ATPase Of Skeletal Muscle Sarcoplasmic Reticulum Research Articles

Sarcolipin uncouples hydrolysis of ATP from accumulation of Ca2+ by the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum.

Sarcolipin (SLN) is a small peptide found in the sarcoplasmic reticulum of skeletal muscle. It is predicted to contain a single hydrophobic transmembrane alpha-helix. Fluorescence emission spectra for the single Trp residue of SLN suggest that SLN incorporates fully into bilayers of dioleoylphosphatidylcholine, but only partially into bilayers of phosphatidylcholines with long (C(22) or C(24)) fatty acyl chains. The fluorescence of SLN is quenched in bilayers of dibromostearoylphosphatidylcholine, also consistent with incorporation into the lipid bilayer. SLN was reconstituted with the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum. Even at a 50:1 molar ratio of SLN/ATPase, SLN had no significant effect on the rate of ATP hydrolysis by the ATPase or on the Ca(2+)-dependence of ATP hydrolysis. However, at a molar ratio of SLN/ATPase of 2:1 or higher the presence of SLN resulted in a marked decrease in the level of accumulation of Ca(2+) by reconstituted vesicles. The effect of SLN was structurally specific and did not result from a breakdown in the vesicular structure or from the formation of non-specific ion channels. Vesicles were impermeable to Ca(2+) in the absence of ATP in the external medium. The effects of SLN on accumulation of Ca(2+) can be simulated assuming that SLN increases the rate of slippage on the ATPase and the rate of passive leak of Ca(2+) mediated by the ATPase. It is suggested that the presence of SLN could be important in non-shivering thermogenesis, a process in which heat is generated by hydrolysis of ATP by skeletal-muscle sarcoplasmic reticulum.

Sarcolipin uncouples hydrolysis of ATP from accumulation of Ca2+ by the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum

Sarcolipin (SLN) is a small peptide found in the sarcoplasmic reticulum of skeletal muscle. It is predicted to contain a single hydrophobic transmembrane α-helix. Fluorescence emission spectra for the single Trp residue of SLN suggest that SLN incorporates fully into bilayers of dioleoylphosphatidylcholine, but only partially into bilayers of phosphatidylcholines with long (C22 or C24) fatty acyl chains. The fluorescence of SLN is quenched in bilayers of dibromostearoylphosphatidylcholine, also consistent with incorporation into the lipid bilayer. SLN was reconstituted with the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum. Even at a 50:1 molar ratio of SLN/ATPase, SLN had no significant effect on the rate of ATP hydrolysis by the ATPase or on the Ca2+-dependence of ATP hydrolysis. However, at a molar ratio of SLN/ATPase of 2:1 or higher the presence of SLN resulted in a marked decrease in the level of accumulation of Ca2+ by reconstituted vesicles. The effect of SLN was structurally specific and did not result from a breakdown in the vesicular structure or from the formation of non-specific ion channels. Vesicles were impermeable to Ca2+ in the absence of ATP in the external medium. The effects of SLN on accumulation of Ca2+ can be simulated assuming that SLN increases the rate of slippage on the ATPase and the rate of passive leak of Ca2+ mediated by the ATPase. It is suggested that the presence of SLN could be important in non-shivering thermogenesis, a process in which heat is generated by hydrolysis of ATP by skeletal-muscle sarcoplasmic reticulum.

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

Accumulation of Ca(2+) by the Ca(2+)-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 Ca(2+) 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 Ca(2+), 10 mol% being the optimum concentration. Cardiolipin or phosphatidylinositol 4-phosphate were more effective than DOPA or DOPS in increasing accumulation of Ca(2+). 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 Ca(2+) from the reconstituted vesicles were slow. The Ca(2+)-accumulation process was simulated assuming either simple passive leak of Ca(2+) from the vesicles or assuming slippage on the ATPase, a process in which the phosphorylated intermediate of the ATPase releases bound Ca(2+) 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.

Influence of dietary n-3 fatty acids on the membrane properties of skeletal muscle in pigs

en

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.

Effects of polycations on Ca2+ binding to the Ca2+-ATPase

Spermine and polyarginine have been shown to increase the rate of dissociation of Ca2+ from the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum. They also decrease the affinity of the ATPase for Mg2+ as detected by changes in the fluorescence intensity of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin (DMC). Polyarginine itself also decreases the fluorescence intensity of DMC-labelled ATPase. These results are consistent with binding of spermine and polyarginine to a gating site controlling the rate of access of Ca2+ to its binding sites on the ATPase. A basic peptide PLN-(1-25) corresponding to residues 1-25 of phospholamban had no effect on the rate of dissociation of Ca2+ or on the fluorescence of DMC-labelled ATPase. Spermine, polyarginine and PLN-(1-25) all increased the equilibrium constant E1/E2, and spermine and polyarginine increased the rate of Ca2+ binding to the ATPase, consistent with an increase in the rate of the E2-->E1 transition. Spermine displaced Tb3+ and Ruthenium Red from the ATPase, consistent with binding in the stalk region of the ATPase. Polyarginine and PLN-(1-25), however, had no effect on Tb3+ or Ruthenium Red binding, suggesting a greater specificity in binding basic peptides to the ATPase than spermine.

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.

Effects of K+ on the binding of Ca2+ to the Ca(2+)-ATPase of sarcoplasmic reticulum.

Equilibrium and kinetic fluorescence methods have been used to characterize the interactions between K+ and the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum. K+ shifts the E2-E1 equilibrium of the ATPase towards E1 and increases the rate of Ca2+ binding to the ATPase, as detected by changes in tryptophan fluorescence intensity, suggesting that K+ increases the rate of the E2-E1 transition. The data are consistent with binding of K+ at the inner Ca(2+)-binding site on the ATPase in competition with H+ and Mg2+, with a higher affinity in the E1 than in the E2 conformation. K+ has no effect on the affinity for Mg2+, as detected by changes in tryptophan fluorescence intensity; since it has been proposed that the changes in tryptophan fluorescence follow from binding to Mg2+ at the outer Ca(2+)-binding site, this suggests that K+ is unable to bind at the outer Ca(2+)-binding site. K+ increases the rate of dissociation of Ca2+ from the Ca(2+)-bound ATPase and reduces the effect of Mg2+ on the fluorescence intensity of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin. It is suggested that these effects of K+ are the result of binding at a 'gating' site on the ATPase, in competition with binding of H+. Binding of K+ at the inner Ca(2+)-binding site and at the gating site account for the observed effects of K+ on the affinity of the ATPase for Ca2+.

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.