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P Champeil

Publications and source records attributed to P Champeil.

At least 37 records · Page 2Linked to original sources
PubMed1994

Rapid kinetics of myo-inositol trisphosphate binding and dissociation in cerebellar microsomes.

Using sheep cerebellum microsomes adsorbed on a filter, we measured the kinetics of [3H]inositol 1,4,5-trisphosphate (InsP3) binding and dissociation on the subsecond time scale during rapid perfusion of the filter with [3H]InsP3-containing or InsP3-free media. At 20 degrees C and pH 7.1, in a cytosol-like medium containing MgCl2, the half-time for InsP3 dissociation was as short as 125 ms. The receptor behaved as a simple target for binding of its ligand, with the rate constant for InsP3 binding increasing linearly with InsP3 concentration. Various modulators of InsP3 binding (KCl, NaCl, pH, Mg2+, and Ca2+) were found to affect the receptor's apparent affinity for InsP3 mainly by altering the rate constant for [3H]InsP3 dissociation. ATP (but not InsP3) also accelerated [3H]InsP3 dissociation. In contrast to these modulators, luminal Ca2+ was found to have no effect on the amount of microsome-bound [3H]InsP3.

Animals↗
PubMed1994

Transient inositol 1,4,5-trisphosphate-induced Ca2+ release: a model based on regulatory Ca(2+)-binding sites along the permeation pathway.

A remarkable property of Ca2+ fluxes through the inositol 1,4,5-trisphosphate (InsP3)-gated Ca2+ channel is that successive increments of InsP3 induce repeated transient release of accumulated Ca2+. The initial aim of this study was to propose a model, based on hypotheses compatible with the current description of this Ca2+ channel, which could account for such experimental observations. The key feature of the model was the assumption that the Ca(2+)-binding sites on the receptor, whose occupancy leads to immediate channel activation but to subsequent slow channel desensitization, were located somewhere along the permeation pathway and were therefore sensitive to the flux of Ca2+ rather than the cytosolic or luminal Ca2+ concentration per se. Simulation showed that, provided Ca2+ bound to both activating and inhibitory sites with adequate cooperativity, addition of submaximal concentrations InsP3 resulted in transient opening well above the stationary state. The model also rationalized the documented existence of a threshold for InsP3 action, the puzzling control of channel sensitivity to InsP3 by luminal and cytosolic Ca2+, as well as the functional heterogeneity of the Ca2+ pools.

Binding Sites↗
PubMed1994

Cancellation of the cooperativity of Ca2+ binding to sarcoplasmic reticulum Ca(2+)-ATPase by the non-ionic detergent dodecylmaltoside.

The perturbation of the kinetics of the sarcoplasmic reticulum (SR) membranous Ca(2+)-ATPase cycle by the non-ionic detergent dodecylmaltoside (DM) has been shown to exhibit specific features which were not observed with the related detergents octa(ethylene glycol) monododecylether and Triton X-100 [de Foresta, B., Henao, F. & Champeil, P. (1992) Eur. J. Biochem. 209, 1023-1034]. This previous study has been completed here by a detailed analysis of the perturbation by DM of the interaction of Ca2+ with membranous ATPase, both in its unphosphorylated and phosphorylated form. Equilibrium binding measurements, performed at pH 7.5 and 20 degrees C, showed that only one 45Ca2+ was bound with high affinity to the ATPase in the presence of maximally perturbing concentrations of DM, as compared to two 45Ca2+ in the absence of detergent. This binding was also assessed by a small decrease in the tryptophan fluorescence intensity. Binding of a second Ca2+ occurred only with a much lower affinity. In the presence of DM, the pCa dependence of the phosphorylation by [gamma-32P]ATP of the ATPase shifted towards 50-fold higher Ca2+ concentrations than in its absence. Furthermore, DM completely inhibited the cooperativity of this dependence. This shift strongly suggests that the phosphorylation of DM-perturbed ATPase requires the binding of this second, low-affinity Ca2+. In order to assess this, samples of ATPase were intramolecularly cross-linked with glutaraldehyde. This treatment stabilized the phosphorylated intermediated with occluded Ca2+ [Ross, D. C., Davidson, G.A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621]. Both in the absence and presence of DM, the cross-linked enzyme occluded close to two Ca2+/phosphorylated molecule. Finally, the pCa dependences of the ATPase hydrolytic activity, measured with two different high-energy substrates, ATP or p-nitrophenylphosphate (PNpP), were also found to shift towards higher Ca2+ concentrations in the presence of DM, which was again consistent with a normal coupling ratio, i.e. two bound Ca2+/substrate hydrolyzed. As compared to other detergents, the maltoside head group of DM might favor a stronger interaction with membranous ATPase, resulting in its high perturbing effect on Ca2+ binding. The loss of cooperativity of Ca2+ binding evidenced here makes DM a useful tool in the analysis of the sequence of events occurring during Ca2+ binding.

Animals↗
PubMed1994

Rapid filtration studies of the effect of cytosolic Ca2+ on inositol 1,4,5-trisphosphate-induced 45Ca2+ release from cerebellar microsomes.

Using microsomal membrane vesicles derived from sheep cerebellum, we measured the rate of inositol 1,4,5-trisphosphate (InsP3)-dependent 45Ca2+ efflux from 45Ca(2+)-loaded compartments during rapid perfusion with a medium containing InsP3 and various concentrations of free 40Ca2+ on the cytosolic side (pH 7.1, 5 mM Mg2+, in the absence of ATP at 20 degrees C). At 0.15 microM InsP3 and pCa 6.5, half-45Ca2+ release was attained within less than 200 ms. At low Ca2+ concentrations, the initial rate of 45Ca2+ release depended smoothly on InsP3 concentration, and InsP3 activated release with moderate positive cooperativity. Preliminary experiments performed at various free 40Ca2+ concentrations were consistent with a bell-shaped 40Ca2+ dependence of 45Ca2+ release. In the range of micromolar or higher free 40Ca2+ concentrations, the apparent inhibition of 45Ca2+ release was dependent on InsP3 concentration, and 45Ca2+ release for intermediate InsP3 concentrations was transient; under selected conditions, a second perfusion period, identical to the first one but separated from it by a short recovery period, was found to allow renewed 45Ca2+ efflux. At high Ca2+ concentration, fast reversible Ca(2+)-dependent desensitization of the channel, and not heterogeneity, was therefore responsible for the termination of InsP3-triggered 45Ca2+ efflux at submaximal concentrations of InsP3. At lower Ca2+ concentrations, a large fraction of the apparent activating effect of submicromolar 40Ca2+ concentrations on 45Ca2+ efflux that we had observed in the preliminary experiments proved to be the artifactual consequence of an inhibitory effect exerted by metal-free Ca2+ chelators on InsP3-dependent efflux at nanomolar 40Ca2+ concentrations. 1,2-Bis(2-aminophenoxy)ethane-N,N,N'-N'-tetraacetic acid, EGTA, and fluo-3 were all effective inhibitors. When this inhibition was taken into account, a rise in free 40Ca2+ concentration from 30 to 300 nM only weakly enhanced 45Ca2+ fluxes in the presence of a low concentration of InsP3. As a result, submicromolar free 40Ca2+ appears to be only a poor activator of InsP3-induced Ca2+ release under these experimental conditions.

Adenosine Triphosphate↗
PubMed1993

Strontium binding to sarcoplasmic reticulum Ca(2+)-ATPase. Spectroscopic differentiation of the substeps involved.

We investigated the consequences of Sr2+ binding to the transport sites of sarcoplasmic reticulum (SR) Ca(2+)-ATPase for two fluorescent conformational probes located in different regions of the ATPase. Using SR vesicles in which Lys-515 in the ATPase had been previously labeled with fluorescein 5'-isothiocyanate (FITC), we found that the Sr(2+)-induced a drop in the fluorescein fluorescence of this FITC-labeled ATPase shifted toward lower Sr2+ concentrations than the Sr(2+)-induced rise in Trp fluorescence for the same FITC-labeled ATPase. The curve describing the Sr(2+)-dependent rise in Trp fluorescence had a characteristic asymmetric shape, and the changes in Trp fluorescence occurred in parallel with the activation by Sr2+ of pNPP hydrolysis by the ATPase. Analysis of these results in terms of the simplest scheme describing the sequential binding of the two Sr2+ ions suggests that under the conditions of these experiments, i.e. at neutral pH in the presence of potassium, the Sr(2+)-induced rise in the Trp fluorescence mainly reflected the formation of ATPase with two ions bound to the transport sites, whereas the binding of a single Sr2+ ion was virtually sufficient to reduce the fluorescence of bound FITC to its minimal level.

Binding Sites↗
PubMed1993

Calcium control on InsP3-induced discharge of calcium from permeabilised hepatocyte pools.

The control exerted by intralumenal and cytosolic Ca2+ on InsP3-induced release of Ca2+ from intracellular Ca2+ pools in suspensions of saponin-permeabilised rat hepatocytes was investigated by combined Quin-2 and 45Ca2+ measurements at 20 degrees C. We failed to detect a major effect of intralumenal Ca2+ in regulating this release, as various manipulations in which the load of the Ca2+ pools was varied by a factor of two did not significantly affect the apparent relative efficiency of InsP3 in releasing Ca2+; these manipulations included loading the Ca2+ pools up to various steady state levels by preliminary equilibration at various external free Ca2+ concentrations, as well as emptying them progressively through the blockade of pump-mediated Ca2+ uptake. As regards Ca2+ on the cytosolic side, in contrast with recent results obtained with other systems, we found that, at maximal doses, InsP3-induced Ca2+ release was not stimulated by raising Ca2+ from very low to submicromolar or micromolar concentrations, and that only relatively high concentrations of free Ca2+ inhibited this release (half-maximal inhibition was between 3 and 15 microM). Such elevated Ca2+ concentrations reduced the size of the InsP3-sensitive Ca2+ pool. We also noted that the apparent cooperativity of InsP3 activation of release at pCa 5 was noticeably less than that observed at pCa 7. As a result, at low InsP3 concentrations, a rise in cytosolic Ca2+ from pCa 7 to pCa 5 stimulated InsP3-mediated Ca2+ release. These results are discussed in the context of the current speculations about tissue specificity, heterogeneity, quantal release, oscillations, and the several different mechanisms that may control InsP3-induced Ca2+ release.

Animals↗
PubMed1992

Kinetic characterization of the perturbation by dodecylmaltoside of sarcoplasmic reticulum Ca(2+)-ATPase.

We investigated the functional aspects of the interaction between the sarcoplasmic reticulum (SR) membranous Ca(2+)-ATPase and the non-ionic detergent dodecylmaltoside, using detergent concentrations allowing perturbation of the membrane but not its solubilization. At pH 7.5, the effects of dodecylmaltoside on ATPase activity and delipidation had previously been shown to resemble, in some respects, those of octa(ethylene glycol) monododecylether (C12E8), an appropriate detergent for ATPase studies. Our aim here was to explore the specific effects of dodecylmaltoside on the different steps in the ATPase catalytic cycle, which may owe their specificity to the difference between the polar head groups of dodecylmaltoside and C12E8. This was done at 20 degrees C, both at pH 6 in the absence of KCl and at pH 7.5 in the presence of 100 mM KCl, two conditions under which the characteristics of unperturbed ATPase have already been well defined. Preliminary estimation of dodecylmaltoside partition between water and SR membranes at pH 6 yielded a partition coefficient K close to 4 x 10(5) (ratio of the molar fraction of dodecylmaltoside in the lipid to that in the aqueous phase at a low detergent concentration, assuming that most of this detergent was present in the lipid phase). At near saturation of SR membranes, bound dodecylmaltoside was roughly equimolar with the constituent phospholipids. Non-solubilizing concentrations of dodecylmaltoside inhibited SR ATPase activity by up to 65-70% at pH 7.5, but not at pH 6, unlike the results of similar experiments with C12E8. The rates of the four main steps in the ATPase catalytic cycle were measured by fast kinetic techniques; they were similarly modified at both pH. Dodecylmaltoside slowed down both the rate of calcium-saturated ATPase phosphorylation and the rate of ATPase isomerization after phosphorylation, two steps which were not targets of perturbation by C12E8. The slowing down of the isomerization step by dodecylmaltoside might well explain why it inhibited overall ATPase activity at pH 7.5. In contrast to C12E8, dodecylmaltoside did not affect the dephosphorylation step, which was the main target of inhibition by C12E8 and the main rate-limiting step at pH 6. However, like C12E8, dodecylmaltoside accelerated the calcium binding-induced transition of nonphosphorylated ATPase. Another striking feature of the perturbation induced by dodecylmaltoside was that it significantly altered the binding of 45Ca2+ to the ATPase and the corresponding conformational changes. At pCa 5-5.5, it almost halved calcium binding to the ATPase but ATPase phosphorylation was unimpaired.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1992

The metal sites on sarcoplasmic reticulum membranes that bind lanthanide ions with the highest affinity are not the ATPase Ca2+ transport sites.

We attempted to establish whether lanthanide ions, when added to sarcoplasmic reticulum (SR) membranes in the absence of nucleotide, compete with Ca2+ for binding to the transport sites of the Ca(2+)-ATPase in these membranes, or whether they bind to different sites. Equilibrium measurements of the effect of lanthanide ions on the intrinsic fluorescence of SR ATPase and on 45Ca2+ binding to it were performed either at neutral pH (pH 6.8), i.e. when endogenous or contaminating Ca2+ was sufficient to nearly saturate the ATPase transport sites, or at acid pH (pH 5.5), which greatly reduced the affinity of calcium for its sites on the ATPase. These measurements did reveal apparent competition between Ca2+ and the lanthanide ions La3+, Gd3+, Pr3+, and Tb3+, which all behaved similarly, but this competition displayed unexpected features: lanthanide ions displaced Ca2+ with a moderate affinity and in a noncooperative way, and the pH dependence of this displacement was smaller than that of the Ca2+ binding to its own sites. Simultaneously, we directly measured the amount of Tb3+ bound to the ATPase relative to the amount of Ca2+ and found that Tb3+ ions only reduced significantly the amount of Ca2+ bound after a considerable number of Tb3+ ions had bound. Furthermore, when we tested the effect of Ca2+ on the amount of Tb3+ bound to the SR membranes, we found that the Tb3+ ions which bound at low Tb3+ concentrations were not displaced when Ca2+ was added at concentrations which saturated the Ca2+ transport sites. We conclude that the sites on SR ATPase to which lanthanide ions bind with the highest affinity are not the high affinity Ca2+ binding and transport sites. At higher concentrations, lanthanide ions did not appear to be able to replace Ca2+ ions and preserve the native structure of their binding pocket, as evaluated in rapid filtration measurements from the effect of moderate concentrations of lanthanide ions on the kinetics of Ca2+ dissociation. Thus, the presence of lanthanide ions slowed down the dissociation from its binding site of the first, superficially bound 45Ca2+ ion, instead of specifically preventing the dissociation of the deeply bound 45Ca2+ ion. These results highlight the need for caution when interpreting, in terms of calcium sites, experimental data collected using lanthanide ions as spectroscopic probes on SR membrane ATPase.

Animals↗
PubMed1992

Do submaximal InsP3 concentrations only induce the partial discharge of permeabilized hepatocyte calcium pools because of the concomitant reduction of intraluminal Ca2+ concentration?

In several types of cells whose cytoplasmic Ca2+ is regulated by inositol phosphate derivatives, low concentrations of InsP3 added to permeabilized cell suspensions induce the rapid discharge of part of the InsPs-sensitive Ca2+ pool instead of slow monophasic release of Ca2+ from the entire pool. As a tentative explanation for this puzzling observation, sometimes called 'quantal release', it was suggested that the reduced intraluminal Ca2+ concentration remaining in the Ca2+ pool after a certain amount of Ca2+ had been released might allosterically reduce the channels' affinity for InsP3 and the corresponding InsP3-dependent Ca2+ efflux, and thus result in partial pool discharge (Irvine, R.F. (1990) FEBS Lett. 263, 5-9). We have tested this hypothesis by manipulating the Ca2+ pool contents with ionophore, and found that the rate of InsP3-dependent Ca2+ efflux after ionophore-induced partial discharge of the Ca2+ pools was much faster than what was predicted on the basis of this hypothesis. Heterogeneity of the Ca2+ pools appears to be a more likely reason for the 'quantal release' behavior.

Animals↗
PubMed1991

Kinetic characterization of the normal and procaine-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump.

We investigated the effect of the local anesthetic procaine on the activity of the calcium pump protein of sarcoplasmic reticulum (SR) vesicles. Procaine slowed down the rate of calcium uptake by SR vesicles without enhancing the vesicles' passive permeability. This slowing of the unidirectional pumping rate was reflected by the inhibition of the maximal rate of the transport-coupled Ca(2+)-ATPase activity. The inhibition was dependent on Mg2+ concentration; at optimal (i.e. low) concentrations of magnesium, half-maximal inhibition occurred with procaine concentrations close to 15-20 mM. Inhibition of ATPase was not mediated by a change in the properties of the bulk lipid phase. Procaine moderately reduced the true affinity of ATPase for ATP, whereas equilibrium binding of calcium to ATPase in the absence of ATP was virtually not modified by procaine. In fast-kinetics studies, we explored the various intermediate steps in the ATPase catalytic cycle, in order to determine which of them were targets for inhibition by procaine. We found that procaine slowed down ATPase dephosphorylation, an effect which is at least partly responsible for the observed inhibition of overall ATPase activity. In contrast, procaine accelerated the calcium-induced transconformation of unphosphorylated ATPase in the absence of ATP, and altered neither the rate of the Ca(2+)-dependent phosphorylation of ATPase, nor the rate of the dissociation of Ca2+ from phosphorylated ATPase towards the SR lumen, a critical step, the rate of which was measured by a novel fast-filtration method. These results are discussed with respect to the possible site(s) of binding of this amphiphile on the ATPase, and in relation to the contribution of individual steps in the catalytic cycle to the rate limitation of unperturbed SR ATPase activity.

Animals↗
PubMed1991

The two calcium ions initially bound to nonphosphorylated sarcoplasmic reticulum Ca(2+)-ATPase can no longer be kinetically distinguished when they dissociate from phosphorylated ATPase toward the lumen.

Using rapid filtration, we investigated the kinetics of release toward the lumen of sarcoplasmic reticulum vesicles of the two Ca2+ ions transported by the Ca(2+)-dependent ATPase of these vesicles. Release rates at 20 degrees C were measured by three methods, with vesicles previously made leaky with an ionophore. First, we measured the rate at which 45Ca2+ bound to ATPase approached its steady-state level after addition of ATP to the 45Ca(2+)-equilibrated ATPase. At pH 6 in the absence of potassium, the observed kinetics did not reveal any very fast phase of 45Ca2+ dissociation from phosphorylated ATPase. Second, we measured the kinetics of 45Ca2+ dissociation from phosphorylated ATPase in a "chase" experiment, by isotopic dilution of calcium under turnover conditions in the presence of potassium. We found that these kinetics were essentially monophasic. Moreover, when they were measured in the presence of a high concentration of calcium, designed to saturate the low-affinity calcium transport sites on the lumenal side of the ATPase, they only departed slightly from monophasic behavior, irrespective of the experimental pH (pH 6, 7, or 9). This small perturbation by high calcium concentrations of the observed dissociation kinetics was attributed to ADP-facilitated rapid exchange of 40Ca2+ for Mg2+ at the catalytic site of phosphorylated ATPase. The third method was based on the fact that phosphorylation-induced 45Ca2+ occlusion occurred faster than 45Ca2+ dissociation from nonphosphorylated ATPase: here, we measured the rate of 45Ca2+ internalization on addition to 45Ca(2+)-saturated ATPase of an unlabeled ATP-containing medium. This method allowed separate observation of the dissociation kinetics of each of the two 45Ca2+ ions bound to phosphorylated ATPase, after either one or the other had been labeled by a preliminary partial isotopic exchange in the non-phosphorylated state of the ATPase. We found that after ATP-induced phosphorylation, the two 45Ca2+ ions dissociated toward the lumenal medium with virtually identical rate constants; this was observed under different ionic and pH conditions and also in the presence of a high Ca2+ concentration. As a control, the same partial isotopic exchange procedure allowed us to confirm that, in contrast, when ATP was absent from the final dissociation medium, the two 45Ca2+ ions dissociated from nonphosphorylated ATPase toward the cytoplasmic medium at different rates, the one bound more deeply only dissociating after a lag period corresponding to dissociation of the superficial one.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1991

Crosslinking the active site of sarcoplasmic reticulum Ca(2+)-ATPase completely blocks Ca2+ release to the vesicle lumen.

Intramolecular crosslinking of the active site of the sarcoplasmic reticulum Ca(2+)-ATPase with glutaraldehyde results in substantial inhibition of ATPase activity and stabilization of the ADP-sensitive E1 approximately P(2Ca) intermediate (E, enzyme) with occluded Ca2+ [Ross, D. C., Davidson, G. A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621]. We show here, using conditions of low passive vesicle permeability and absence of ADP, that Ca2+ "deoccludes" more rapidly than it leaks out of the vesicle lumen. Deocclusion is paralleled by dephosphorylation. Therefore, turnover of crosslinked E1 approximately P(Ca) (approximately 5 nmol/min per mg of protein at 25 degrees C) involves Ca2+ release to the vesicle exterior and concomitant phosphoenzyme hydrolysis. Ca2+ release to the lumen, the normal pathway, is apparently blocked completely. In the presence of ADP, Ca2+ is also released to the vesicle exterior, and this release is coupled to the synthesis of ATP. The results suggest that a tertiary structural change at the active site follows phosphorylation and is an absolute requirement for Ca2+ release from the native enzyme to the vesicle lumen.

Animals↗
PubMed1991

Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase.

We investigated the kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum Ca2(+)-ATPase by combining fast filtration with stopped-flow fluorescence measurements. At pH 6 and 20 degrees C, in the absence of potassium and in the presence of 20 mM MgCl2, isotopic exchange of bound calcium exhibited biphasic kinetics, with two phases of equal amplitude, regardless of the initial extent of binding site saturation. The rapidly exchangeable site, whose occupancy by calcium controlled the rate constant of the slow phase, had an apparent affinity for calcium of about 3-6 microM. A similar high affinity was also deduced from measurements of the calcium dependence of the rate constant for ATPase fluorescence changes. This affinity was higher than the overall affinity for calcium deduced from the equilibrium binding measurements (dissociation constant of 15-20 microM); this was consistent with the occurrence of cooperativity (Hill coefficient of 1.6-1.8). The drop in intrinsic fluorescence observed upon chelation of calcium was always slightly faster than the dissociation of calcium itself, although the rates for both this drop in fluorescence and calcium dissociation varied slightly from one preparation to the other. This fluorescence drop was therefore mainly due to dissociation of the bound ions, not to slow transconformation of the ATPase. Dissociation of the two bound calcium ions in a medium containing EGTA exhibited monophasic kinetics in the presence of a calcium ionophore, with a rate constant about half that of the fast phase of isotopic exchange. This particular pattern was observed over a wide range of experimental conditions, including the presence of KCl, dimethyl sulfoxide, 4-nonylphenol, or a nucleotide analogue, at pH 6 or 7, and at various temperatures. The kinetics of calcium dissociation under the above various conditions were not correlated with the ATPase affinity for calcium deduced from equilibrium measurements under the same conditions. These results are consistent with sequential dissociation of calcium from a narrow binding pocket inside which a single calcium ion can move fairly easily. Escape of calcium might be controlled by a structural compartment acting as a gate.

Adenosine Triphosphate↗
PubMed1991

Detection of Ca(2+)-binding proteins by electrophoretic migration in the presence of Ca2+ combined with 45Ca2+ overlay of protein blots.

When high affinity Ca(2+)-binding proteins like calmodulin, or proteins with a high Ca(2+)-binding capacity like calsequestrin, underwent sodium dodecyl sulfate-gel electrophoresis in Laemmli systems, their electrophoretic migration rates were much higher in gels containing 1 mM Ca2+ than in gels containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Replacement of EGTA by Ca2+ in the gel, combined with the blotting of electrophoretically separated proteins on polyvinylidene difluoride membranes and subsequent 45Ca2+ overlay, proved a very effective means of detecting Ca(2+)-binding proteins. This combined approach is important since artifacts occur in both techniques when used separately. We found that the usual procedure of adding Ca2+ to the sample before electrophoresis without including it in the gel itself (C.B. Klee, T. H. Crouch, and M. H. Krinks, 1979, Proc. Natl. Acad. Sci. USA 76, 6270-6273) permitted the detection of only very high affinity Ca(2+)-binding proteins.

Adenosine Triphosphatases↗
PubMed1990

Different classes of tryptophan residues involved in the conformational changes characteristic of the sarcoplasmic reticulum Ca2(+)-ATPase cycle.

Various classes of tryptophan residues in the Ca2(+)-ATPase of sarcoplasmic reticulum membranes have been distinguished on the basis of their sensitivities to certain fluorescence quenchers: the brominated phospholipid 1,2-bis(9,10-dibromostearoyl)-sn-glycero(3)phosphocholine, the calcium ionophore calcimycin (A23187) and its brominated analog (4-bromo-A23187), and the nucleotide analog 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate. We show that tryptophans located at the protein-lipid interface are the main contributors to the well-known fluorescence intensity change occurring in parallel with the conformational rearrangement induced by addition of calcium to the ATPase or its removal; Trp-794 on the ATPase chain may be one of these tryptophans. We also show that tryptophans more deeply embedded in the transmembrane protein structure contribute to the fluorescence change observed upon phosphorylation from inorganic phosphate of the calcium-free ATPase. This phosphorylation step involves opposite changes in the fluorescence quantum yield of tryptophans located in the membrane and in the cytoplasmic regions of the ATPase. This result is in agreement with models in which phosphorylation from inorganic phosphate not only changes the ATPase conformation locally around the catalytic center, but also reorganizes the membrane portion of the ATPase by long-range action, allowing, for instance, the calcium sites to become accessible from the luminal medium.

Animals↗
PubMed1990

Effects of phospholipids on binding of calcium to (Ca2(+)-Mg2(+)-ATPase.

The (Ca2(+)-Mg2(+)-ATPase purified from skeletal muscle sarcoplasmic reticulum binds two Ca2+ ions per ATPase molecule. On reconstitution into bilayers of dioleoylphosphatidylcholine [C18:1)PC) or dinervonylphosphatidylcholine [C24:1)PC) the stoichiometry of binding remains unchanged, but when the ATPase is reconstituted into bilayers of dimyristoleoylphosphatidylcholine [C14:1)PC) the stoichiometry changes to one Ca2+ ion per ATPase molecule. Nevertheless, the level of phosphorylation is the same for the ATPase reconstituted with (C18:1)PC or (C14:1)PC. The effect of (C14:1)PC on the stoichiometry of Ca2+ binding is prevented by androstenol at a 1:1 molar ratio with the phospholipid.

Androstenols↗