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F Alvarado

Publications and source records attributed to F Alvarado.

At least 37 records · Page 2Linked to original sources
PubMed1992

Trans-potassium effects on the chloride/proton symporter activity of guinea-pig ileal brush-border membrane vesicles.

To investigate the inhibitory effect of trans potassium on the Cl-/H+ symporter activity of brush-border membrane vesicles from guinea pig ileum, we measured both 36Cl uptake and, by the pyranine fluorescence method, proton fluxes, in the presence of appropriate H+ and K+ gradients. In the absence of valinomycin, a time-dependent inhibitory effect of chloride uptake by trans K+ was demonstrated. This inhibition was independent of the presence or absence of any K+ gradient. Electrical effects cannot be involved to explain these inhibitions because the intrinsic permeability of these vesicles to Cl- and K+ is negligibly small. Rather, our results show that, in the absence of valinomycin, the inhibitory effect of intravesicular K+ involves an acceleration of the rate of dissipation of the proton gradient through an electroneutral exchange of trans K+ for cis H+, catalyzed by the K+/H+ antiporter also present in these membranes. Valinomycin can further accelerate the rate of pH gradient dissipation by facilitating an electrically-coupled exchange between K+ and H+. To evaluate the apparent rate of pH-dissipating, downhill proton influx, we measured chloride uptake by vesicles preincubated in the presence of alkaline-inside pH gradients (pHout/pHin = 5.0/7.5), charged or not with K+. In the absence of intravesicular K+, proton influx exhibited monoexponential kinetics with a time constant k = 11 s-1. Presence of 100 mM K+ within the vesicles significantly increased the rate of pH gradient dissipation which, furthermore, became bi-exponential and revealed the appearance of an additional, faster proton influx component with k = 71 s-1. This new component we interpret as representing the sum of the electroneutral and the electrically-coupled exchange of trans K+ for cis H+, mentioned above. Finally, by using the pH-sensitive fluorophore, pyranine, we demonstrate that, independent of the absence or presence of a pH gradient, either vesicle acidification or alkalinisation can be generated by adding, respectively, Cl- or K+ to the extravesicular medium. Such results confirm the independent existence of both Cl-/H+ symporter and K+/H+ antiporter activities in our vesicle preparations, the relative activity of the former being larger under the conditions of the present experiments. The possible interplay of these two proton-transfer mechanisms in the regulation of the intracellular pH is discussed.

Animals↗
PubMed1992

Chloride transport in control and cystic fibrosis human skin fibroblast membrane vesicles.

Plasma membrane vesicles were isolated from either cystic fibrosis (CF) or non-CF cultured fibroblasts derived from skin biopsies of either foetus, child or adolescent human donors. The total membrane yield was essentially identical for either CF or control membranes. By using a rapid filtration technique, 36Cl uptake by these vesicles was quantitated in the absence and presence of alkali-metal ion-, electrical- and/or pH gradients. In the absence of a pH gradient (pHout = pHin = 7.5), Cl uptake took place downhill in both cases. Either cis K+, cis Na+ or an equimolar mixture of cis Na+ plus K+ caused Cl uptake activation. In the presence of an alkaline-inside pH gradient (pHout/pHin = 5.5/7.5), Cl uptake exhibited an apparent overshoot independently of the presence or absence of any metal-ion gradient. The observed potassium-, sodium- and proton-dependent Cl influx rates were all unaffected by voltage clamping, indicating the existence in these vesicles of electroneutral symport systems of the type Cl-/H+, Cl-/K+ and/or Cl-/Na+; but not 2 Cl-/Na+/K+. In the presence of an inward-directed K+ gradient, valinomycin further increased Cl uptake, both in the presence and in the absence of a pH gradient, indicating the presence of a rheogenic Cl uniport. In absolute quantitative terms, the two different modes (rheogenic and electroneutral) of Cl transport evinced in these vesicles were about 45% lower in CF than in control skin fibroblasts. However, qualitatively, there was no difference between normal and CF cells. The evidence obtained indicates that the CF defect, which is expressed in fibroblast plasma membranes, does not affect specifically either the rheogenic or the electroneutral Cl transport systems. Rather, the CF cells appear to give a smaller yield of closed, functional vesicles, reflected by a significantly smaller apparent intravesicular volume. Because it also affects the transport of D-glucose and L-alanine, this anomaly could be the consequence of a generalized membrane defect characterizing CF fibroblasts.

Adolescent↗
PubMed1992

A four-proton-families model for pH-dependent enzyme activation: application to intestinal brush border sucrase.

Current concepts of pH-dependent enzyme function are expanded to consider enzymes with up to four key proton families. In an earlier paper the authors extended classical theory to explain the existence, in the acid ionization reaction, of two functionally distinct, V and K, proton families, exemplified by the 1988 sucrase three-proton-families model of Vasseur et al. They now propose that enzymes having two distinguishable proton families at each side of the pH-activity curves exist in nature although there is no previously published evidence of their existence. The resulting, more general, four-proton-families model is treated as a useful framework from which submodels can be derived by simplification, the simplest being the 1911 linear model of Michaelis and Davidsohn, which took into account two proton families out of the theoretical maximum of four proposed here. It is shown that whether a three-proton-families or a four-proton-families model can explain sucrase better is not merely a question of theory but also involves the practical question of having enough data, at each side of the pH spectrum, to permit making an unequivocal choice between the two alternatives. The paper concludes with a discussion of substrate-induced pK shifts according to both models.

Animals↗
PubMed1992

Safety and immunogenicity of the synthetic malaria vaccine SPf66 in a large field trial.

In the first field trial with synthetic malaria vaccine SPf66 in a large population naturally exposed to malaria, 9957 persons greater than 1 year old and residing on the Colombian Pacific coast received three doses of the vaccine. To evaluate vaccine safety, clinical observations were made 30 min and 48 h after each immunization. There were no adverse reactions in 95.7% of cases. In the 4.3% of cases with adverse reactions, local induration and erythema were the most frequent. In a randomly selected group of vaccinees, anti-SPf66 antibody titers were measured after the third dose: 93% of the vaccinees raised antibodies to SPf66. Among these, 55% had titers greater than 1:1600. These results demonstrate the safety and immunogenicity of the SPf66 vaccine in a large field trial.

Adolescent↗
PubMed1991

Carrier-mediated L-lactate transport in brush-border membrane vesicles from rat placenta during late gestation.

The mechanism for L-lactate transport across microvillous membrane vesicles prepared from rat placenta was examined. Uptake of L-lactate into these vesicles was mainly the result of transport into the intravesicular (osmotically active) space. The initial rate of L-lactate uptake was not affected by the presence of an inward gradient of either Na+ or K+. In the presence of an inward-directed proton gradient, L-lactate uptake was markedly stimulated, accumulating at concentrations 6-7-fold higher than the equilibrium. Lower transmembrane pH gradients were associated with slower initial uptakes and smaller overshoots. L-Lactate uptake determined under an inside-directed pH gradient was strongly inhibited by p-chloromercuriphenylsulphonic acid, a protein-thiol oxidizing agent. L-Lactate uptake was: (1) saturable as a function of the concentration of L-lactate, (2) inhibited by monocarboxylic acids such as pyruvate, D-lactate, beta-hydroxybutyrate and alpha-cyano-4-hydroxycinnamic acid, and (3) temperature-dependent. When present inside the vesicles, L-lactate, pyruvate and beta-hydroxybutyrate caused trans-stimulation of L-lactate uptake both in the presence and in the absence of an inside-directed pH gradient, indicating that L-lactate transport is a reversible process that can be shared by other monocarboxylic acids. There were no significant changes in maximal initial rate or in the kinetic parameters of L-lactate transport during the last 3 days of gestation.

4-Chloromercuribenzenesulfonate↗
PubMed1990

pH-dependent inhibitory effects of tris and lithium ion on intestinal brush-border sucrase.

Tris and two of its hydroxylated amine analogs were examined in a metal-free, universal n-butylamine buffer, for their interaction with intestinal brush border sucrase. Our recent three-proton-families model (Vasseur, van Melle, Frangne and Alvarado (1988) Biochem. J., 251, 667-675) has provided the sucrase pK values necessary to interpret the present work. At pH 5.2, 2-amino-2-methyl-l-propanol (PM) causes activation whereas Tris has a concentration-dependent biphasic effect, first causing activation, then fully competitive inhibition. The amine species causing activation is the protonated, cationic form. The difference between the two amines is related to the fact that Tris has a much lower pKa value than PM (respectively, 8.2 and 9.8). Even at pH 5.2, Tris (but not PM) exists as a significant proportion of the free base which, by inhibiting the enzyme fully competitively, overshadows the activating effect of the cationic, protonated amine. Above pH 6.8, both Tris and PM act as fully competitive inhibitors. These inhibitions increase monotonically between pH 6.5 and 8.0 but, above pH 8, inhibition by 2.5 mM Tris tends to diminish whereas inhibition by 40 mM PM increases abruptly to be essentially complete at pH 9.3 and above. As pH increases from 7.6 to 9.0, the apparent affinity of the free amine bases decreases whereas that of the cationic, protonated amines, increases. In this way, the protonated amines replace their corresponding free bases as the most potent inhibitors at high pH. The pH-dependent inhibition by 300 mM Li+ is essentially complete at pH 8, independent of the presence or absence of either 2.5 mM Tris or 40 mM PM. Even at pH 7.6, an excess (300 mM) of Li+ causes significant increases in the apparent Ki value of each Tris, PD (2-amino-2-methyl-1-3-propanediol) and PM, suggesting the possibility of a relation between the effects of Li+ and those of the hydroxylated amines which in fact are mutually exclusive inhibitors. The inhibitory results are interpreted in terms of a mechanistic model in which the free bases bind at two distinct sites in the enzyme's active center. Binding at the glucosyl sub-site occurs through the amine's free hydroxyl groups. This positioning facilitates the interaction between the lone electron pair of the deprotonated amino group with a proton donor in the enzyme's active center, characterized by a pK0 around 8.1. When this same group deprotonates, then the protonated amines acting as proton donors replace the free bases as the species giving fully competitive inhibition of sucrase.

Animals↗
PubMed1989

Electroneutral, HCO3(-)-independent, pH gradient-dependent uphill transport of Cl- by ileal brush-border membrane vesicles. Possible role in the pathogenesis of chloridorrhea.

By applying a rapid filtration technique to isolated brush border membrane vesicles from guinea pig ileum, 36Cl uptake was quantified in the presence and absence of electrical, pH and alkali-metal ion gradients. A mixture of 20 mM-Hepes and 40 mM-citric acid, adjusted to the desired pH with Tris base, was found to be the most suitable buffer. Malate and Mes could be used to replace the citrate, but succinate, acetate and maleate proved to be unsuitable. In the absence of a pH gradient (pHout:pHin = 7.5:7.5), Cl- uptake increased slightly when an inside-positive membrane potential was applied, but uphill transport was never observed. A pH gradient (pHout:pHin = 5.0:7.5) induced both a 400% increase in the initial Cl- influx rate and a long-lasting (20 to 300 s) overshoot, indicating that a proton gradient can furnish the driving force for uphill Cl- transport. Under pH gradient conditions, initial Cl- entry rates had the following characteristics. (1) They were unaffected by cis-Na+ and/or -K+, indicating the absence of Cl-/K+, Cl-/Na+ or Cl-/K+/Na+ symport activity. (2) Inhibition by 20-100 mM-trans-Na+ and/or -K+ occurred, independent of the existence of an ion gradient. (3) Cl- entry was practically unaffected by short-circuiting the membrane potential with equilibrated potassium and valinomycin. (4) Carbonyl cyanide m-chlorophenylhydrazone was strongly inhibitory and so, to a lesser extent, was 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid [(SITS)], independent of the sign and size of the membrane potential. (5) Cl- entry was negligibly increased (less than 30%) by either trans-Cl- or -HCO3-, indicating the absence of an obligatory Cl-/anion antiport activity. In contrast, the height of the overshoot at 60 s was increased by trans-Cl-, indicating time-dependent inhibition of 36Cl efflux. That competitive inhibition of 36Cl fluxes by anions is involved here is supported by initial influx rate experiments demonstrating: (1) the saturability of Cl- influx, which was found to exhibit Michaelis-Menten kinetics; and (2) competitive inhibition of influx by cis-Cl- and -Br-. Quantitatively, the conclusion is warranted that over 85% of the total initial Cl- uptake energized by a pH gradient involves an electroneutral Cl-/H+ symporter or its physicochemical equivalent, a Cl-/OH- antiporter, exhibiting little Cl- uniport and either Cl-/Cl- or Cl-/HCO3- antiport activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Substrate- and alkali-metal-ion-induced pK shifts in intestinal brush-border sucrase, according to the three-protons model.

To define adequately enzyme activation/inhibition mechanisms as a function of pH, it is necessary to characterize the effector-induced pK shifts on both the free enzyme and on the enzyme-substrate complex. On the basis of our recent three-protons model for sucrase [Vasseur, van Melle, Frangne & Alvarado (1988) Biochem. J. 251, 667-675], we show how the 'fundamental' pK values, deduced from the classical double-logarithmic transformations, are insufficient to generate the required information. This insufficiency derives from the fact that, for sucrase, the acid ionization constant, K1, is a molecular constant that involves complex, V-type plus K-type, activatory and inhibitory kinetic effects. As a consequence, substrate-induced pK shifts cannot be interpreted correctly only by using the fundamental pK approach, because an unequal number of key protons is involved, depending on whether the free enzyme or the enzyme-substrate complex is considered. We demonstrate how this problem can be solved by using the 'theoretical' pK values, derived from the reciprocals of the Michaelis pH functions, i.e. Cha's fractional concentration factors. The procedure we propose, which is general, has the advantage of yielding all the macroscopic pK values for any given model, as calculated from the microscopic pK values. Furthermore, it permits predicting pK shifts as a function of [S] and/or [A] (where S is the substrate and A is the allosteric modifier), an objective that cannot be attained by using the double-logarithmic plot approach. Finally, we describe the relation existing between the fundamental and the theoretical pK values.

Animals↗
PubMed1989

A method for writing enzyme rate equations: application to the estimation of the number and size of key proton families.

We develop a method to derive the rate equation for enzyme models that include pH-dependent activation. Our presentation is based on a kinetic model recently described for sucrase, the three-key-proton model of Vasseur and coworkers, which considers the existence, in the acid ionization reaction, of two functionally distinct prototropic groups, respectively responsible for either V-type or K-type kinetic effects. In contrast, as concerns the basic ionization reaction, the model conforms to classical concepts of pH-dependent activation, whereby a single proton participates in either V-type or K-type effects but not in both at the same time. Enzymes with more than three key protons have been described, indicating that, rather than isolated protons, groups of protons should be considered, and therefore the model can be better described as a three-proton-family model, where a proton family is defined as one or several protons that are gained or lost as a block and perform the same kinetic function. The resulting model is treated here as a useful framework upon which other models can be built. To facilitate the writing of the rate equations, we define two new entities: (1) intralevel coefficients, which describe the various combinations of the enzyme with either the substrate(s), the allosteric effector(s), or both at a given protonation level, and (2) interlevel coefficients, which describe the interplay between the various protonation levels. The resulting rate equation can be used in a global fit procedure permitting in a single computer run the estimation of (1) the entire set of dissociation and microscopic ionization constants of the model, (2) the number and kinetic function of proton families characterizing the enzyme under consideration, and (3) the number of key protons constituting each family, which is derived from the derivatives of the kinetic parameters, Vm/Km, Vm, and Km.

Enzymes↗
PubMed1988

Alkali-metal-ion- and H+-dependent activation and/or inhibition of intestinal brush-border sucrase. A model involving three functionally distinct key prototropic groups.

For rabbit intestinal brush-border sucrase, a model based on classical Michaelis-Dixon theory cannot fully explain the peculiar antagonistic relationship existing between the substrate and one key proton, Hx, which at acid pH values behaves as a fully competitive inhibitor. In the same pH range, a second proton, Hy, is responsible for changes in catalytic activity and behaves as a mixed-type partially non-competitive inhibitor [Vasseur, Tellier & Alvarado (1982) Arch. Biochem. Biophys. 218, 263-274]. Although involved in the same ionization reaction, these two protons have different kinetic functions, since they are responsible for affinity-type and capacity-type effects respectively. Depending on whether Hx is bound or not, we postulate the enzyme to alternate between two distinct forms differing in their binding properties. The alkali-metal ions Na+ and Li+ have a concentration-dependent biphasic effect on this equilibrium. At low concentrations they facilitate the release of Hx, resulting in K-type activation. At higher concentrations they favour enzyme reprotonation, causing K-type inhibition. On the basic side of the pH spectrum, our results confirm the existence of separate non-competitive effects of the alkali-metal ions, particularly Li+ [Alvarado & Mahmood (1979) J. Biol. Chem. 254, 9534-9541]. To explain the molecular mechanisms underlying the alkali-metal-ion- and H+-dependent effects, we formulate a sucrase model, the three-protons model, in which the acid and basic ionization constants involve respectively two and one key prototropic groups that are functionally distinguishable. A global iterative fit of the relevant general equation to our whole set of data has permitted us to estimate the numerical value of each of the constants constituting the model.

Animals↗
PubMed1988

Independent modulation by food supply of two distinct sodium-activated D-glucose transport systems in the guinea pig jejunal brush-border membrane.

D-glucose transport across the intestinal brush-border membrane involves two transport systems designated here as systems 1 and 2. Kinetic properties for both D-glucose and methyl alpha-D-glucopyranoside transport were measured at 35 degrees C by using brush-border membrane vesicles prepared from either control, fasted (48 hr), or semistarved (10 days) animals. The results show the following: (i) The sugar influx rate by simple diffusion was identical under either altered condition. (ii) Semistarvation stimulated D-glucose uptake by system 2 (both its Vmax and Km increased), whereas system 1 was untouched. (iii) Fasting increased the capacity of system 1 without affecting either Km of system 1 or Vmax and Km of system 2. The effect of fasting on Vmax of system 1 cannot be attributed to indirect effects from changes in ionic permeability because the kinetic difference between control and fasted animals persisted when the membrane potential was short-circuited with equilibrated K+ and valinomycin. This work provides further evidence for the existence of two distinct sodium-activated D-glucose transport systems in the intestinal brush-border membrane, which adapt independently to either semistarvation or fasting.

Animals↗
PubMed1987

Characterization of the D-glucose/Na+ cotransport system in the intestinal brush-border membrane by using the specific substrate, methyl alpha-D-glucopyranoside.

By using isolated membrane vesicles, we have investigated the tenet that D-glucose transport across the intestinal brush-border membrane involves at least two distinct, Na+-activated agencies (D-glucose transport systems S-1 and S-2), only one of which (S-1) can use methyl alpha-D-glucopyranoside (methyl alpha-glucoside) as a substrate. Our results with this glucose analogue show that: (a) As a function of time, methyl alpha-glucoside uptake exhibits a typical overshoot, similar to but smaller than that given by D-glucose with the same vesicle batch. (b) Nonlinear regression analysis of substrate-saturation curves reveals that, contrary to D-glucose, methyl alpha-glucoside transport involves a single transport system which we have identified as S-1. (c) Methyl alpha-glucoside exhibits an apparent affinity (defined as the reciprocal of Km) 4-times smaller than that of D-glucose for S-1 (Km(Dglucose) = 0.5 mM; Km(methyl alpha-glucoside) = 2 mM). However, methyl alpha-glucoside has a Vmax (230 pmol/mg protein per s) identical to that characterizing D-glucose transport by this system. (d) In the absence of Na+, methyl alpha-glucoside uptake is indistinguishable from simple diffusion, confirming that Na+ is an obligatory activator of S-1. (e) Phlorizin behaves as a fully competitive inhibitor of methyl alpha-glucoside transport (Ki = 18 microM), again indicating that S-1 is involved. (f) Neither phloretin nor cytochalasin B affects methyl alpha-glucoside uptake. We conclude that methyl alpha-glucoside is a substrate specific for S-1, which permits study of the properties of this system without interference by substrate fluxes taking place through any other channel.

Animals↗
PubMed1987

Kinetic evidence for separate systems in transport of D- and L-methionine by rat small intestine.

The kinetics of D- and L-methionine uptake by rings of everted intestine in vitro are consistent with a saturable Michaelis-Menten component (Km = 11.7 and 1.7 mM; Vmax = 0.53 and 0.74 mumol X g-1 X min-1 for D- and L-methionine, respectively) plus a linear, diffusional one. All the data could be fit with a diffusion constant (Kd = 3.2 microliters X g-1 X min-1), which was essentially the same, independent of whether it was estimated by iteration or by using the extracellular marker, inulin. Similar results were obtained from in vivo perfusion experiments, except that the diffusional term was negligible. D-Methionine was found to inhibit L-methionine uptake by intestinal rings according to fully noncompetitive kinetics (Ki = 45 mM). Another set of experiments with jejunal brush-border membrane vesicles showed that D-methionine uptake is dependent on a Na+ gradient and is significantly inhibited by L-methionine and L-proline, but not by beta-alanine and alpha-methylaminoisobutyric acid. Our results indicate that, in rat jejunum, D-methionine is taken up through a Na+-dependent pathway distinct from the neutral amino acid (L-methionine) carrier and from the imino acid (L-proline, alpha-methylaminoisobutyric acid, beta-alanine) carrier.

Animals↗