PubMed Health⌕ Search

Biomedical subjects

L De Meis

Publications and source records attributed to L De Meis.

At least 19 recordsLinked to original sources
PubMed1998

Control of heat production by the Ca2+-ATPase of rabbit and trout sarcoplasmic reticulum.

The sarcoplasmic reticulum Ca2+-ATPase of rabbit skeletal muscle can convert the energy derived from a Ca2+ gradient into heat (L. de Meis, M. L. Bianconi, and V. A. Suzano. FEBS Lett. 406: 201-204, 1997). In this report, it is shown that this conversion varies depending on the temperature and on whether rabbit (endotherm) or trout (poikilotherm) sarcoplasmic reticulum vesicles are used. The gradient doubled the yield of heat produced during ATP hydrolysis and the calorimetric enthalpy of ATP hydrolysis (DeltaHcal) value found with both rabbit and trout varied between -10 and -12 kcal/mol in leaky vesicles (no gradient) and between -20 and -22 kcal/mol with intact vesicles (gradient). For the rabbit, the difference of DeltaHcal measured with and without gradient was detected in the range of 30-35 degrees C and disappeared when the temperature was decreased below 30 degrees C. For the trout, the difference was detected between 20 and 25 degrees C and disappeared below 20 degrees C. The effect of the gradient on the DeltaHcal for ATP hydrolysis was modified by DMSO, trifluoperazine, and heparin sodium.

Adenosine Triphosphate↗
PubMed1997

Uncoupling of Ca2+ transport ATPase in muscle and blood platelets by diacylglycerol analogues and cyclosporin A antagonism.

The possibility that diacylglycerol analogues might have a wider spectrum of intracellular targets than the well-known protein kinase C was investigated with vesicles containing the Ca2+-ATPase derived from the dense tubular system in platelets and from the sarcoplasmic reticulum of skeletal muscle. The diacylglycerol analogues PMA and 1-oleoyl-2-acetyl-rac-glycerol (OAG) inhibited Ca2+ accumulation by these vesicles, an effect that was antagonized by cyclosporin A. The inhibitory activity of PMA and OAG resulted from the uncoupling of the Ca2+-ATPase, characterized by a pronounced inhibition of Ca2+ uptake accompanied by a discrete decrease in ATPase activity and by the inhibition of the enzyme's phosphorylation by Pi, leading to both a decrease in ATP synthesis and an enhancement of Ca2+ efflux. The inhibition of Ca2+ uptake by PMA was found to decrease as the Ca2+ concentration in the medium was raised from 0.1 to 10.0 microM. This was observed with muscle, but not with platelet vesicles. In contrast, the ability of cyclosporin A to antagonize the inhibition of Ca2+ uptake by PMA also increased when the Ca2+ concentration in the medium was raised from 0.1 to 10.0 microM, but this was observed with both muscle and platelet vesicles. The fact that phospholipase C activity and products from the inositol metabolism have been described as localized in regions of the sarcoplasmic reticulum where Ca2+-ATPase and Ca2+ channels are found suggests a possible physiological role for these products in the regulation of cytosolic Ca2+ levels.

Adenosine Triphosphate↗
PubMed1996

Pharmacological differentiation between intracellular calcium pump isoforms.

We studied the Ca(2+)-ATPase isoforms of sarco/endoplasmic reticulum (SERCA) derived from cerebellum, cardiac muscle, and skeletal muscle. The Mg2+ dependence varied among the three enzyme preparations. The Ca2+ transport in skeletal muscle vesicles, but not in cerebellar or cardiac vesicles, was activated by free Mg2+ concentrations varying from 0.1 to 0.3 mM. Concentrations of Mg2+ of > 1 mM inhibited Ca2+ transport in all three vesicle preparations but with more pronounced effect in cerebellar and cardiac vesicles. At 10-80 microM, trifluoperazine activated Ca2+ uptake in cerebellar and cardiac vesicles but not in skeletal muscle vesicles. The activation was due to an increase in the coupling ratio between Ca2+ transport and ATP hydrolysis and was observed only in the presence of ATP concentrations of > 100 microM. The Ca2+ transport in all three vesicle preparations was inhibited by trifluoperazine concentrations of > 100 microM. The inhibition promoted by trifluoperazine was prevented by the addition of dimethylsulfoxide (10% v/v) to the medium. The Ca2+ efflux from loaded vesicles was increased by arsenate and even more by trifluoperazine. In skeletal muscle vesicles, the efflux promoted by arsenate was several-fold faster than that promoted in vesicles derived from cerebellum or cardiac muscle. In skeletal muscle, the enhancement of Ca2+ efflux promoted by both arsenate and trifluoperazine was antagonized by thapsigargin, Ca2+, Mg2+, and K+. These agents partly antagonized the enhancement of Ca2+ efflux promoted by trifluoperazine in cardiac vesicles but had little or no effect in the cerebellar vesicles. Finally, Mg.Pi and Mg.ATP, the two substrates that phosphorylate the Ca(2+)-ATPase, antagonized the effect of trifluoperazine in all of the preparations tested. The concentration of ATP needed was in the same range as that of the second K(m) value for ATP (50-300 microM) of the SERCA isoforms. The results indicate that the effect of the drugs on the cytosolic Ca2+ homeostasis may vary depending on the target tissue.

Animals↗
PubMed1993

Modulation by fatty acids of Ca2+ fluxes in sarcoplasmic-reticulum vesicles.

The fatty acids palmitic (C16:0), stearic (C18:0), arachidic (C20:0) and arachidonic (C20:4) acids inhibit Ca2+ uptake and enhance Ca2+ efflux measured in vesicles derived from the sarcoplasmic reticulum of skeletal muscle. These effects of the fatty acids are impaired by the Ca(2+)-ATPase ligands Mg2+, Ca2+ and K+, and by drugs that block the leakage of Ca2+ through the Ca(2+)-ATPase such as Ruthenium Red, spermine [de Meis (1991) J. Biol. Chem. 266, 5736-5742] and thapsigargin [de Meis and Inesi (1992) FEBS Lett. 299, 33-35].

Adenosine Triphosphate↗
PubMed1991

Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase.

The first step towards ATP synthesis by the Ca2-ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35 degrees C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10-fold, whereas it had no effect on the apparent affinity for Mg2+. In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations. Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+. The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.

Adenosine Triphosphate↗
PubMed1990

Modification of ATP regulatory function in sarcoplasmic reticulum Ca2(+)-ATPase by hydrophobic molecules.

The effects of the three hydrophobic molecules triphenylphosphine, trifluoperazine and 3-nitrophenol on Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles was investigated. When ATP was the substrate, triphenylphosphine (3 microM) increased the amount of Ca2+ accumulated by the vesicles. At high concentrations triphenylphosphine inhibited Ca2+ uptake. This effect varied depending on the ATP concentration and the type of nucleotide used. With ITP there was only inhibition and no activation of Ca2+ uptake by triphenylphosphine. On the other hand, trifluoperazine inhibited Ca2+ accumulation regardless of whether ATP or ITP was used as substrate. When 5 mM oxalate was included in the medium in order to avoid binding of Ca2+ to the low-affinity Ca2(+)-binding sites of the enzyme, both stimulation by triphenylphosphine and inhibition by trifluoperazine were reduced. In leaky vesicles at low Ca2+ concentrations, triphenylphosphine and 3-nitrophenol were competitive inhibitors of ATPase activity at the regulatory site of the enzyme (0.1-1 mM ATP). A striking difference was observed when both the high- and low-affinity Ca2(+)-binding sites were saturated. In this condition, triphenylphosphine and 3-nitrophenol promoted a 3-4-fold increase in the apparent affinity for ATP at its regulatory site.

Adenosine Triphosphate↗
PubMed1989

Effect of ADP on the rate of acetyl phosphate hydrolysis by the Ca2+-ATPase of sarcoplasmic reticulum.

Hydrolysis of acetyl phosphate is inhibited by high concentrations of Pi and MgCl2, probably due to an increase in the steady-state level of phosphoenzyme formed from Pi in the medium. A dual effect of ADP during steady-state hydrolysis of acetyl phosphate was observed. ADP inhibited hydrolysis in the presence of 5 mM MgCl2 and no added Pi, whereas it stimulated hydrolysis when phosphoenzyme formation by Pi was favored by including 6 mM Pi and 20 mM MgCl2 in the assay medium. ATP inhibited acetyl phosphate hydrolysis in both of these assay media. When phosphoenzyme formation by Pi in the presence of acetyl phosphate was stimulated at Ca2+ concentrations sufficient to saturate the low-affinity Ca2+-binding sites, ADP stimulated acetyl phosphate hydrolysis and also promoted ATP synthesis by reversal of the catalytic cycle. The rate of ATP synthesis was dependent on ADP, Pi and Ca2+. Phosphoenzyme formation by Pi and MgCl2, whether in the absence of Ca2+ and acetyl phosphate, or during acetyl phosphate hydrolysis, was inhibited by ADP and ATP. These results suggest that ADP interacts with different intermediates of the catalytic cycle and that expression of inhibition or activation of acetyl phosphate hydrolysis depends on the steady-state level of phosphoenzyme formed by Pi.

Adenosine Diphosphate↗
PubMed1988

Rate of calcium release and ATP synthesis in sarcoplasmic reticulum vesicles.

Sarcoplasmic reticulum vesicles can catalyze the synthesis of ATP coupled to the efflux of calcium. The rate of this reaction is much faster when the vesicles are loaded in a medium containing phosphate than when oxalate is the precipitating agent. Two components of ATP synthesis can be observed when vesicles loaded with calcium phosphate are used. In the millisecond range and when the loaded vesicles are phosphorylated by Pi, the addition of ADP leads to an initial burst of ATP synthesis and after 50 ms approximately 3.0 nmol of ATP/mg protein are synthesized. This burst is not inhibited by ATP and is enhanced by physiological concentrations of KCl. The slow component of ATP synthesis is inhibited by both ATP and 100 mM KCl. In the physiological pH range, betaine, a trimethylamine present in different tissues, increases the level of phosphoenzyme formed by Pi and enhances the amount of ATP synthesized during the first turn of the reversal of the calcium pump.

Adenosine Triphosphate↗
PubMed1988

Inhibition of mitochondrial F1 ATPase and sarcoplasmic reticulum ATPase by hydrophobic molecules.

The hydrophobic nature of the active site of two energy-transducing ATPases was explored by comparing interactions between Pi and each of three hydrophobic drugs in the absence and presence of organic solvents. The drugs tested were the Fe . bathophenanthroline complex and the anticalmodulin drugs, calmidazolium and trifluoperazine. All inhibit the Pi in equilibrium with ATP exchange reaction catalyzed by submitochondrial particles and the ATPase activity of both submitochondrial particles and soluble F1 ATPase. The inhibition by the three drugs is reversed by either raising the Pi concentration or by adding organic solvent (dimethylsulfoxide, ethyleneglycol or methanol) to the medium. The inhibition of the Pi in equilibrium with ATP exchange by trifluoperazine becomes more pronounced when the electrochemical proton gradient formed across the membrane of the submitochondrial particles is decreased by the addition to the medium of the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum vesicles are inhibited by the Fe . bathophenanthroline complex, calmidazolium and trifluoperazine. Phosphorylation of the ATPases by Pi, synthesis of ATP from ADP and Pi and the fast efflux of Ca2+ observed during reversal of the Ca2+ pump are inhibited by the three drugs. The inhibition is reversed by raising the concentration of Pi or dimethylsulfoxide. The three drugs tested appear to compete with Pi for a common binding site on the Ca2+-ATPase. The data presented are interpreted according to the proposal that the catalytic site of an enzyme involved in energy transduction undergoes a hydrophobic-hydrophilic transition during the catalytic cycle.

Biological Transport, Active↗
PubMed1985

Synthesis of pyrophosphate by chromatophores of Rhodospirillum rubrum in the light and by soluble yeast inorganic pyrophosphatase in water-organic solvent mixtures.

Chromatophores of Rhodospirillum rubrum contain a membrane-bound pyrophosphatase that synthesizes pyrophosphate when an electrochemical H+ gradient is formed across the chromatophore membrane upon illumination. In this report it is shown that MgCl2 and Pi have different effects on the synthesis of pyrophosphate in the light depending on whether initial velocities or steady-state levels are examined. When the water activity of the medium is reduced by the addition of organic solvents, soluble yeast inorganic pyrophosphatase (no H+ gradient present) synthesizes pyrophosphate in amounts similar to those synthesized by the chromatophores in totally aqueous medium during illumination, (H+ gradient present). The pH, MgCl2 and Pi dependence for the synthesis of pyrophosphate by the chromatophores at steady-state is similar to that observed at equilibrium with the soluble enzyme in the presence of organic solvents. The possibility is raised that a decrease in water activity may play a role in the mechanism by which the energy derived from the electrochemical H+ gradient is used for the synthesis of pyrophosphate in chromatophores of R. rubrum.

Bacterial Chromatophores↗
PubMed1982

Exchange between inorganic phosphate and adenosine triphosphate in (Na+,K+)-ATPase.

(Na+,K+)-ATPase is able to catalyze a continuous ATP in equilibrium Pi exchange in the presence of Na+ and in the absence of a transmembrane ionic gradient. At pH 7.6 the Na+ concentration required for half-maximal activity is 85 mM and at pH 5.1 it is 340 mM. In the presence of optimal Na+ concentration, the rate of exchange is maximal at pH 6.0 and varies with ADP and Pi concentration in the assay medium. ATP in equilibrium Pi exchange is inhibited by K+ and by ouabain.

Adenosine Diphosphate↗
PubMed1977

Effects of anions, pH and magnesium on calcium accumulation and release by sarcoplasmic reticulum vesicles.

In the absence of oxalate, Ca2+ accumulation by isolated sarcoplasmic reticulum vesicles may show a transient behavior in which the vesicles accumulate during the first 2 min of incubation as much as twice the amount of Ca2+ which is retained after 5-7 min, when Ca2+ accumulation approaches a steady state. Before Ca2+ release begins, the Ca2+ accumulation can reach 200-250 nmol/mg protein. The spontaneous release of the "extra" Ca2+ initially accumulated appears to be triggered by the attainment of a sufficiently high concentration of free Ca2+ inside the vesicles. The amplitude of the transient phase of Ca2+ accumulation reaches a high value near pH 6.0 and is increased by free Mg2+. At optimal concentrations of H+ and Mg2+, the amount of Ca2+ accumulated during the transient is augmented by various anions, in the order maleate > or = propionate > or = succinate > chloride > sulfate > acetylglycine. The divalent anions have their maximum effects at 20-40 mM and the monovalent anions, at 40-200 mM. At 200 mM, all of the carboxylic anions tested significantly reduce the amount of Ca2+ retained in the steady state.

Adenosine Triphosphate↗
PubMed1976

ADP-activated calcium ion exchange in sarcoplasmic reticulum vesicles.

(1) Ca2+ efflux from rabbit skeletal muscle sarcoplasmic reticulum vesicles pre-loaded with 45Ca2+ was studied in the presence and in the absence of external Ca2+. (2) In the absence of Ca2+ in the assay media, ADP activates the Ca2+ efflux. The increment of Ca2+ efflux requires Pi, is coupled to ATP synthesis, and is inhibited by external Ca2+ (Ki 0.1-0.2 muM). (3) When Ca2+ is added to the assay media, ADP alone activates the Ca2+ efflux, but this is coupled to a Ca2+ influx of the same magnitude. It is therefore an exchange of internal for external Ca2+ in a 1:1 ratio. (4) The ADP-activated Ca2+ exchange requires external Ca2+ with an apparent Km of 0.1-0.2 muM, does not require the addition of Pi or Mg2+, although 3-10 mM MgCl2 activates it. It is not inhibited by the removal of contaminating ATP with hexokinase plus glucose. (5) It seems likely that Ca2+ can be translocated across sarcoplasmic reticulum membrane without the formation of a phosphorylated intermediate.

Adenosine Diphosphate↗
PubMed1975

Ca-2+-dependent inhibitory effects of Na+ and K+ on Ca-2+ transport in sarcoplasmic reticulum vesicles.

Effects of Na+ and K+ on Ca-2+ transport by sarcoplasmic reticulum vesicles were studied in a medium containing high Mg-2+ and ATP (2mM) and low Ca-2+ (0.44muM) concentrations. Under these conditions, Na+ and K+ inhibit Ca-2+ uptake, ATPase activity and membrane phosphorylation by ATP. Since the concentrations of ATP and Ca-2+ used are consistent with relaxation in vivo, the results suggest that under physiological resting conditions the Ca-2+ pump of the sarcoplasmic reticulum operates below its maximal capacity.

Adenosine Triphosphate↗