RESUMEN
The activation of oxidized phosphoribulokinase either "free" or as part of a bi-enzyme complex by reduced thioredoxins during the enzyme reaction was studied. In the presence of reduced thioredoxin, the product of the reaction catalyzed by phosphoribulokinase within the bi-enzyme complex does not appear in a linear fashion. It follows a mono-exponential pattern that suggests a slow dissociation process of the bi-enzyme complex in the assay cuvette. A plot of the steady state of product appearance against thioredoxin concentration gave a sigmoid curve. On the basis of our experimental results, we propose a minimum model of the activation of phosphoribulokinase by reduced thioredoxin. Reduced thioredoxin may act on the phosphoribulokinase, either within the complex or in the dissociated metastable form. However, the time required to activate the enzyme as part of the complex is shorter (about 20 s) than that required to activate the dissociated form (about 10 min). This might be of physiological relevance, and we discuss the role of the interactions between phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase in the regulation of the Calvin cycle.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Cloroplastos/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Tiorredoxinas/metabolismo , Animales , Activación Enzimática , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Cinética , Modelos QuímicosRESUMEN
Genes CIT1 and CIT2 from Saccharomyces cerevisiae encode mitochondrial and peroxisomal citrate synthases involved in the Krebs tricarboxylic acid (TCA) cycle and glyoxylate pathway, respectively. A Deltacit1 mutant does not grow on acetate, despite the presence of Cit2p that could, in principle, bypass the resulting block in the TCA cycle. To elucidate this absence of cross-complementation, we have examined the ability of Cit1p to function in the cytosol, and that of Cit2p to function in mitochondria. A cytosolically localized form of Cit1p was also incompetent for restoration of growth of a Deltacit1 strain on acetate, suggesting that mitochondrial localization of Cit1p is essential for its function in the TCA cycle. Cit2p was able, when mislocalized in mitochondria, to restore a wild-type phenotype in a strain lacking Cit1p. We have purified these two isoenzymes as well as mitochondrial malate dehydrogenase, Mdh1p, and have shown that Cit2p was also able to mimic Cit1p in its in vitro interaction with Mdh1p. Models of Cit1p and Cit2p structures generated on the basis of that of pig citrate synthase indicate very high structural and electrostatic surface potential similarities between the two yeast isozymes. Altogether, these data indicate that metabolic functions may require structural as well as catalytic roles for the enzymes.
Asunto(s)
Citrato (si)-Sintasa/metabolismo , Mitocondrias/enzimología , Peroxisomas/enzimología , Saccharomyces cerevisiae/enzimología , Acetatos/metabolismo , Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/genética , Ciclo del Ácido Cítrico , Citosol/enzimología , Activación Enzimática/genética , Malato Deshidrogenasa/metabolismo , Mitocondrias/genética , Modelos Moleculares , Peroxisomas/genética , Plásmidos/biosíntesis , Plásmidos/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Electricidad EstáticaRESUMEN
The phosphoribulokinase, when it is in a reduced state in a bi-enzyme complex, is more active than when it is oxidized. This complex dissociates upon dilution to give a metastable reduced form of phosphoribulokinase, which differs from the stable form isolated beside the complex. The kinetic parameters of the reduced stable phosphoribulokinase and those of the complex are very similar, unlike those of the metastable form. Although the kinetic mechanism of the reduced stable form is ordered, with ribulose-5-phosphate binding first, ATP binds first to the phosphoribulokinase in the complex and to the metastable form. Therefore, phosphoribulokinase bears an imprint from glyceraldehyde-3-phosphate dehydrogenase after disruption of the complex. Dissociation of phosphoribulokinase from the complex also enhances its flexibility. The imprinting and greater flexibility result in the catalytic constant of dissociated phosphoribulokinase being 10-fold higher than that of the enzyme in the complex. Imprinting corresponds to stabilization-destabilization energies resulting from conformation changes generated by protein-protein interactions. The energy stored within the metastable phosphoribulokinase is mainly used to decrease the energy barrier to catalysis.
Asunto(s)
Chlamydomonas reinhardtii/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasas/fisiología , Complejos Multienzimáticos/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Animales , Metabolismo Energético/fisiología , Cinética , Especificidad por SustratoRESUMEN
BACKGROUND: Silymarin is a well-known hepatoprotective agent. Tacrine, the first drug marketed for Alzheimer's disease (AD), induces an elevation of serum liver transaminase prohibiting an effective dosage in many patients. This 12-week randomised, double-blind, placebo-controlled study was undertaken to evaluate the ability of silymarin to antagonise or prevent the hepatotoxic effects of tacrine and to analyse its action on tacrine efficacy and tolerability. METHODS: Outpatients suffering from mild-to-moderate dementia of the Alzheimer type were randomly assigned to two treatment groups: tacrine + silymarin and tacrine + placebo. The study was double-blind for silymarin and open for tacrine and was conducted in 22 French neurology and geriatric centres. Silymarin (420 mg/day) was given first (1 week) and tacrine was added at 40 mg/day for 6 weeks, then increased to 80 mg/day (6 weeks). Serum ALAT was the main evaluation criterion (> upper limit of normal, ULN). Serum ASAT as well as adverse side effects and cognitive performance assessed by MMSE and the Syndrome Kurtz test (SKT) were secondary evaluation criteria. Null hypotheses were evaluated with Fisher's exact test. FINDINGS: 222 patients were recruited and received silymarin and tacrine (110 patients) or placebo and tacrine (112 patients). 28 patients dropped out; 217 were included in the intent-to-treat analysis. No statistical difference was observed between the two groups for serum ALAT (p = 0.39). Fewer patients had ALAT levels >5 ULN in the silymarin group (-33.3%). Side effects and notably gastrointestinal disorders were much less frequent in the silymarin group. Cognitive performance remained unchanged in both groups. INTERPRETATION: Silymarin does not prevent tacrine-induced ALAT elevation but does reduce the rate of gastrointestinal and cholinergic side effects without any impact on cognitive status. As a consequence, silymarin (420 mg/day) could be co-administered with tacrine to improve tolerability in the initial phases of AD treatment.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Inhibidores de la Colinesterasa/farmacología , Sustancias Protectoras/farmacología , Silimarina/efectos adversos , Silimarina/sangre , Tacrina/antagonistas & inhibidores , Tacrina/sangre , Transaminasas/sangre , Transaminasas/efectos de los fármacos , Anciano , Enfermedad Hepática Inducida por Sustancias y Drogas , Cognición/efectos de los fármacos , Trastornos del Conocimiento/diagnóstico , Método Doble Ciego , Humanos , Pruebas NeuropsicológicasRESUMEN
We describe the characterization and a functional analysis in Xenopus development of RalB, a small G protein. RalB RNA and protein are detectable during oogenesis and early development, but the gene is expressed only weakly in adult tissues. The RalB transcripts are processed by poly(A) extension during oocyte maturation and up to the gastrulation stage. Microinjection of wild-type or mutant RalB RNAs was performed in fertilized eggs in order to gain insight into the function of RalB during development. We show that during cleavage stages the activated GTP form of RalB specifically induces a cortical reaction that affects the localization of pigment granules. The use of different drugs suggests that this reaction is dependent on the outer cortical actin array. The relation between F-actin and RalB was shown by confocal analysis. Injection of mRNAs encoding the mutated activated form of RalB leads, at dependent doses, to a blocking of gastrulation or defects in closing of neural folding structures. In contrast, the inactivated form blocks only the closing of neural tube. Altogether, these observations suggest that RalB is part of a regulatory pathway that may affect the blastomere cytoskeleton and take part in early development.
Asunto(s)
Actinas/metabolismo , Proteínas de Unión al GTP/fisiología , Proteínas de Xenopus , Xenopus laevis/genética , Proteínas de Unión al GTP ral , Secuencia de Aminoácidos , Animales , Polaridad Celular , Citocalasina B/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Proteínas de Unión al GTP/genética , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica , Guanosina Trifosfato/metabolismo , Microinyecciones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sistema Nervioso/embriología , Oocitos/metabolismo , Oogénesis , Pigmentos Biológicos/metabolismo , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Xenopus laevis/embriología , Cigoto/metabolismoRESUMEN
Mitochondrial malate dehydrogenase and citrate synthase are sequential enzymes in the Krebs tricarboxylic acid cycle. We have shown [Lindbladh, C., Rault, M., Hagglund, C., Small, W. C., Mosbach, K., Bülow, L., Evans, C., and Srere, P.A (1994) Biochemistry 33, 11692-11698] that a fusion protein of yeast mitochondrial citrate synthase and yeast mitochondrial malate dehydrogenase channels oxaloacetate between the active sites. A Brownian dynamics simulation model of porcine mitochondrial enzymes of citrate synthase and malate dehydrogenase was used [Elcock, A. H., and McCammon, A. M. (1996) Biochemistry 35, 12652-12658], showing that a positive electrostatic surface potential between the active sites of the fusion protein could account for the channeling of oxaloacetate we observed with the yeast fusion protein. Since the data were established with a yeast fusion protein and the model was with porcine fusion protein, we have now prepared and studied the porcine fusion protein. The channeling of the oxaloacetate intermediate was the same for the porcine fusion protein as it was for the yeast fusion protein. This channeling behavior is eliminated at high ionic strength. A fusion protein of porcine citrate synthase and porcine cytosolic malate dehydrogenase does not exhibit any channeling of oxaloacetate. A model of the fusion protein with the cytosolic malate dehydrogenase shows no clear positive electrostatic potential surface between the two active sites, thus distinguishing it from the fusion protein with the mitochondrial malate dehydrogenase. These results establish the electrostatic nature of channeling in mitochondrial fusion proteins.
Asunto(s)
Citrato (si)-Sintasa/química , Malato Deshidrogenasa/química , Mitocondrias Hepáticas/enzimología , Ácido Oxaloacético/química , Proteínas Recombinantes de Fusión/química , Animales , Catálisis , Citrato (si)-Sintasa/genética , Citrato (si)-Sintasa/metabolismo , Simulación por Computador , Citosol/enzimología , Cinética , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Modelos Moleculares , Concentración Osmolar , Ácido Oxaloacético/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Electricidad Estática , PorcinosRESUMEN
Methodology used for the development of anti-Alzheimer's disease (AD) drugs raises specific problems which are rarely examined in the literature. While the general development scheme is similar to that required for most drugs, some specific aspects must be analyzed, highly dominated by the dual goal of pharmacology, i.e., to obtain both symptomatic and etiopathogenic drugs. During preclinical studies, aged or lesioned animals are mainly useful for symptomatic drugs, whereas transgenic models or neurodegeneration-induced techniques would probably lead to etiopathogenic drugs potentially slowing down the process of AD. The first administrations of a new compound to human beings raise the question of the activity measurement techniques. Psychometry remains the most informative procedure to detect and analyze the activity of the drugs on the different components of cognition. Electrophysiology and neuroimaging need some complementary studies before they can be proposed as surrogate criteria in phase III trials. At this stage of development, American and the recently published European guidelines are of great help while insisting on long-term (6 months) placebo controlled trials with the use of the triple efficacy criterion: an objective cognition scale, a global assessment, and the opinion of the caregiver. In the long term, pharmacoepidemiology and pharmacoeconomy will have to confirm the rationale of this recent progress in the methodology of anti-AD drug development.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Envejecimiento/metabolismo , Envejecimiento/patología , Envejecimiento/fisiología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Ensayos Clínicos como Asunto/métodos , Modelos Animales de Enfermedad , Drogas en Investigación/farmacología , Drogas en Investigación/uso terapéutico , HumanosRESUMEN
A bienzyme complex made up of phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase has been isolated and purified from chloroplasts of Chlamydomonas reinhardtii. The complex contains four phosphoribulokinase and eight glyceraldehyde-3-phosphate dehydrogenase polypeptide chains. As phosphoribulokinase is dimeric and glyceraldehyde-3-phosphate dehydrogenase tetrameric, it is concluded that the complex comprises two phosphoribulokinase and two glyceraldehyde-3-phosphate dehydrogenase molecules. Its overall molecular mass is 460 kDa, which is in excellent agreement with its stoichiometry. Moreover, owing to the nature of the two enzymes, this complex must catalyse two nonconsecutive reactions. The bienzyme complex tended to spontaneously dissociate into the free enzymes upon dilution. This dissociation process was considerably promoted by reducing agents such as dithiothreitol or reduced thioredoxin. The kinetics of the dissociation process induced by dithiothreitol or reduced thioredoxin were paralleled by an increase of activity of phosphoribulokinase. The dissociation of the complex was reversible. If oxidized phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase were mixed, a certain amount of the complex was formed. The reconstituted complex displayed properties that were indistinguishable from those of the native complex extracted from chloroplasts of Chlamydomonas reinhardtii. These results suggest that the concentration of the complex in vivo must vary depending on the light intensity.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Complejos Multienzimáticos/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Animales , Western Blotting , Cloroplastos/enzimología , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Ditiotreitol/farmacología , Durapatita , Gliceraldehído-3-Fosfato Deshidrogenasas/aislamiento & purificación , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Peso Molecular , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , NAD/metabolismo , NADP/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Conformación Proteica , Tiorredoxinas/farmacologíaRESUMEN
Oxidized, free, stable phosphoribulokinase from Chlamydomonas reinhardtii was almost completely devoid of catalytic activity (0.06 s(-1)/site). However, when it was bound to glyceraldehyde-3-phosphate dehydrogenase from the same organism, it displayed significant activity (3.25 s(-1)/site). Moreover, this complex tended to spontaneously dissociate upon dilution; the isolated phosphoribulokinase activity increased up to 56 s(-1)/site, subsequently decreased, and finally became almost completely inactive. Its intrinsic kinetic properties (Km and k(cat)) changed with the variation of the overall activity. These effects were paralleled by changes of conformation of the enzyme as revealed by fluorescence analysis. A model is proposed that allows quantitative expression of the dynamics of the dissociation of the oxidized bienzyme complex and the effects of either of the two substrates, ATP and ribulose 5-phosphate, on this dissociation process. Whereas ATP destabilized the complex and promoted its dissociation, ribulose 5-phosphate tended to stabilize this complex. Inactive, stable, oxidized phosphoribulokinase may form a complex with glyceraldehyde-3-phosphate dehydrogenase regaining its catalytic activity. In this case, glyceraldehyde-3-phosphate dehydrogenase acts in a manner similar, but not identical to a chaperonin. The information content of the phosphoribulokinase gene, as defined by the sequence of its base pairs, was therefore not sufficient to specify full enzyme activity. It needed the presence of glyceraldehyde-3-phosphate dehydrogenase to give the oxidized phosphoribulokinase a conformation competent for its activity. The potential biological significance of these effects remains to be discovered.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Complejos Multienzimáticos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Cloroplastos/enzimología , Activación Enzimática , Estabilidad de Enzimas , Fluorescencia , Glutatión/farmacología , Cinética , Modelos Químicos , Oxidación-Reducción , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Conformación Proteica , Ribulosafosfatos/metabolismo , TriptófanoRESUMEN
Oxidized phosphoribulokinase is almost inactive in its isolated state but becomes active when associated with glyceraldehyde-3-phosphate dehydrogenase. There is therefore an information transfer that takes place between these two enzymes. However, when the complex dissociates, free oxidized phosphoribulokinase is even more active than when it is associated with glyceraldehyde-3-phosphate dehydrogenase. This means that glyceraldehyde-3-phosphate dehydrogenase exerts an imprinting effect upon phosphoribulokinase which persists for a while after the parting of the two proteins. Various methods derived from statistical thermodynamics can be used to estimate the fraction of energy transferred from glyceraldehyde-3-phosphate dehydrogenase to phosphoribulokinase and which alters the kinetic parameters of the latter enzyme. In the complex, the decrease of the free energy associated with the binding of ribulose 5-phosphate is larger than that of ATP. This implies that the mutual association of the two enzymes facilitates the binding of the former substrate but is without effect on that of the latter. The main effect exerted by the association of the two enzymes is to decrease by about 10 kJ/mol the height of the energy barrier of the catalytic process. Phosphoribulokinase keeps an imprinting effect exerted by glyceraldehyde-3-phosphate dehydrogenase after the parting of the two enzymes. Part of the energy transferred from one protein to the other is used to decrease slightly the apparent binding free energy of the two substrates of phosphoribulokinase by about 1.5 kJ/mol. Whereas the previous association of the two enzymes does not significantly alter substrate binding to phosphoribulokinase, it greatly affects catalysis and decreases by about 16 kJ/mol the height of the energy barrier pertaining to this step. Therefore, within multienzyme complexes, information and energy can be transferred between proteins. Statistical thermodynamics offers the possibility of estimating how this energy is used to alter the various kinetic parameters of the reaction.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Termodinámica , Animales , Cloroplastos/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Cinética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Conformación ProteicaRESUMEN
A mutant phosphoribulokinase has been isolated from the 12-2B mutant of Chlamydomonas reinhardtii. In this mutant, Arg64 has been replaced by Cys. The enzyme, which may exist in the dimeric and tetrameric states, is almost devoid of activity. Neither of these enzymes is able to form a complex with glyceraldehyde-3-phosphate dehydrogenase. The phosphoribulokinase gene has been expressed in Escherichia coli. The resulting recombinant protein, after isolation and purification, is apparently identical to the native enzyme extracted from the chloroplast. Three mutants have been generated by site directed mutagenesis. Arg64 has been replaced by Ala, Lys or Glu. With the exception of the latter, the two other mutants, [A64]phosphoribulokinase and [K64]phosphoribulokinase, are active when they are reduced, and nearly totally inactive in their oxidized state. Their activity, however, is decreased relative to that of the native, or to that of the wild-type recombinant phosphoribulokinase. Both the catalytic constant and the apparent affinity of ribulose 5-phosphate are decreased relative to the corresponding values obtained for the wild-type, the native or the recombinant enzyme. Whereas the [A64]phosphoribulokinase is unable to form a complex with glyceraldehyde-3-phosphate dehydrogenase, [K64]phosphoribulokinase does, but the stability of the resulting complex is much decreased relative to that of the wild-type complex. The oxidized mutant [K64]phosphoribulokinase becomes active in the presence of glyceraldehyde-3-phosphate dehydrogenase but this activity is smaller than that of the corresponding wild-type enzyme. Taken together, these results show that Arg64 plays a major role in the association of the two enzymes and in the information transfer that takes place between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase. As this residue also appears to be important for catalytic activity, it may be tempting to consider that it stabilizes a conformation that is required for both the catalytic activity and the formation of the bienzyme complex.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Animales , Arginina , Cloroplastos/enzimología , Escherichia coli/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Cinética , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
OBJECTIVE: This study was conducted to assess nutrient intake outside the home of 629 people representative of the French population. SUBJECTS: The study population consisted of 629 people aged 15 years and over. They were recruited in a randomized way with two levels (town and household). METHOD: Food intake outside the home was assessed by self-completed estimated record for 7 d. Individuals referred to photographs to estimate portions. Nutrient intake has been calculated for energy, protein, carbohydrate, fat and some minerals (calcium, phosphorus, magnesium, iron). RESULTS: Lunches and dinners eaten out are on average too rich in protein (20% of the energy), too high in fat (40-43% of the energy) and do not contain enough carbohydrate. The percentage of energy from sugars varies between 11% for lunch and 30% for breakfast. Mean intake of nutrients by beverages drunk outside the home decrease with the age of consumers. CONCLUSION: This study shows that foods and drinks consumed outside the home in France are on average too rich in fat and protein.
Asunto(s)
Servicios de Alimentación , Alimentos , Fenómenos Fisiológicos de la Nutrición , Adolescente , Adulto , Envejecimiento , Bebidas , Registros de Dieta , Carbohidratos de la Dieta/administración & dosificación , Grasas de la Dieta/administración & dosificación , Proteínas en la Dieta/administración & dosificación , Ingestión de Energía , Femenino , Francia , Humanos , Masculino , Persona de Mediana Edad , Minerales/administración & dosificación , Valor Nutritivo , Restaurantes , Caracteres SexualesRESUMEN
The menopause was considered in the past as a critical age during which women who no more can procreate stop being a real female. Actually it is estimated as a situation of hormonal deficiency which should be heated with the well-known. But limitation of this period to exclusive hormonal went seems reducing. At the menopause women have to manage the bereaving a part of their peculiar identity, the real and imaginary fertility. That needs a mental reorganization which may be difficult to be faced and may be in origin of neurosis and various somatic events. Therapeutics are now able to approach this period in comfortable conditions as well on the somatic than the functional level. But it is undeniable that during the consultation something else than only the hormonal aspects of the woman's story is getting in stage.