PubMed Health⌕ Search

Biomedical subjects

E I Canela

Publications and source records attributed to E I Canela.

At least 19 recordsLinked to original sources
PubMed1992

A model for adenosine transport and metabolism.

1. A model is presented for adenosine transport and metabolism in different steady states. The model considers steady-state equations for metabolic enzymes based on information from the literature on their kinetic behaviour. 2. Assuming that extracellular adenosine and inosine are translocated by three transporters, we have devised rate equations for these nucleoside transporters which are valid when both nucleosides are present. Since the Na(+)-independent transporter can either incorporate nucleosides into the cell or release them, various conditions have been simulated in which inosine was either incorporated or released. 3. Control analyses are reported which show that the fluxes towards intracellular adenine nucleosides are controlled by ecto-5'-nucleotidase in some circumstances and by the nucleoside transporters in others. The nucleoside transporter is responsible for five fluxes (two Na+ dependent adenosine transport mechanisms, a Na(+)-dependent inosine transport, a Na(+)-independent adenosine transport and a Na(+)-independent inosine influx or efflux) but the control is not always positive for all these fluxes. The control patterns of these five fluxes indicate that, in the presence of extracellular adenosine and inosine, the intracellular metabolism of adenine derivatives would be highly dependent on the extracellular and intracellular concentrations of both nucleosides, on the ectoenzymes (5'-nucleotidase and adenosine deaminase) and on the transporter. 4. Predictions of the model were examined. The results indicate that a change in one independent variable (extracellular AMP concentration) makes the system evolve towards a new steady state which is far from the initial one and has a different control pattern. In contrast, simulation of inhibition of the carriers produces only slight modification of the fluxes since the concentrations of the metabolites change to counteract the effect. Thus, for instance, a 50% inhibition of the three carriers does not affect the flux towards intracellular adenine nucleotides. Finally, our model has confirmed that the evolution of the concentration of extracellular adenosine, when an increase in extracellular AMP is produced, agrees with the behaviour expected for a neurohormone.

Adenosine↗
PubMed1992

Optimal association-saturation procedure for estimating association and dissociation rate parameters in receptor studies. Application to solubilized A1 adenosine receptors.

A method of obtaining estimates of the maximum binding and association and dissociation rate constants for a receptor-ligand interaction is described. This new procedure, the association-saturation method, is based on an exact mathematical equation which defines the model without simplifications. The method proposed is readily applicable to any system consisting of one ligand and one receptor. With only four determinations, each involving one ligand concentration and an association time, it is possible to determine the number of specific binding sites, the dissociation and association rate constants and the equilibrium dissociation constant with accuracy. No dissociation curve is required, and only one point of the association curve for each ligand concentration is necessary. In addition to the higher confidence in the estimates of the parameter values obtained, this method leads to important savings in radiolabelled compounds and experimental time. The results were compared with those obtained with standard association-dissociation curves and saturation isotherms. The optimum number of replicates of each experimental point to obtain reliable estimates of different parameters is discussed on the basis of simulation studies. The performance of the procedure is analysed by means of association-saturation experiments with the agonist [adenine-2,8-3H,ethyl-2-3H]N6-phenylisopropyladenosine and solubilized A1 adenosine receptors from pig brain cortex.

Animals↗
PubMed1992

Modulation of adenosine agonist [3H]N6-(R)-phenylisopropyladenosine binding to pig brain cortical membranes by changes of membrane fluidity and of medium physicochemical characteristics.

The binding of [3H]N6-(R)-phenylisopropyladenosine ([3H]R-PIA) to pig brain cortical membranes was investigated as a function of membrane fluidity and of the physicochemical characteristics of the medium. The two affinity states of the A1 adenosine receptor behave differently in the experiments performed. Increases (up to 6.7 poises) or reduction (up to 1.3 poises) in membrane microviscosity did not lead to significant variation of equilibrium parameter values except for a slight modification of the low-affinity Kd value at 1.3 poises. Addition of cholesterol to raise the microviscosity up to 9.0 poises led to a marked decrease of binding to the high-affinity state without modifying the Kd. Furthermore, the Kd value for the low-affinity state rose markedly. Addition of sucrose, which modifies the conductance, viscosity and density of the aqueous medium, did not lead to significant changes when used at a concentration of 0.25 M, either in isolation or in radioligand binding assay medium. The presence of 0.32 M sucrose in the isolation medium led to a 30% fall in the total binding without affecting the distribution and Kd values for either affinity state very much. However, the presence of greater than or equal to 0.32 M sucrose in the binding assay medium produced the disappearance of the low-affinity states and the appearance of high-affinity states; only one affinity state was found to have a somewhat increased Kd value. On the other hand, increases of the medium conductance did not lead to the disappearance of the low-affinity state although some decrease of the binding was observed at high Tris concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The molybdoenzymes xanthine oxidase and aldehyde oxidase contain fast- and slow-DTNB reacting sulphydryl groups.

The reactivities with an excess of 5-5'-dithiobis (2-nitrobenzoic) acid (DTNB) of sulphydryl residues present in xanthine oxidase and aldehyde oxidase were studied and compared. The results show that two classes of sulphydryl groups with quite different reactivities exist in both enzymes either native or denatured. Some of the available sulphydryl residues thus react instantaneously with the DTNB, whereas the others react very slowly following pseudo-first-order kinetics. The number of sulphydryl residues of each class and the rate constant of slowly reacting groups are, respectively, 1.7 and 0.8 in native xanthine oxidase and 1.6 and 1.7 in native aldehyde oxidase. In denatured enzymes, the number of fast- and slow-reacting sulphydryl residues obtained are, respectively, 13.9 and 7.9 in xanthine oxidase and 5.7 and 5.4 in aldehyde oxidase. Analogously, the rate constant for the slowly reacting groups is similar for the two native enzymes, but in denatured aldehyde oxidase it is double that of denatured xanthine oxidase.

Aldehyde Oxidase↗
PubMed1992

The adenosine receptors present on the plasma membrane of chromaffin cells are of the A2b subtype.

The adenosine receptors in the plasma membrane of chromaffin cells from bovine adrenal medulla were characterized. The presence of A1 receptors was discounted owing to the absence of R-[3H]phenylisopropyladenosine (R-PIA) and [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) binding. The binding of the specific A2a ligand CGS-21680 was low. In contrast, the binding of 5'-(N-[3H]-ethylcarboxamidoadenosine ([3H]NECA) was relatively high (1.7 pmol/mg of protein at a ligand concentration up to 90 nM). This binding did not correspond to non-adenosine receptor NECA binding sites because the specific [3H]-NECA binding was similar when unlabeled adenosine, NECA, or R-PIA was used to measure the nonspecific binding. The rank order of potency of different ligands for the displacement of specific [3H]NECA binding was DPCPX greater than NECA greater than chloroadenosine greater than R-PIA greater than theophylline = CGS-21680. These results indicate that the receptors present on the plasma membrane of chromaffin cells are exclusively of the A2b subtype.

2-Chloroadenosine↗
PubMed1992

Characterization of adenosine receptors in brush-border membranes from pig kidney.

1. The adenosine receptors from pig kidney proximal tubules have been studied in membrane vesicle preparations derived from either luminal (brush-border membranes-BBM-) or basolateral (BL) sides. There was a substantial amount of A2-like NECA binding in both preparations, but the A1 subtype of adenosine receptors was not found in either BBM or BL membranes. The use of [3H]-CGS21680 which is a more specific ligand for A2a receptors revealed true adenosine receptors in the BBM. 2. The kinetic parameters for [3H]-CGS21680 binding to pig renal BBM were: Bmax = 1.48 pmol mg-1 protein and Kd = 150 nM. In the presence of Gpp(NH)p the affinity decreased (Kd = 220 nM), whereas the addition of Mg2+ induced a marked increase in affinity (Kd = 83 nM). These equilibrium constants are higher than those found for the A2a adenosine receptors present in pig brain striatal membranes (Kd = 12 nM), and are close to those found in rat renal BBM (Kd = 90 nM). 3. The order of potency of agonist and antagonists was not consistent with the presence of either A1 or A2 receptors, but it was very similar to the agonist order of potency for the A3 receptor subtype. Furthermore, the blockade of the [3H]-CGS21680 binding by both cholera and pertussis toxin further supports the view that the subtypes present in BBM are neither A1 nor A2. 4. Overall the results suggest the presence in BBM of an A3 receptor, or of a new subtype of adenosine receptor, which is linked to G proteins sensitive to both cholera and pertussis toxins.

Adenosine↗
PubMed1991

N-ethylmaleimide affects agonist binding to A1 adenosine receptors differently in the presence than in the absence of ligand.

The effect of sulphydryl reagents (N-ethylmaleimide-NEM-4- hydroxymercuriobenzoate-HMB- and 5-5'-dithio-bis-2-nitrobenzoate-DTNB-) on agonist and antagonist binding to A1 adenosine receptors from pig brain was studied. The action of the mercurial agent HMB was found to be strong and seemed to be nonspecific. The effects of either NEM or DTNB were milder and more specific. The characterization of the agonist binding in membranes pretreated with moderate concentrations of DTNB and NEM led to reduced affinities for both high- and low-affinity sites without marked modifications of maximal binding or of proportion of affinity states. These results for NEM are surprising since the compound is usually used to mimick the effects of Gpp(NH)p, i.e. to shift high-affinity states to low-affinity states. It was found that this Gpp(NH)p-like effect of NEM is only possible when the compound is included in the assay medium. Similarly, Gpp(NH)p produces the uncoupling of the receptor molecule from G protein if included in the assay medium. Thus, membranes pretreated with Gpp(NH)p exhibited both affinity states and with similar equilibrium binding parameter values to those of the crude membranes.

Adenine↗
PubMed1991

The binding of [3H]R-PIA to A1 adenosine receptors produces a conversion of the high- to the low-affinity state.

Kinetic evidence for negative cooperativity on the binding of [3H]R-PIA to A1 adenosine receptors was obtained from dissociation experiments at different ligand concentrations and from the equilibrium isotherm. The dissociation curves indicate that there is an apparent ligand-induced transformation of high- to low-affinity states of the receptor. At concentrations of 18.2 nM R-PIA or higher there was only found the low-affinity state of the receptor. In view of these results equilibrium binding data were analyzed by the usual two-state model (assuming that there is an interconversion between them) and by the negative cooperativity model employing the Hill equation.

Animals↗
PubMed1991

Biochemical systems theory: increasing predictive power by using second-order derivatives measurements.

Models based on the power-law formalism provide a useful tool for analyzing metabolic systems. Within this methodology, the S-system variant furnishes the best strategy. In this paper we explore an extension of this formalism by considering second-order derivative terms of the Taylor series which the power-law is based upon. Results show that the S-system equations which include second-order Taylor coefficients give better accuracy in predicting the response of the system to a perturbation. Hence, models based on this new approach could provide a useful tool for quantitative purposes if one is able to measure the required derivatives experimentally. In particular we show the utility of this approach when it comes to discriminating between two mechanisms that are equivalent in the S-system a representation based on first-order coefficients. However, the loss of analytical tractability is a serious disadvantage for using this approach as a general tool for studying metabolic systems.

Kinetics↗
PubMed1991

Fitting integrated enzyme rate equations to progress curves with the use of a weighting matrix.

A method is presented for fitting the pairs of values product formed-time taken from progress curves to the integrated rate equation. The procedure is applied to the estimation of the kinetic parameters of the adenosine deaminase system. Simulation studies demonstrate the capabilities of this strategy. A copy of the FORTRAN77 program used can be obtained from the authors by request.

Adenosine Deaminase↗
PubMed1991

Control analysis of transition times. Extension of analysis and matrix method.

The present theoretical basis of Control Analysis is extended with the definition of Transition Time Response Coefficients. Some new relationships between local and global coefficients defined in Control Analysis are presented. These relationships are in the form of matrix products constructed in a priori form. The use of these straightforward relationships is shown in an exemplary application corresponding to an experimental system consisting of the glycolytic degradation from glucose to glyceraldehyde-3-phosphate.

Animals↗
PubMed1991

Effect of phospholipases and proteases on the [3H]N6-(R)-phenylisopropyladenosine ([3H]R-PIA) binding to A1 adenosine receptors from pig cerebral cortex.

The effect of phospholipases and proteases on the membrane-bound and solubilized A1 adenosine receptor has been studied. Phospholipids modulate the [3H]N6-(R)-phenylisopropyladenosine binding to A1 adenosine receptors in crude membranes and in soluble preparations, because changes in the phospholipid environment decrease both the binding capacity and the affinity for the ligand. It has become clear that 1) there is co-solubilization of receptor and phospholipids; 2) the phospholipid requirements are different for the coupled and the uncoupled receptor; 3) a net charge in the polar head produced by phospholipase D prevents the agonist binding to the receptor-G protein complex; alternatively, when the whole polar head is removed by phospholipase C the uncoupled receptor is altered; and 4) the protease action upon the receptor suggests that receptor coupled to G protein is more protected by the membrane than the uncoupled receptor. In kinetic experiments performed on membranes it was demonstrated that phospholipase C and trypsin increased the Kd value of the high-affinity state by modifying both k1 and k-1. In contrast they only modified the dissociation constant of the low-affinity state. In conclusion it should be noted that phospholipids play a key role for the binding of R-PIA to A1 adenosine receptor. Also, a different disposition within the membrane of the coupled and uncoupled receptor is encountered.

Animals↗
PubMed1991

An improved purification procedure for sulfite oxidase from bovine liver.

Sulfite oxidase (Sulfite:O2 oxidoreductase, EC 1.8.3.1) has been purified 2,440-fold from bovine liver. The procedure developed was used to isolate the enzyme from 1,000 g of liver and permitted the rapid isolation of enzyme with a very high specific activity (40,405 mU/mg). The enzyme preparations obtained have been characterized by electrophoretic and spectrophotometric analysis and the molecular mass and the Stokes radius of the enzyme have been determined.

Acetone↗
PubMed1991

Adenosine receptors in myelin fractions and subfractions: the effect of the agonist (R)-phenylisopropyladenosine on myelin membrane microviscosity.

In this article the existence of A1 adenosine receptors and the absence of A2 adenosine receptors in myelin membranes purified from pig brain white matter are demonstrated. The characterization of (R)-[3H]phenylisopropyladenosine ([3H]R-PIA) binding to purified myelin fractions was performed. The distribution of high- and low-affinity species of the A1 adenosine receptor was different in heavy, medium, and light myelin. The fluidity of myelin subfractions and of pig brain cortical membranes was estimated; the microviscosity of heavy myelin (5.4 poises) and of cortical membranes (5.1 poises) was similar and less than that of medium (7.8 poises) and light (8.2 poises) myelin. It was also demonstrated that the agonist R-PIA modifies the microviscosity of myelin membranes and that the degree of modification depends on the fluidity of the membrane assayed. These results suggest that adenosine receptors may have an important role in the functionality of myelin membranes.

Animals↗
PubMed1990

Preparative purification of adenosine deaminase from human erythrocytes by affinity chromatography.

The purification of adenosine deaminase from human erythrocytes is reported. By means of classical procedures and by using affinity chromatography as the last step, the enzyme is purified 760,000-fold with a yield of 32%. The affinity resin is composed of purine riboside (nebularine) linked to Sepharose CL6B. Since the compound has no leaving group at the C-6 position the affinity gel is stable and the chromatography can be repeated several times (up to fifteen times in eight months). Purine riboside was chosen because its potency as a reversible inhibitor of adenosine deaminase is greater than that of inosine (a low-affinity inhibitor), but lower than that of erythro-9-(2-hydroxy-3-nonyl)adenine (a high-affinity inhibitor).

Adenosine Deaminase↗
PubMed1990

Control analysis of systems having two steps catalyzed by the same protein molecule in unbranched chains.

The analysis of the control of a metabolic pathway having an enzyme catalyzing two different reactions (or a protein displaying two different activities) has been performed. For such systems although the summation theorems are valid, the flux and concentration connectivity theorems of the metabolic control analysis are not valid. Another general relationship of control analysis is shown to be more widely obeyed and holds in these systems. An exemplary case, where the enzyme catalyzes two irreversible reactions, demonstrates that the level of one internal intermediate is constant, i.e. it does not depend upon the independent variables of the system.

Catalysis↗