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J C Gomez-Fernandez

Publications and source records attributed to J C Gomez-Fernandez.

At least 19 recordsLinked to original sources
PubMed1995

Functional properties of a sarcoplasmic reticulum Ca(2+)-ATPase with an altered Ca(2+)-binding mechanism.

Treatment of sarcoplasmic reticulum vesicles with diethylpyrocarbonate in the presence of a large excess of reagent, at pH 6.2 and at room temperature, reveals both a fast- and a slow-reacting population of protein residues. The loss of the Ca(2+)-ATPase activity is mainly associated with the fast-reacting population being partially sensitive to hydroxylamine. There is also an effect on the Ca(2+)-binding mechanism. Shorter derivatization times (5 min) produce a loss of the positive cooperativity of Ca2+ binding. When the treatment was prolonged for 30 min there was an additional decrease in the overall Ca2+ affinity. Curve-fitting procedures applied to the non-cooperative binding isotherms provide the equilibrium constants for the two Ca2+ sites, although they cannot discriminate between interacting and independent site mechanisms. Prestationary kinetics assays show 2 Ca2+:1 ATP ratios, at any extent of Ca2+ saturation, indicating that the Ca2+ sites are not independent. The Ca2+ dissociation profile after derivatization shows a decrease in the dissociation constant for the release of the second Ca2+, which is consistent with interacting sites. Isotopic exchange experiments show fast and slow components of equal amplitude even at subsaturating Ca2+ concentrations, which is incompatible with independent binding sites. The experimental data suggest a modification of the equilibrium binding constants making them more similar, but keeping the interacting character. The structural position of the external (cytoplasmic) and the internal (lumenal) Ca2+ sites remains unaltered in the absence of positive cooperativity.

Adenosine Triphosphate↗
PubMed1995

Drug action of ritodrine on the sarcoplasmic reticulum Ca(2+)-ATPase from skeletal muscle.

Ritodrine inhibits the steady-state Ca(2+)-ATPase activity of isolated sarcoplasmic reticulum vesicles in a dose-dependent manner. The observed K0.5 value for inhibition (approximately 3 mM) gives proof of a low-affinity interaction. Ritodrine also interferes with the steady-state Ca2+ transport ability decreasing the maximal rate without modification of the Ca2+ or ATP affinity for the enzyme. This is consistent with an absence of competition for the transport and the catalytic sites. Analysis of the catalytic and transport cycle shows that: (i) ritodrine inhibits the phosphorylation partial reaction. This is supported by pre-steady-state kinetic experiments of Ca2+ transport and also by the temperature dependence of the phosphoenzyme level. (ii) At high ritodrine concentrations the dephosphorylation step becomes rate-limiting. This is suggested by the biphasic profile (V-shape) of phosphoenzyme accumulation at different ritodrine concentrations. This was also confirmed by chase experiments of radioactive phosphoenzyme decomposition at steady state. These data reveal a complex pattern of inhibition involving two sites for interaction with low and high ritodrine concentrations. It is envisaged that ritodrine does not exert any direct effect on the smooth muscle sarcoplasmic reticulum Ca(2+)-ATPase when used in the treatment of preterm birth.

Adenosine Triphosphate↗
PubMed1994

Interdependence of H+ and K+ fluxes during the Ca(2+)-pumping activity of sarcoplasmic reticulum vesicles.

The release of H+ during the oxalate-supported Ca2+ uptake in sarcoplasmic reticulum vesicles is kinetically coincident with the initial phase of Ca2+ accumulation. The Ca2+ uptake is increased and the H+ release is decreased in the presence of KCl and other monovalent chloride salts as expected for a H(+)-monovalent cation exchange. The functioning of the Ca(2+)-pump is disturbed by the presence of potassium gluconate and to a lesser extent, of choline chloride. These salts do not inhibit the ATPase activity of Ca(2+)-permeable vesicles, suggesting a charge imbalance inhibition which is specially relevant in the case of gluconate. Therefore, K+, and also Cl-, appear to be involved in secondary fluxes during the active accumulation of Ca2+. The microsomal preparation seems homogeneous with respect to the K(+)-channel, showing an apparent rate constant for K+ release of approximately 25 s-1 measured with the aid of 86Rb+ tracer under equilibrium conditions. A Rb+ efflux, sensitive to Ca(2+)-ionophore, can be also detected during the active accumulation of Ca2+. The experimental data suggest that both monovalent cations and anions are involved in a charge compensation during the Ca2+ uptake and H+ release. Fluxes of these highly permeable ions would contribute to cancel the formation of a resting membrane potential through the sarcoplasmic reticulum membrane.

Animals↗
PubMed1993

Intramolecular distances within the Ca(2+)-ATPase from sarcoplasmic reticulum as estimated through fluorescence energy transfer between probes.

Fluorescence energy transfer measurements have been carried out to estimate intramolecular distances between probes bound to Ca(2+)-transporting ATPase (Ca(2+)-ATPase) as well as distances between these probes and the phospholipid headgroup. The nucleotide binding site was monitored by using 1,N6-ethenoadenosine 5'-triphosphate, a fluorescent analogue of ATP, and also by labelling Lys515 with fluorescein 5'-isothiocyanate. Three different cysteine residues were individually labelled using the following probes: 5-[(2-iodoacetyl)aminoethyl]amino-naphthalene-1-sulfonic acid (I-AEDANS), 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) and fluorescent maleimides. The surface of the membrane was labelled by reconstitution with fluorescent phospholipids (fluorescein and rhodamine derivatives). We found a distance of 4.1 nm from the nucleotide binding site to NBD (at Cys344), and the same distance to fluorescent maleimides (at Cys364). The AEDANS label (at Cys670,672) was found separated 3.5 nm from NBD, 4.4 nm from fluorescent maleimides, and 3.9 nm from the lipid matrix. The NBD label was 3.2 nm apart from fluorescent maleimides and 2.2 nm from the lipid matrix. Finally, fluorescent maleimides were found to be located 4.2 nm above the membrane surface. All these distances agree with a molecular model in which NBD is located in the stalk portion of the Ca(2+)-ATPase, near the surface of the membrane, and the rest of the probes are above it, in the globular domain of the protein.

4-Chloro-7-nitrobenzofurazan↗
PubMed1993

Biophysical studies of the Pf1 coat protein in the filamentous phage, in detergent micelles, and in a membrane environment.

During the assembly of the Pf1 phage, the membrane-bound coat proteins convert into subunits of the filamentous phage. Fourier-transform infrared (FT-IR) transmission spectroscopy has been applied to a study of the secondary structure of these coat proteins when present (a) in the phage, (b) in detergent micelles, and (c) in a phospholipid membrane aqueous system. Suspensions of the Pf1 phage in H2O and 2H2O show an amide I band at 1652 cm-1, indicative of a high content of the alpha-helical structure present. Oriented films of the Pf1 phage studied by polarized FT-IR transmission spectroscopy indicate that the alpha-helical structures as well as the tyrosine residues (band at 1515 cm-1) are both aligned along the axis of the phage. When the Pf1 coat protein is present in sodium dodecyl sulfate detergent micelles and in lipid membrane systems, the FT-IR spectra show an amide I band at 1657-1658 cm-1, also indicative of a predominantly alpha-helical secondary structure. H/2H amide proton exchange studies show that when present in a phospholipid membrane system some 50-60% of the Pf1 protein exchanges rapidly, while the rest undergoes slow exchange. This is consistent with a proportion of the protein being exposed to the solvent and the other being embedded in the lipid bilayer. The presence of a band at 1630-1640 cm-1 is indicative of the presence of random structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1993

Limited carbodiimide derivatization modifies some functional properties of the sarcoplasmic reticulum Ca2+ release channel.

Sarcoplasmic reticulum membrane derived from the terminal cisternae region reacts with the carboxyl reagent N,N'-dicyclohexylcarbodiimide. The extension of this reaction is dependent on the reagent/protein ratio. By using a low ratio (10 microM reagent and 1 mg of protein/mL), we can selectively prevent the closure of the 450-kDa Ca2+ channel. Rapid filtration experiments indicate no alteration in the activating mechanism of Ca2+ release induced by Ca2+ or Sr2+ whereas the Ca2+ efflux inhibition by Ca2+, Mg2+, or ruthenium red disappears after the chemical treatment. The activating/inhibitory effect of ryanodine on the Ca2+ channel does not appear to be perturbed by N,N'-dicyclohexylcarbodiimide. The negligible incorporation of the 14C radioactive reagent to the 450-kDa band (the Ca2+ channel subunit) indicates the possibility of protein cross-linking in addition to simple derivatization. The functional alterations produced by this reagent suggest the presence of critical acidic residue(s) in a hydrophobic environment which are involved in the low-affinity cationic binding site. They can be tentatively associated with hydrophobic domains of the channel subunits contributing to the lining of the pore for Ca2+ release. The data also indicate that the channel activation by micromolar Ca2+ occurs in a different protein domain which is carbodiimide-insensitive under the experimental conditions tested.

Animals↗
PubMed1993

Diffusivity and structural polymorphism in some model stratum corneum lipid systems.

Mixtures of model stratum corneum lipids were prepared in water from cholesterol, six fatty acids and ceramides. The influence of composition on the polymorphism of these mixtures and also on the diffusivity of a model drug within them, Dlip, was determined. The former was obtained from X-ray diffraction and Fourier transform infrared spectrometry, and the latter from a diffusional release model. An L beta structure was formed for the composition approximating that of the extracellular lipids in intact human abdominal stratum corneum. Dlip was independent of water content in the range 20-40% w/w, with the bilayers showing one dimensional swelling without lateral expansion. Although removal of the ceramides did not result in a significant alteration in Dlip, crystalline cholesterol now appeared. The ceramides were, therefore, necessary for solubilization within the fatty acid bilayers of the large proportion of cholesterol present in the lipid fraction of intact SC. They were also responsible for a thermal L alpha-HII transition observed at approx. 68 degrees. At the concentration in which it exists in intact SC, cholesterol also had only a minimal effect on Dlip, but was necessary to suppress HII phase formation within the fatty acids and ensure an L beta structure. All lipid mixtures that had an L beta structure presented a diffusional barrier approx. 1 order of magnitude greater than that of an unstructured, isotropic lipid mixture. HII structures formed at cholesterol/fatty acid proportions less than approx 8:92 mol% and appeared more permeable than L beta ones. All the results indicate that the diffusional barrier within the model lipid mixtures is guaranteed essentially by the presence of an L beta phase. Although the ceramides and cholesterol exert no intrinsic influence on the magnitude of Dlip, their presence in necessary for the existence of an L beta phase at 33 degrees that is free of both crystalline cholesterol and HII character.

Ceramides↗
PubMed1992

Characterization of ruthenium red-binding sites of the Ca(2+)-ATPase from sarcoplasmic reticulum and their interaction with Ca(2+)-binding sites.

Sarcoplasmic reticulum Ca(2+)-ATPase has previously been shown to bind and dissociate two Ca2+ ions in a sequential mode. This behaviour is confirmed here by inducing sequential Ca2+ dissociation with Ruthenium Red. Ruthenium Red binds to sarcoplasmic reticulum vesicles (6 nmol/mg) with a Kd = 2 microM, producing biphasic kinetics of Ca2+ dissociation from the Ca(2+)-ATPase, decreasing the affinity for Ca2+ binding. Studies on the effect of Ca2+ on Ruthenium Red binding indicate that Ruthenium Red does not bind to the high-affinity Ca(2+)-binding sites, as suggested by the following observations: (i) micromolar concentrations of Ca2+ do not significantly alter Ruthenium Red binding to the sarcoplasmic reticulum; (ii) quenching of the fluorescence of fluorescein 5'-isothiocyanate (FITC) bound to Ca(2+)-ATPase by Ruthenium Red (resembling Ruthenium Red binding) is not prevented by micromolar concentrations of Ca2+; (iii) quenching of FITC fluorescence by Ca2+ binding to the high-affinity sites is achieved even though Ruthenium Red is bound to the Ca(2+)-ATPase; and (iv) micromolar Ca2+ concentrations prevent inhibition of the ATP-hydrolytic capability by dicyclohexylcarbodi-imide modification, but Ruthenium Red does not. However, micromolar concentrations of lanthanides (La3+ and Tb3+) and millimolar concentrations of bivalent cations (Ca2+ and Mg2+) inhibit Ruthenium Red binding as well as quenching of FITC-labelled Ca(2+)-ATPase fluorescence by Ruthenium Red. Studies of Ruthenium Red binding to tryptic fragments of Ca(2+)-ATPase, as demonstrated by ligand blotting, indicate that Ruthenium Red does not bind to the A1 subfragment. Our observations suggest that Ruthenium Red might bind to a cation-binding site in Ca(2+)-ATPase inducing fast release of the last bound Ca2+ by interactions between the sites.

Animals↗
PubMed1992

Effect of diethylstilbestrol and related compounds on the Ca(2+)-transporting ATPase of sarcoplasmic reticulum.

Diethylstilbestrol is a potent inhibitory agent of the Ca(2+)-ATPase activity of sarcoplasmic reticulum membranes. Other structurally related molecules, such as dienestrol or hexestrol having hydroxyl groups at para positions of the two benzene rings produce similar effects. The absence or derivatization of the hydroxyl groups as occurs with trans-stilbene or diethylstilbestrol dipropionate converts the structure in an activating agent of the enzyme. The Ca2+ transport profiles in the presence of the referred drugs reproduces the same behavior observed for the hydrolytic activity. There is also a clear indication of a membrane-mediated mechanism of these drugs. Ligand binding experiments at equilibrium indicate that diethylstilbestrol decreases the affinity for Ca2+ of the high affinity Ca2+ sites. Functional studies reveal that the activation/inhibition induced by these drugs is correlated with decreased levels of phosphoenzyme at steady state, and these levels are sensitive to the Ca2+ concentration. Chase experiments of [32P]phosphoenzyme and 45Ca2+ indicate a slight activation effect of diethylstilbestrol dipropionate on Ca2+ dissociation during the enzyme turnover. The use of different anthroyloxy derivatives of stearic acid as a fluorescent probe suggest that diethylstilbestrol and other inhibitory agents could be located close to the polar region of the lipid bilayer, which interferes with the Ca(2+)-binding sites, whereas the activators trans-stilbene and diethylstilbestrol dipropionate may have a deeper position into the membrane, which accelerates the Ca2+ translocation process.

Adenosine Triphosphate↗
PubMed1992

The Ca2+ release channel in junctional sarcoplasmic reticulum: gating and blockade by cations.

1. By using a sarcoplasmic reticulum preparation containing feet structures and the 45Ca2+/filtration technique, the opening and closing response of the Ca(2+)-channel was studied. 2. Extravesicular Sr2+ can activate the channel even though this cation is less efficient than Ca2+ in stimulating the Ca2+ release. Higher Sr2+ concentrations display inhibitory action. 3. By studying the closing response high- and low-affinity cations can be distinguished, according to the concentration range required to exert their effect. 4. The synergistic behavior observed by combining high- and low-affinity blocking cations suggest that they interact through the same binding site. 5. The high-and low-affinity cations are noncompetitive blockers of the activating Ca2+ suggesting the existence of an inhibitory site which is different to the activating site.

Animals↗
PubMed1990

Characterization of the steady-state calcium fluxes in skeletal sarcoplasmic reticulum vesicles. Role of the Ca2+ pump.

Unidirectional Ca2+ fluxes (influx and efflux), supported by ATP as a phosphate-donor substrate, were measured without alteration of the lumenal Ca2+ content in longitudinal sarcoplasmic reticulum vesicles. The referred fluxes are dependent on extravesicular Ca2+, ATP and ADP. They are unaffected by ruthenium red but inhibited by quercetin. The Ca2+ fluxes at steady state are drastically diminished when ATP is substituted by acetylphosphate although the addition of 10 microM ADP increases the apparent rate constants more than eight fold. The observed fluxes appear to be dependent on Ca2(+)-ATPase phosphoenzyme transitions. The results indicate that: (a) the slow Ca2+ release, due to the passive permeability of the membrane, is a minor component of the total Ca2+ efflux, and (b) the ATPase protein is basically operating as a Ca2+/Ca2+ exchanger at steady state. Kinetic resolution of the Ca2+ fluxes, measured by isotopic tracer and rapid filtration techniques can be recreated by computer simulation of the ATPase reaction cycle featuring some modifications to account for the fast Ca2+/Ca2+ exchange and the uncoupling effect observed at steady state.

Animals↗
PubMed1989

Quinacrine inhibits the calcium-induced calcium release in heavy sarcoplasmic reticulum vesicles.

Quinacrine is a fluorescence probe useful for studying the effect of local anesthetics. The interaction of quinacrine and sarcoplasmic reticulum membranes measured by fluorescence spectroscopy indicates the presence of a saturable binding site. Typical local anesthetics are able to displace quinacrine bound to heavy sarcoplasmic reticulum membranes. The effectiveness of that displacement decreases in the order dibucaine greater than tetracaine greater than benzocaine greater than lidocaine greater than procaine greater than procainamide, indicating that the size and hydrophobicity of quinacrine are major determinants in the binding process. The use of radioactive tracer and a rapid filtration technique reveals that quinacrine interacts, at lower concentrations, with sarcoplasmic reticulum membranes by blocking the Ca2+-induced Ca2+ release. Higher quinacrine concentrations also affect the Ca2+-pump activity.

Adenosine Triphosphatases↗
PubMed1989

Characterization of the tetraphenylboron-induced calcium release from skeletal sarcoplasmic reticulum.

Release of Ca2+ from skeletal sarcoplasmic reticulum vesicles was studied by the spectrophotometric stopped-flow technique using tetraphenylboron as a releasing agent. The extent of Ca2+ release shows a sigmoidal response, with respect to the tetraphenylboron concentration, being dependent on Ca2+ preloading and Ca2+-ATPase activity, since these experiments were performed on actively loaded vesicles. The release process has a rapid component with an apparent rate constant of 6-8 s-1, showing a linear relationship between the rapid rate of Ca2+ release and the Ca2+ content of the vesicles. The release is not mediated by the reversal of the Ca2+ pump. Since the amphipathic anion tetraphenylboron was unable to elicit a Ca2+-release response when added to a preparation of sarcoplasmic reticulum phospholipid vesicles, it is suggested that there may be an interaction with some membrane protein(s) at the hydrophobic/hydrophilic interface leading to the opening of some specific Ca2+-release pathway.

Animals↗
PubMed1989

Fourier transform infrared spectroscopic studies on the secondary structure of the Ca2+-ATPase of sarcoplasmic reticulum.

Fourier transform infrared spectroscopy has been applied to the study of the secondary structure of the Ca2+-ATPase of sarcoplasmic reticulum. An attempt is made to quantitatively assess the various secondary structures present. Values of 45% alpha-helix, 32% beta-sheet and 23% turns were obtained. A comparison is made of these results and those obtained using other techniques such as CD and Raman spectroscopy. The various assumptions inherent in the present procedure are discussed. The effect of various ligands, e.g. Ca2+, vanadate, ATP and phosphate, upon the structure were investigated. Upon binding these ligands no marked spectral changes were observed.

Animals↗