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F Henao

Publications and source records attributed to F Henao.

At least 19 recordsLinked to original sources
PubMed2004

Inhibition of mouth skeletal muscle relaxation by flavonoids of Cistus ladanifer L.: a plant defense mechanism against herbivores.

Cistus ladanifer exudate is a potent inhibitor of the sarcoplasmic reticulum Ca2+-ATPase (Ca2+-pump) of rabbit skeletal muscle, a well-established model for active transport that plays a leading role in skeletal muscle relaxation. The low concentration of exudate needed to produce 50% of the maximum inhibition of the sarcoplasmic reticulum Ca2+-ATPase activity, 40-60 microg/ml, suggests that eating only a few milligrams of C. ladanifer leaves can impair the relaxation of the mouth skeletal muscle of herbivores, as the exudate reaches up to 140 mg/g of dry leaves in summer season. The flavonoid fraction of the exudate accounts fully for the functional impairment of the sarcoplasmic reticulum produced by the exudate (up to a dose of 250-300 microg/ml). The flavonoids present in this exudate impair the skeletal muscle sarcoplasmic reticulum function at two different levels: (i) by inhibition of the Ca2+-ATPase activity, and (ii) by decreasing the steady state ATP-dependent Ca2+-accumulation. Among the exudate flavonoids, apigenin and 3,7-di-O-methyl kaempferol are the most potent inhibitors of the skeletal muscle sarcoplasmic reticulum. We conclude that the flavonoids of this exudate can elicit an avoidance reaction of the herbivores eating C. ladanifer leaves through impairment of mouth skeletal muscle relaxation.

Animals↗
PubMed2001

Phosphorylated Ca2+-ATPase stable enough for structural studies.

The atomic structure of sarcoplasmic reticulum Ca(2+)-ATPase, in a Ca(2+)-bound conformation, has recently been elucidated (Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature 405, 647-655). Important steps for further understanding the mechanism of ion pumps will be the atomic structural characterization of different key conformational intermediates of the transport cycle, including phosphorylated intermediates. Following our previous report (Champeil, P., Henao, F., Lacapère, J.-J. & McIntosh, D. B. (2000) J. Biol. Chem. 276, 5795-5803), we show here that it is possible to prepare a phosphorylated form of sarcoplasmic reticulum Ca(2+)-ATPase (labeled with fluorescein isothiocyanate) with a week-long stability both in membranes and in mixed lipid-detergent micelles. We show that this phosphorylated fluorescein isothiocyanate-ATPase can form two-dimensional arrays in membranes, similar to those that were used previously to reconstruct from cryoelectron microscopy images the three-dimensional structure of Ca(2+)-free unphosphorylated ATPase. The results also provide hope that crystals of phosphorylated Ca(2+)-ATPase suitable for x-ray crystallography will be achieved.

Animals↗
PubMed2001

The content of glycogen phosphorylase and glycogen in preparations of sarcoplasmic reticulum-glycogenolytic complex is enhanced in diabetic rat skeletal muscle.

AIMS/HYPOTHESIS: We have examined the effect of diabetes and pharmacological insulin treatment on the content of glycogen phosphorylase and glycogen associated with the sarcoplasmic reticulum-glycogenolytic complex from rat skeletal muscle. METHODS: Diabetes was induced in rats by streptozotocin injection. Enzymatic activities were measured using spectrophotometric methods. Glycogen phosphorylase was determined measuring the pyridoxal-5' -phosphate content and using polyacrylamide gel electrophoresis. Glycogen content was measured by enzymatic and the phenol sulfuric methods. RESULTS: The content of glycogen phosphorylase associated with the sarcoplasmic reticulum glycogenolytic complex gradually arises after diabetes induction. The content of glycogen phosphorylase was restored to a control value by pharmacological insulin treatment. In addition, the content of glycogen in preparations of sarcoplasmic reticulum-glycogenolytic complex of diabetic animals was also increased, whereas the content of glycogen in total muscle of diabetic rats was similar to that of the control rats. The absolute and relative amount of glycogen associated with sarcoplasmic reticulum seemed to increase in diabetic animals. These effects on the compartmentalisation of glycogen were suppressed by insulin treatment. Additionally, the rate of conversion of glycogen phosphorylase b to a, an index of the phosphorylase kinase activity, was 50 % lower in diabetic rats, increasing the dephosphorylated form of glycogen phosphorylase and, as a consequence, its association with sarcoplasmic reticulum membranes. CONCLUSION/INTERPRETATION: These results suggest that under diabetic conditions, both glycogen phosphorylase and a small percentage of muscle glycogen are relocalized in the sarcoplasmic reticulum-glycogenolytic complex.

Animals↗
PubMed2000

A remarkably stable phosphorylated form of Ca2+-ATPase prepared from Ca2+-loaded and fluorescein isothiocyanate-labeled sarcoplasmic reticulum vesicles.

After the nucleotide binding domain in sarcoplasmic reticulum Ca2+-ATPase has been derivatized with fluorescein isothiocyanate at Lys-515, ATPase phosphorylation in the presence of a calcium gradient, with Ca2+ on the lumenal side but without Ca2+ on the cytosolic side, results in the formation of a species that exhibits exceptionally low probe fluorescence (Pick, U. (1981) FEBS Lett. 123, 131-136). We show here that, as long as the free calcium concentration on the cytosolic side is kept in the nanomolar range, this low fluorescence species is remarkably stable, even when the calcium gradient is subsequently dissipated by ionophore. This species is a Ca2+-free phosphorylated species. The kinetics of Ca2+ binding to it indicates that its transport sites are exposed to the cytosolic side of the membrane and retain a high affinity for Ca2+. Thus, in the ATPase catalytic cycle, an intrinsically transient phosphorylated species with transport sites occupied but not yet occluded must also have been stabilized by fluorescein isothiocyanate (FITC), possibly mimicking ADP. The low fluorescence mainly results from a change in FITC absorption. The Ca2+-free low fluorescence FITC-ATPase species remains stable after addition of thapsigargin in the absence or presence of decavanadate, or after solubilization with dodecylmaltoside. The remarkable stability of this phosphoenzyme species and the changes in FITC spectroscopic properties are discussed in terms of a putative FITC-mediated link between the nucleotide binding domain and the phosphorylation domain in Ca2+-ATPase, and the possible formation of a transition state-like conformation with a compact cytosolic head. These findings might open a path toward structural characterization of a stable phosphorylated form of Ca2+-ATPase for the first time, and thus to further insights into the pump's mechanism.

Biological Transport, Active↗
PubMed1999

Plausible stoichiometry of the interacting nucleotide-binding sites in the Ca(2+)-ATPase from sarcoplasmic reticulum membranes.

The Ca(2+),Mg(2+)-ATPase from sarcoplasmic reticulum couples ATP hydrolysis to Ca(2+) transport toward the lumen of the muscular vesicular system. Combined structural and functional studies suggest that the Ca(2+) binding sites are formed by six amino acids of the same polypeptide and that cation translocation may take place through a channel inside a monomer of the ATPase. However, calorimetric, fluorescent, and kinetic studies suggest that the ATPase may assemble into functional oligomers of as yet unknown stoichiometry. We have addressed this question and attempted to determine the ATPase stoichiometry using a biophysical approach based on the analysis of the ATPase inhibition by fluorescein 5'-isothiocyanate in the presence of increasing ATP concentrations. For native SR membranes, our inhibition data are well described by a model consisting of two interacting nucleotide-binding sites per oligomer. This stoichiometry was disrupted in detergent C(12)E(8)-solubilized ATPase. Thus, these findings suggest that interacting nucleotide binding sites of the ATPase may appear as dimers, and imply that interactions of the globular cytoplasmic domains would play a modulatory role of the protein enzymatic activity.

Animals↗
PubMed1998

Characterization of a protease-resistant domain of the cytosolic portion of sarcoplasmic reticulum Ca2+-ATPase. Nucleotide- and metal-binding sites.

Treatment of rabbit sarcoplasmic reticulum Ca2+-ATPase with a variety of proteases, including elastase, proteinase K, and endoproteinases Asp-N and Glu-C, results in accumulation of soluble fragments starting close to the ATPase phosphorylation site Asp351 and ending in the Lys605-Arg615 region, well before the conserved sequences generally described as constituting the "hinge" region of this P-type ATPase (residues 670-760). These fragments, designated as p29/30, presumably originate from a relatively compact domain of the cytoplasmic head of the ATPase. They retain two structural characteristics of intact Ca2+-ATPase as follows: high sensitivity of peptidic bond Arg505-Ala506 to trypsin cleavage, and high reactivity of lysine residue Lys515 toward the fluorescent label fluorescein 5'-isothiocyanate. Regarding functional properties, these fragments retain the ability to bind nucleotides, although with reduced affinity compared with intact Ca2+-ATPase. The fragments also bind Nd3+ ions, leaving open the possibility that these fragments could contain the metal-binding site(s) responsible for the inhibitory effect of lanthanide ions on ATPase activity. The p29/30 soluble domain, like similar proteolytic fragments that can be obtained from other P-type ATPases, may be useful for obtaining three-dimensional structural information on the cytosolic portion of these ATPases, with or without bound nucleotides. From our findings we infer that a real hinge region with conformational flexibility is located at the C-terminal boundary of p29/30 (rather than in the conserved region of residues 670-760); we also propose that the ATP-binding cleft is mainly located within the p29/30 domain, with the phosphorylation site strategically located at the N-terminal border of this domain.

Adenosine Triphosphate↗
PubMed1997

Structural changes of the sarcoplasmic reticulum Ca(II)-ATPase nucleotide binding domain by pH and La(III).

The Ca(2+)-ATPase from sarcoplasmic reticulum couples the hydrolysis of one molecule of ATP to the transport of two Ca2+ ions in skeletal muscle fibers. Here, we study the accessibility of the fluorescein covalently attached to the Lys515 at the nucleotide binding domain of the ATPase to the small collisional quencher iodide at pH 6 and 8, as well as the effect of ligand binding (La3+, La(3+)-nucleotide, and Ca2+). Our results indicate that bound fluorescein is significantly more accessible at pH 6 than at pH 8, suggesting that pH modulates the structure of the nucleotide binding domain of the ATPase. This notion was further substantiated by the finding that La(3+)-nucleotide only interacted with the catalytic center at acidic pH. Notably, the differential accessibility of the nucleotide binding domain at acidic and basic pH cannot be rationalized in terms of the ATPase E1/E2 conformational equilibrium since a shift of the ATPase toward the E1 (plus Ca2+) or E2 (plus EGTA) did not affect the accessibility of fluorescein-labeled ATPase to the quencher. Taken together, these findings show the presence of structural flexibility in the FITC binding site and suggest a structural modulation of the Ca(2+)-ATPase nucleotide binding domain by pH and La3+ binding through long-range link-age mechanisms.

Adenosine Diphosphate↗
PubMed1997

Dissociation of Ca2+ from sarcoplasmic reticulum Ca2+-ATPase and changes in fluorescence of optically selected Trp residues. Effects of KCl and NaCl and implications for substeps in Ca2+ dissociation.

Sequential dissociation of the two Ca2+ ions bound to non-phosphorylated sarcoplasmic reticulum Ca2+-ATPase was triggered by addition, in a stopped-flow experiment, of quin2, which acted both as a high-affinity chelator and as a Ca2+-sensitive fluorescent probe. The kinetics of Ca2+ dissociation were deduced from the observed changes in quin2 fluorescence in the visible region (with lambdaex = 313 nm), while fluorescence detection in the UV region (with lambdaex = 290 nm) made it possible to monitor the tryptophan fluorescence changes accompanying this dissociation under the same ionic conditions. In the absence of KCl or NaCl, at pH 6 or 7, the observed changes in quin2 fluorescence were monoexponential, with rate constants very close to those of the changes in ATPase tryptophan fluorescence, which also appeared monophasic. In the presence of 100 mM KCl, quin2 fluorescence changes, although still monoexponential, were faster than in the absence of the monovalent ions but distinctly slower than the changes in tryptophan fluorescence, which were accelerated to a larger extent. In addition, the apparent kinetics of the Trp fluorescence changes depended on the excitation wavelength. Using an excitation wavelength of 296 nm, the Trp fluorescence drop was still faster than with an excitation wavelength of 290 nm, and in the presence of NaCl it even displayed a clear undershoot. We conclude that in the presence of KCl or NaCl and with an excitation wavelength of 290 nm, the rapid drop in tryptophan fluorescence mainly monitors the dissociation of the first of the two Ca2+ ions to be released from Ca2+-ATPase, while excitation at 296 nm optically selects a subpopulation of Trp residues whose fluorescence level is lower in the ATPase species with one Ca2+ ion bound than in the Ca2+-deprived ATPase species. The latter conditions result in an initial drop in Trp fluorescence whose apparent rate constant (in single-exponential analysis) is faster than the true rate of dissociation of the first Ca2+ ion and in a subsequent slower rise related to dissociation of the second Ca2+ ion. The difference between results obtained in the absence and in the presence of K+ or Na+ is due to an antagonizing effect of these cations on proton-induced conformational rearrangement of Ca2+-free ATPase, a conformational rearrangement which changes the ATPase Trp fluorescence level and significantly affects the cooperativity of Ca2+ binding at equilibrium.

Aminoquinolines↗
PubMed1995

Interaction between glycogen phosphorylase and sarcoplasmic reticulum membranes and its functional implications.

Skeletal muscle glycogen phosphorylase b binds to sarcoplasmic reticulum (SR) membranes with a dissociation constant of 1.7 +/- 0.6 mg of phosphorylase/ml at 25 degrees C at physiological pH and ionic strength. Raising the temperature to 37 degrees C produced a 2-3-fold decrease in the dissociation constant. The SR membranes could bind up to 1.1 +/- 0.1 mg of glycogen phosphorylase b/mg of SR protein, whereas liposomes prepared with endogenous SR lipids and reconstituted Ca(2+)-ATPase were unable to bind glycogen phosphorylase. Binding of glycogen phosphorylase b to SR membranes is accompanied by inhibition of its activity in the presence of AMP. The Vmax for glycogen phosphorylase b associated with SR membranes is 40 +/- 5% of that for purified glycogen phosphorylase and shows a decreased affinity for its allosteric activators, AMP and IMP. These kinetic effects are also observed with purified glycogen phosphorylase b when starch or alpha-amylose is used as substrate instead of glycogen. Treatment of SR membranes with alpha-amylase produced dissociation of glycogen phosphorylase b from the SR membranes. Thus, linear polysaccharide fragments of glycogen bound to the SR membranes are likely mediating the binding of glycogen phosphorylase b to these membranes.

Animals↗
PubMed1994

Quantification and removal of glycogen phosphorylase and other enzymes associated with sarcoplasmic reticulum membrane preparations.

The enzymatic characterization of sarcoplasmic reticulum membrane fragments from rabbit skeletal muscle presented in this paper shows that glycogen phosphorylase, as well as other enzymes (e.g., creatine kinase, myokinase, phosphorylase kinase, glycosidase, AMP-deaminase, phosphoglucomutase) are associated with these membrane preparations. Amongst these enzymes, the highest activity associated with sarcoplasmic reticulum membranes is that of glycogen phosphorylase, which is mostly (at least 95%) in its b form (dephosphorylated form), since its activity in sarcoplasmic reticulum membranes is largely dependent upon AMP. A protocol is presented to quantify the amount of phosphorylase bound to sarcoplasmic reticulum membranes from fluorimetric measurements of the content of its coenzyme, pyridoxal 5'-phosphate. The content of phosphorylase ranged from 0.03 to 0.37 mg phosphorylase per mg of membrane protein, in sarcoplasmic reticulum membrane preparations made following several of the protocols most commonly used and also depending upon the length of the starvation period of the animal before killing. We also show that dilution of sarcoplasmic reticulum membranes to 0.1-0.2 mg protein per ml in a buffer containing 50 mM Tes-KOH (pH 7.4), 0.1 M KCl and 0.25 M sucrose removes at least 95% of glycogen phosphorylase from these membrane fragments, as well as other enzymes like myokinase and glycosidase. On these grounds, we suggest to introduce a final dilution step as indicated above in protocols of sarcoplasmic reticulum membrane preparations.

AMP Deaminase↗
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↗
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↗
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↗
PubMed1990

Distances between functional sites of the Ca2+ + Mg2(+)-ATPase from sarcoplasmic reticulum using Co2+ as a spectroscopic ruler.

Cobalt ion inhibits the Ca2+ + Mg2(+)-ATPase activity of sealed sarcoplasmic reticulum vesicles, of solubilized membranes and of the purified enzyme. To use Co2+ appropriately as a spectroscopic ruler to map functional sites of the Ca2+ + Mg2(+)-ATPase, we have carried out studies to obtain the kinetic parameters needed to define the experimental conditions to conduct the fluorimetric studies. 1. The apparent K0.5 values of inhibition of this ATPase are 1.4 mM, 4.8 mM and 9.5 mM total Co2+ at pH 8.0, 7.0 and 6.0, respectively. The inhibition by Co2+ is likely to be due to free Co2+ binding to the enzyme. Millimolar Ca2+ can fully reverse this inhibition, and also reverses the quenching of the fluorescence of fluorescein-labeled sarcoplasmic reticulum membranes due to Co2+ binding to the Ca2+ + Mg2(+)-ATPase. Therefore, we conclude that Co2+ interacts with Ca2+ binding sites. 2. Co2+.ATP can be used as a substrate by this enzyme with Vmax of 2.4 +/- 0.2 mumol ATP hydrolyzed min-1 (mg protein)-1 at 20-22 degrees C and pH 8.0, and with a K0.5 of 0.4-0.5 mM. 3. Co2+ partially quenches, about 10 +/- 2%, the fluorescence of fluorescein-labeled sarcoplasmic reticulum Ca2+ + Mg2(+)-ATPase upon binding to this enzyme at pH 8.0. From the fluorescence data we have estimated an average distance between Co2+ and fluorescein in the ATPase of 1.1-1.8 nm or 1.3-2.1 nm for one or two equidistant Co2+ binding sites, respectively. 4. Co2+.ATP quenches about 20-25% of the fluorescence of fluorescein-labeled Ca2+ + Mg2(+)-ATPase, from which we obtain a distance of 1.1-1.9 nm between Co2+ and fluorescein located at neighbouring catalytic sites.

Animals↗
PubMed1990

Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital.

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.

Adenosine Triphosphate↗