PubMed Health⌕ Search

Biomedical subjects

M Vasseur

Publications and source records attributed to M Vasseur.

At least 37 records · Page 2Linked to original sources
PubMed1993

Buffer-dependent pH sensitivity of the fluorescent chloride-indicator dye SPQ.

The fluorescence intensity of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) in an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) 2-(N-morpholino)ethanesulfonic acid (MES)-Tris(hydroxymethyl)aminomethane buffer, pH 7.0, decreased as a function of Cl- concentration and/or gluconate concentration, as expected. Contrary to expectation, however, the fluorescence intensity progressively increased as the pH decreased, independently of the presence of gluconate. Consequently, the modulation of SPQ fluorescence by commonly used buffers was investigated as a function of pH. Titration curves demonstrated SPQ quenching and yielded pK values characteristic of each buffer. from here, pH-independent Stern-Volmer constants, KQbase, were calculated for each of the morpholine derivatives, MES and 3-(N-morpholino)-2-hydroxypropanesulfonic acid. In contrast, HEPES and piperazine-N,N'-bis(2-ethanesulfonic acid), which are piperazine derivatives, exhibited an additional pH-independent "molecular" quenching constant KmQ throughout the pH range 3-10. To study chloride fluxes, therefore, what counts is the apparent Cl-Stern-Volmer constant KappCl, which is a function of both pH and buffer composition. Equations describing these relationships are presented. In conclusion, unless both pH and the buffer composition are taken into account, SPQ is unsuitable for studying the concomitant transmembrane fluxes of Cl- and H+.

Alkanesulfonates↗
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↗
PubMed1991

Self-cleavage of a 71 nucleotide-long ribozyme derived from hepatitis delta virus genomic RNA.

Self-cleavage efficiency of ribozymes derived from hepatitis delta virus (HDV) has been shown to depend on the RNA structure, which in turn may be determined by the length of the considered sequences. Here we describe the construction and functional analysis of a 71 nucleotide-long RNA genomic fragment, Rz71, which carries an 18 nucleotide deletion in a very stable GC-rich stem-loop (stem IV), predicted to be present in several computer-derived secondary structure models. Rz71 is able to undergo self-cleavage under non-denaturing conditions (the t1/2 of the reaction at 37 degrees C is 3 min). The deletion, however, is not neutral, since under the same conditions the non-deleted ribozyme cleaves to 50% in less than 15 sec. Therefore, stem-loop IV seems to play a structural role, not being directly involved in the catalytic reaction, but contributing to the correct positioning of the catalytic core of the HDV ribozyme. Rz71 is the smallest self-cleaving sub-fragment of HDV genomic RNA reported so far.

Base Sequence↗
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

Purification of the rabbit small intestinal sucrase-isomaltase complex: separation from other maltases.

The rabbit intestinal sucrase-isomaltase complex has been purified to homogeneity after solubilization with Triton X 100 followed by chromatography on DEAE Sepharose CL 6B and a second solubilization with papain. After hydrophobic chromatography on Octyl Sepharose CL 6B, separation from other contaminating maltases was achieved by gel filtration on Ultrogel ACA 22. The final enzyme was purified 390 fold, with a specific activity of about 10 units per mg protein.

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↗
PubMed1989

Posttranslational regulation of keratins: degradation of mouse and human keratins 18 and 8.

Human keratin 18 (K18) and keratin 8 (K8) and their mouse homologs, Endo B and Endo A, respectively, are expressed in adult mice primarily in a variety of simple epithelial cell types in which they are normally found in equal amounts within the intermediate filament cytoskeleton. Expression of K18 alone in mouse L cells or NIH 3T3 fibroblasts from either the gene or a cDNA expression vector results in K18 protein which is degraded relatively rapidly without the formation of filaments. A K8 cDNA containing all coding sequences was isolated and expressed in mouse fibroblasts either singly or in combination with K18. Immunoprecipitation of stably transfected L cells revealed that when K8 was expressed alone, it was degraded in a fashion similar to that seen previously for K18. However, expression of K8 in fibroblasts that also expressed K18 resulted in stabilization of both K18 and K8. Immunofluorescent staining revealed typical keratin filament organization in such cells. Thus, expression of a type I and a type II keratin was found to be both necessary and sufficient for formation of keratin filaments within fibroblasts. To determine whether a similar proteolytic system responsible for the degradation of K18 in fibroblasts also exists in simple epithelial cells which normally express a type I and a type II keratin, a mutant, truncated K18 protein missing the carboxy-terminal tail domain and a conserved region of the central, alpha-helical rod domain was expressed in mouse parietal endodermal cells. This resulted in destabilization of endogenous Endo A and Endo B and inhibition of the formation of typical keratin filament structures. Therefore, cells that normally express keratins contain a proteolytic system similar to that found in experimentally manipulated fibroblasts which degrades keratin proteins not found in their normal polymerized state.

Amino Acid Sequence↗
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

Nucleotide sequence of the human vimentin gene and regulation of its transcription in tissues and cultured cells.

We have previously reported the identification and isolation by mRNA selection/translation of a recombinant clone containing 80% of the human vimentin gene sequence [Lilienbaum et al., EMBO J. 5 (1986) 2809-2814]. We present here the nucleotide sequence of this genomic clone including the 3' untranslated region. To complete the coding sequence, we have isolated cDNA recombinant clones (1.1 kb) of vimentin from human libraries constructed in lambda gt11. Comparison of the coding sequence between human and hamster shows 90% homology at the nucleotide level and four differences out of 353 amino acid residues, as deduced from the nucleotide sequences. In addition to the extensive homology previously reported between the coding sequences of hamster and human vimentin genes [Ferrari et al., Mol. Cell. Biol. 6 (1986) 3614-3620], we observed that the positions of the noncoding regions are also conserved and that the 3' nontranslated region includes two canonic poly(A) signals. Hybridization of the clones to mRNA from different mammalian sources revealed a single species of 2 kb and confirmed that the length of the untranslated and coding sequences are conserved. Quantitative estimations of the mRNA levels in mammalian cells and tissues of various origins are consistent with transcriptional regulation.

Amino Acid Sequence↗
PubMed1988

Sequence analysis of murine cytokeratin endo A (no. 8) cDNA. Evidence for mRNA species initiated upstream of the normal 5' end in PCC4 cells.

Keratin 8 (Endo A) is expressed in simple epithelia, together with keratin 18 (Endo B). Filaments formed by this keratin pair are the first cytoskeletal elements induced during mouse embryogenesis. We have isolated Endo A cDNA clones from lambda gt11 libraries prepared with mRNA isolated from PCC4 embryonal carcinoma (EC) cells. Sequencing of three overlapping cDNAs and of a genomic clone allowed us to determine the complete sequence of the Endo A message. Analysis of the protein sequence deduced showed that the Endo A protein presented all the characteristics of intermediate filaments, including an alpha-helical central rod domain and nonhelical N- and C-termini. In the rod domain, the degree of similarity to the other members of the basic keratin family was high. A high degree of homology to keratin 8 of other species was observed, even in the non-helical domains. During these analyses, we found clones extending upstream of the normal 5' end of the mRNA. Sequence comparison between these cDNAs and the 5' upstream region of the Endo A gene suggested that they corresponded to transcripts initiated at an upstream alternative promoter. These observations supported previous results showing the presence of Endo A transcripts initiated upstream of the normal 5' end in mouse morulae and blastocysts.

Amino Acid Sequence↗