activity followed from a decrease in the rate of the Ca2+ transport step (E1PCa2-->E2P) as observed with the hydrophilic domain PLB(1-25). The concentration dependences of the effects of the hydrophobic domain and of the whole PLB molecule were very similar, suggesting that the hydrophilic domain made little contribution to the affinity of the ATPase for PLB. The effect of PLB on the ATPase was dependent on the molar ratio of phospholipid to ATPase, suggesting partition of the PLB between its binding site on the ATPase and the bulk lipid phase in the membrane. Neither PLB nor its hydrophobic domain affected the rates of phosphorylation or dephosphorylation of the ATPase. Despite their effects on the apparent affinity of the ATPase for Ca2+, neither PLB nor its hydrophobic domain had any effect on the true affinity of the ATPase for Ca2+, as measured from changes in the tryptophan fluorescence of the ATPase. The effects of PLB on the activity of the ATPase are the sum of the effects of its hydrophilic and hydrophobic domains.

Animals↗

PubMed1996

The effect of N-terminal acetylation on Ca(2+)-ATPase inhibition by phospholamban.

The peptide Ac-MEKVQYLTRSAIRRASTIEMPQQAR (Ac-PLB(1-25)) representing residues 1-25 of phospholamban (PLB) inhibited the maximal activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum by about 53%, with a Kd value of 5 microM; the equivalent non-acetylated peptide PLB(1-25) had no effect. However, it was found that the non-acetylated peptide increased the effective Kd value for inhibition by Ac-PLB(1-25) consistent with competitive binding to the ATPase, with a Kd value of 8 microM for PLB(1-25). The non-acetylated peptide must therefore be able to bind to the ATPase, but in a conformation that does not lead to inhibition of the ATPase. The identity of the N-terminal residue is important in determining the strength of binding; replacement of the Met residue by Ile led to fourfold weaker binding, again with only binding of the acetylated peptide leading to inhibition of ATPase activity.

Acetylation↗

PubMed1996

The mechanism of inhibition of the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum by the cross-linker o-phthalaldehyde.

Labelling the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum with o-phthalaldehyde (OPA) results in loss of ATPase activity at a 1:1 molar ration of label to ATPase. The affinity of the ATPase for CA2+ is unaffected, as is the E1/E2 equilibrium constant. The rate of dissociation of Ca2+ from the Ca(2+)-bound ATPase is also unaffected and Mg2+ increases the rate of dissociation, as for the unlabelled ATPase. Effects of Mg2+ on the fluorescence intensity of the ATPase labelled with 4-(bromo-methyl)-6,7-dimethoxycoumarin are also unaffected by labelling with OPA, consistent with the fluorescence change reporting on Mg2+ binding at the gating site on the ATPase. The affinity of the ATPase for ATP is reduced by labelling, as is the rate of phosphorylation. The rate of phosphorylation is independent of the concentration of ATP above 25 microM ATP, so that the slow step is the first-order rate constant for phosphorylation by bound ATP. The rate of the back reaction between phosphorylated ATPase and ADP is little affected, suggesting that the slow step in phosphorylation could be the slow conformation step before phosphoryl transfer. The rate of dephosphorylation of the phosphorylated ATPase is also decreased, suggesting that a similar conformation change could be involved in the dephosphorylation step. The rate of the Ca(2+)-transport step appears to be unaffected by labelling. The net result of these changes is that the labelled ATPase is present predominantly in a Ca(2+)-free, phosphorylated form at steady state in the presence of ATP.

Adenosine Triphosphate↗

PubMed1995

The hydrophilic domain of phospholamban inhibits the Ca(2+)-ATPase--the importance of the method of assay.

The peptide MEKVQYLTRSAIRRASTIEMPQQAR-Cys representing residues 1-25 of phospholamban (PLN) decreases by 40% the maximal state rate of ATP hydrolysis by the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR), measured at saturating concentrations of Ca2+. The pattern of Ca2+ uptake by SR vesicles in the presence of oxalate is complex, with an initial fast phase being followed by a lag phase and a second, slower phase of Ca2+ accumulation. PLN(1-25) reduces the rate of the slower phase of Ca2+ accumulation by 30%. However, if the level of accumulation of Ca2+ is measured after 2 min., the effect of PLN(1-25) is much less marked. It is concluded that PLN(1-25) inhibits the ATPase, but that the effects of this inhibition are not apparent under some assay conditions.

Amino Acid Sequence↗

PubMed1995

Effects of phospholipid fatty acyl chain length on phosphorylation and dephosphorylation of the Ca(2+)-ATPase.

The kinetics of the Ca(2+)-ATPase purified from sarcoplasmic reticulum have been studied after reconstitution into bilayers of dimyristoleoylphosphatidylcholine [di(C14:1)PC], dioleoylphosphatidylcholine[di(C18:1)PC] and dinervonylphosphatidylcholine [di(C24:1)PC]. In di(C24:1)PC the rate of phosphorylation of the ATPase by ATP was comparable with that in di(C18:1)PC (about 70 s-1), but in di(C14:1)PC the rate was much lower (21 s-1). Fluorescence responses of the ATPase suggest changes in the phosphoryl-transfer step rather than in the preceding conformational change E1Ca2ATP<-->E1'Ca2ATP. The rate of dephosphorylation of the phosphorylated ATPase was found to decrease in the order di(C24:1)PC < di(C14:1)PC < di(C18:1)PC. For the ATPase in di(C24:1)PC the rate of dephosphorylation (3.3 s-1) was slow enough to be the rate-limiting step for ATP hydrolysis; in di(C14:1)PC, it is suggested that both phosphorylation and dephosphorylation contribute to rate limitation. Phosphorylation of the ATPase in di(C24:1)PC by Pi was normal, but no phosphoenzyme could be detected in di(C14:1)PC. The rate of the Ca(2+)-transport step was normal in di(C24:1)PC, suggesting that the single Ca2+ ion bound to the ATPase in di(C24:1)PC could be transported.

Animals↗

PubMed1995

Phosphatidylinositol 4-phosphate increases the rate of dephosphorylation of the phosphorylated Ca(2+)-ATPase.

Incubation of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum with ATP in the absence of Ca2+ leads to phosphorylation of phosphatidylinositol (PtdIns) to phosphatidylinositol 4-phosphate (PtdIns-4P) and to a doubling of ATPase activity. Similarly, reconstitution of the ATPase with mixtures of dioleoylphosphatidylcholine and PtdIns-4P also led to a doubling of activity; ATPase activity increased with increasing PtdIns-4P content, up to 10% beyond which no further increase was observed. Reconstitution with PtdIns had a much smaller effect on activity. Changes in the Ca2+ affinity of the ATPase following incubation with ATP or reconstitution with PtdIns-4P were small. The rates of phosphorylation of the ATPase by ATP and of the Ca2+ transport step were unaffected, but the rate of dephosphorylation of the phosphorylated ATPase increased by a factor of 2 either following incubation with ATP or following reconstitution with PtdIns-4P. Activation of the ATPase led to a decrease in the level of phosphorylation of the ATPase by Pi corresponding to a 10-fold decrease in the equilibrium constant E2PMg/E2PiMg.

Adenosine Triphosphate↗

PubMed1995

Evidence that the effects of phospholipids on the activity of the Ca(2+)-ATPase do not involve aggregation.

The Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum, solubilized in monomeric from in C12E8, has been reconstituted by dialysis into sealed vesicles of dioleoyl phosphatidylcholine [di(C18:1)PC], dimyristoleoyl phosphatidylcholine [di(C14:1)PC], dinervonyl phosphatidylcholine [di(C24:1)PC] or dipalmitoyl phosphatidylcholine [di(C16:0)PC] in the gel phase, at a phospholipid/ATPase molar ratio of 10,000: 1. Cross-linking experiments show that ATPase molecules are present in these reconstituted vesicles as isolated monomeric species. ATPase activities for the reconstituted vesicles are about half of those for the ATPase reconstituted with the same lipid in unsealed membrane fragments, attributed to a close to random orientation for the ATPase molecules in the reconstituted vesicles. ATPase activities for the ATPase in reconstituted vesicles of di(C14:1)PC or di(C24:1)PC are less than in vesicles of di(C18:1)PC, and no activity could be detected for the ATPase in di(C16:0)PC in the gel phase. It is concluded that effects of lipids on the activity of the ATPase are independent of any changes in the state of aggregation of the ATPase. Inhibition of ATPase activity by spermine and by the hydrophilic domain of phospholamban are observed both for the unreconstituted ATPase and for the ATPase in reconstituted vesicles, so that inhibition is independent of any aggregation caused by these polycationic species. Stimulation of ATPase activity by jasmone is also observed both for the unreconstituted ATPase and for the ATPase in reconstituted vesicles, so that stimulation of the ATPase also does not follow from any change in the state of aggregation of the ATPase.

Animals↗

PubMed1995

Effects of gel phase phospholipid on the Ca(2+)-ATPase.

ATPase activities for the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum reconstituted in dimyristoylphosphatidylcholine [di(C14:0)PC] or dipalmitoylphosphatidylcholine [di(C16:0)PC] are very low at temperatures below 25 and 30 degrees C, respectively. The stoichiometry of Ca2+ binding to the ATPase is 1 Ca2+ ion bound per ATPase molecule in di(C14:0)PC in both gel and liquid-crystalline phases; addition of cholesterol at a 1:1 molar ratio with di(C14:0)PC increases Ca2+ binding to two Ca2+ ions bound per ATPase molecule. The affinity of the ATPase for Ca2+ is slightly higher in di(C16:0)PC in the gel phase than in the liquid-crystalline phase, consistent with a shift in the E1/E2 equilibrium toward E1 in gel phase lipid. The rates of dissociation of Ca2+ from the ATPase in gel and liquid-crystalline phase lipids are the same in the absence of Mg2+, but whereas addition of Mg2+ to the ATPase in liquid-crystalline lipid increases the rate of dissociation in liquid-crystalline phase lipid, Mg2+ has no effect in gel phase lipid. The fluorescence intensity of the Ca(2+)-ATPase labeled with 4-(bromomethyl)-6,7-dimethoxycoumarin decreases on addition of Mg2+ in liquid-crystalline phase lipid, but is unaffected by Mg2+ in gel phase lipid. The rate of phosphorylation of the ATPase in gel phase lipid is very slow, and rates of dephosphorylation of the phosphorylated ATPase are also very slow. p-Nitrophenolphosphatase activity is also very low in gel phase lipid. Binding of ATP results in the same changes in the fluorescence of the ATPase labeled with IAEDANS in gel and liquid-crystalline phase lipids, but changes in tryptophan fluorescence intensity are different.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1995

Lipid structure and Ca(2+)-ATPase function.

Effects of lipid structure on the function of the Ca(2+)-ATPase of skeletal muscle of sarcoplasmic reticulum are reviewed. Binding of phospholipids to the ATPase shows little specificity. Phosphatidylcholines with short (C14) or long (C24) fatty acyl chains have marked effects on the activity of the ATPase, including a change in the stoichiometry of Ca binding. Low ATPase activity in gel phase lipid follows from low rate of phosphorylation. Phosphatidylinositol 4-phosphate increases ATPase activity by increasing the rate of dephosphorylation of the phosphorylated ATPase. Stimulation is not seen with other anionic phospholipids; phosphatidic acid decreases ATPase activity in a Mg(2-)-dependent manner.

Animals↗

PubMed1994

Characterization of the single Ca(2+)-binding site on the Ca(2+)-ATPase reconstituted with short- or long-chain phosphatidylcholines.

On reconstitution of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum into bilayers of dimyristoleoylphosphatidylcholine [di(C14:1)PC] or dinervonylphosphatidylcholine [di(C24:1)PC] the stoichiometry of Ca2+ binding changes from the usual two Ca2+ ions bound per ATPase molecule to one Ca2+ ion bound per ATPase molecule. For the ATPase in di(C24:1)PC, removal of Ca2+ from the Ca(2+)-bound ATPase results in a decrease in tryptophan fluorescence intensity, as observed for the ATPase in dioleoylphosphatidylcholine [di(C18:1)PC]. For the ATPase in di(C14:1)PC removal of Ca2+ results in no change in tryptophan fluorescence intensity. In the presence of Mg2+, removal of Ca2+ from the ATPase in di(C18:1)PC or di(C24:1)PC results in a decrease in tryptophan fluorescence intensity, but for the ATPase in di(C14:1)PC this results in an increase in intensity. Fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) is the same for the ATPase in di(C18:1)PC or di(C24:1)PC, but is markedly greater in di(C14:1)PC, consistent with a 4-fold increase in the E1/E2 equilibrium constant. Addition of Mg2+ to NBD-labelled ATPase in di(C18:1) PC or di(C24:1)PC results in an increase in NBD fluorescence, attributed to stronger binding of Mg2+ to the E1 than to the E2 conformation; addition of Mg2+ had no effect on the fluorescence of the NBD-labelled ATPase in di(C14:1)PC. In the absence of Ca2+, Mg2+ increased the tryptophan fluorescence of the ATPase in di(C14:1)PC, di(C18:3)PC or di(C24:1)PC, with the same binding-constant for Mg2+ in all three lipids. Addition of Mg2+ to the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin resulted in a decrease in fluorescence in di(C18:1)PC or di(C24:1)PC but had no effect in di(C14:1)PC. These effects are interpreted in terms of binding of Ca2+ at a single outer Ca2+ binding-site on the ATPase in di(C14:1)PC and di(C24:1)PC, in a conformation in which the inner site is occluded [in di(C14:1)PC] or modified in its affinity for Ca2+ [in di(C24:1)PC]. Thapsigargin binds to the ATPase, reducing its affinity for Ca2+ both in di(C14:1)PC and di(C24:1)PC.

Binding Sites↗

PubMed1994

Mechanism of inhibition of the Ca(2+)-ATPase by spermine and other polycationic compounds.

The ATPase activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by a variety of polyamines, including spermine, spermidine, and poly(arginine). The effects of spermine on the ATPase are highly specific. It has no effect on the affinity of the ATPase for Ca2+ or ATP, and no effect on the rate of phosphorylation by ATP. When the ATPase is phosphorylated with Pi in the presence of dimethyl sulfoxide at pH 6.0, and then dephosphorylation is induced by dilution in buffer at pH 7.5 in the absence of dimethyl sulfoxide, spermine is found to have no effect on the rate of dephosphorylation. If the ATPase is phosphorylated with [gamma-32P]ATP and the rate of loss of radiolabeled phosphoenzyme is measured following the addition of unlabeled ATP, spermine is found to decrease the rate of loss of radiolabel, consistent with an effect of spermine on the rate of the Ca2E1P-->E2P step. Direct measurement confirms that spermine decreases the rate of dissociation of Ca2+ from the phosphorylated ATPase (Ca2E1P-->E2P), with the decrease in the rate of this step explaining the inhibition of ATPase activity. Spermine also increases the equilibrium constant E1/E2 and inhibits phosphorylation of the ATPase by Pi by competition with the Mg2+ essential for the reaction. It is suggested that spermine could bind to the site on the Ca(2+)-ATPase that interacts with phospholamban.

Amino Acid Sequence↗

PubMed1994

Binding sites for cholesterol on Ca(2+)-ATPase studied by using a cholesterol-containing phospholipid.

Phosphatidylcholines have been synthesized containing a cholesterol moiety at the 2-position of the glycerol backbone. Fluorescence quenching studies show that cholesterol-containing phosphatidylcholines can bind at the lipid-protein interface of the Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum, with an affinity half that of dioleoylphosphatidylcholine. The ATPase activity measured for the ATPase reconstituted with the cholesterol-containing phosphatidylcholine containing an oleoyl fatty acyl chain, (C18:1, CHS)PC, is less than that measured for the ATPase reconstituted with dioleoylphosphatidylcholine. The activity measured for the ATPase reconstituted with the cholesterol-containing phosphatidylcholine containing a myristoleoyl fatty acyl chain, (C14:1, CHS)PC, is less than that measured in (C18:1,CHS)PC and is comparable to that measured in dimyristoleoylphosphatidylcholine (di(C14: 1)PC. The stoichiometry of Ca2+ binding to the ATPase is two Ca2+ ions bound per ATPase molecule in the native membrane or in (C18:1,CHS)PC, but one bound per ATPase molecule in di(C14:1)PC or (C14: 1,CHS)PC. Addition of cholesterol to the ATPase in di(C14:1)PC or (C14:1,CHS)PC increases the Ca2+ binding stoichiometry to the usual 2:1, but the binding stoichiometry remains 1:1 in mixtures of di(C14: 1)PC and (C14:1,CHS)PC. Removal of Ca2+ from the Ca(2+)-bound ATPase results in a decrease in tryptophan fluorescence intensity for the ATPase in the native membrane, but an increase in fluorescence intensity for the ATPase in di(C14:1)PC or (C14:1,CHS)PC. Addition of cholesterol to the ATPase in di(C14:1)PC or (C14:1,CHS)PC reverses this change. It is concluded that cholesterol linked to a phospholipid molecule can interact with the ATPase only at the lipid-protein interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1994

Mechanism of stimulation of the calcium adenosinetriphosphatase by jasmone.

The ATPase activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is increased ca. 3-fold at 25 degrees C and pH 7.2 by jasmone at a concentration of 100 microM, concentrations above 10 mM resulting in reduced stimulation. Stimulation by methyl jasmonate, menthol, or menthone requires much higher concentrations. Effects of jasmone are much less marked at 37 degrees C than at 25 degrees C, and much higher concentrations of jasmone are required to stimulate ATPase activity at pH 6.0 than at pH 7.2. The effects of jasmone on the ATPase are highly specific. Jasmone has no effect on the E1<-->E2 equilibrium constant for the ATPase or on Ca2+ binding. The rate of phosphorylation by ATP is unaffected by jasmone, and only small effects are seen on the reaction of the phosphorylated ATPase with ADP. Jasmone does, however, increase the rate of dephosphorylation by a factor of 2 and the rate of dissociation of Ca2+ from the phosphorylated ATPase by a factor of 3. Jasmone decreases the level of phosphorylation of the ATPase by P(i) in the absence of Ca2+ consistent with a decrease in the equilibrium constant E2P(i)Mg<-->E2PMg. Reconstitution of the ATPase with dimyristoleoylphosphatidylcholine decreases the stoichiometry of Ca2+ binding from the usual 2:1 to 1:1. Unlike other hydrophobic molecules, jasmone failed to reverse this effect. Further, jasmone had very similar effects on the activity of the ATPase reconstituted with either dimyristoleoylphosphatidylcholine or dioleoylphosphatidylcholine, whereas other hydrophobic molecules caused a much greater stimulation of activity for the ATPase reconstituted with the short-chain lipid.

Adenosine Diphosphate↗

PubMed1994

Binding of Ca2+ to the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum: kinetic studies.

Stop-flow fluorescence and rapid-filtration methods have been used to establish the kinetics of Ca2+ binding to, and dissociation from, the (Ca(2+)-Mg2+)-ATPase of skeletal-muscle sarcoplasmic reticulum and to define the effects of H+ and Mg2+ on Ca2+ binding and dissociation rates. The kinetics have been interpreted in terms of the scheme: E2 E2<==>E1<==>E1Ca<==>E1'Ca<==>E1'Ca2. The kinetics of the E2<==>E1 E1 transition have been determined by measuring the rate of change of the fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole after a pH jump or the addition of Ca2+ to the labelled ATPase in the presence of thapsigargin or thapsivillosin A. It has been shown that Mg2+ has a marked effect on Ca2+ dissociation at pH 7.2 and that changes in the tryptophan fluorescence of the ATPase follow the same time course as the dissociation of 45Ca2+. It is proposed that the effect of Mg2+ follows from binding to a 'gating' site, as detected by changes in the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin. The rate of dissociation of Ca2+ from the ATPase increases with increasing pH. The rate of dissociation of Ca2+ decreases with increasing Ca2+ concentration in the medium, with an apparent affinity for Ca2+ greater than that seen for the change in fluorescence amplitude. It is shown that this follows if the first, inner, Ca(2+)-binding site on the ATPase has a lower affinity for Ca2+ than the second, outer, site. Effects of H+ and Mg2+ on Ca2+ dissociation can be treated by the quasiequilibrium approach. Mg2+ and H+ also affect the rate of Ca2+ binding to the ATPase, and effects of H+ and Mg2+ on the E2<==>E1 equilibrium explain the results of experiments in which the concentrations of H+ and Mg2+ are jumped.

Animals↗