RESUMO
Galactosemia is an autosomal recessive disorder with a wide range of clinical abnormalities. Cellular oxidative stress is considered as one of the pathogenic mechanisms of galactosemia. In this study, we examined the activity of NADPH oxidase (NOX), a major superoxide-generating enzyme system, in peripheral blood lymphocytes (PBL) from galactosemia patients. PBL were isolated from galactosemia patients and healthy control subjects and used for cell culture studies and biochemical assays. PBL were cultured in the presence or absence of galactose or galactose-1-phosphate (Gal-1-P), and enzyme activities and/or gene expression of NOX, catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in the cell homogenates. PBL isolated from galactosemia patients showed significantly reduced (P < 0.01) activities of catalase and GPx; however SOD activity remained unaltered. Galactosemia patients were found to have significantly (P < 0.01) increased levels of malondialdehyde (MDA) in blood lymphocytes. Enzymatic activity of NOX was significantly (P < 0.001) reduced in galactosemia patients; however, Western blotting revealed that NOX-1 protein was not significantly altered. Interestingly, levels of NOX activity in lymphocytes isolated from galactosemia patients significantly increased but remained subnormal when cultured in galactose-deficient medium for two weeks, indicating a galactose-mediated inhibition of NOX. Lymphocytes isolated from control subjects were found to have significantly (P < 0.01) reduced NOX activity when cultured in the presence of galactose or Gal-1-P for two weeks. These results show that galactose-induced cellular oxidative stress is not NOX mediated. However, impairment of the NOX system might be responsible for some of the clinical complications in galactosemia patients.
Assuntos
Galactosemias/sangue , Galactosemias/enzimologia , NADPH Oxidases/sangue , Antioxidantes/metabolismo , Western Blotting , Estudos de Casos e Controles , Separação Celular , Células Cultivadas , Pré-Escolar , Galactose/farmacologia , Galactosefosfatos/metabolismo , Galactosefosfatos/farmacologia , Glucosefosfato Desidrogenase/metabolismo , Humanos , Lactente , Peroxidação de Lipídeos/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Linfócitos/patologia , Malondialdeído/metabolismo , NADPH Oxidase 1 , NADPH Oxidases/metabolismo , Nitritos/metabolismo , Fosfoproteínas/metabolismoRESUMO
UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.
Assuntos
Hordeum/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/química , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Galactosefosfatos/metabolismo , Galactosefosfatos/farmacologia , Glucofosfatos/metabolismo , Glucofosfatos/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína/efeitos dos fármacos , Uridina Difosfato Glucose/metabolismo , Uridina Difosfato Glucose/farmacologia , Uridina Trifosfato/metabolismo , Uridina Trifosfato/farmacologiaRESUMO
Inhibition of Na+,K+-ATPase activity causes edema and cell death in central nervous system. We determined the in vitro effects of galactose-l-phosphate (Gal-1-P), galactitol (Galtol) and galactose (Gal) (mix A = classical galactosaemia) or Galtol and Gal (mix B = galactokinase deficiency galactosaemia), on Na+,K+-ATPase activity in suckling rat brain frontal cortex, hippocampus or hypothalamus homogenates. Gal-1-P or Galtol alone at different concentrations, significantly inhibited Na+,K+-ATPase whereas Gal activated the enzyme in all investigated brain regions. Both mix A and mix B inactivated the enzyme by 20-30% (p < 0.001) in all studied areas. L-Cysteine (Cys) and glutathione (GSH) supplementation in mix B not only reversed the enzyme inhibition but also resulted in an activation of 50-60%, (p < 0.001) in all brain areas. Their presence in mix A also activated the inhibited Na+,K+-ATPase in hippocampus and hypothalamus to a lower degree, whereas Cys reversed the frontal cortex enzyme activity to control value only. These findings indicate that oxidation of the enzyme critical groups may be involved in galactosaemia, producing inhibitory effect. This phenomenon is reversed by antioxidants Cys and GSH, implying that free radicals may be implicated in the observed enzyme inactivation.
Assuntos
Encéfalo/metabolismo , Cisteína/metabolismo , Galactosemias/metabolismo , Glutationa/metabolismo , Estresse Oxidativo/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Animais Lactentes , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Química Encefálica/efeitos dos fármacos , Química Encefálica/fisiologia , Cisteína/farmacologia , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Feminino , Radicais Livres/metabolismo , Galactitol/metabolismo , Galactitol/farmacologia , Galactose/metabolismo , Galactose/farmacologia , Galactosemias/complicações , Galactosefosfatos/metabolismo , Galactosefosfatos/farmacologia , Glutationa/farmacologia , Técnicas In Vitro , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismoRESUMO
UNLABELLED: Mg2+-ATPase activity is implicated with Mg2+ homeostasis, maintaining high brain intracellular Mg2+ content. We determined the in vitro effects of galactose-1-phosphate (Gal-1-P), galactitol (Galtol) and galactose (Gal) {mix A=Gal-1-P(2 mM)+Galtol(2 mM)+Gal(4 mM) concentrations commonly found in patients with classical galactosaemia} or Galtol and Gal {mix B=Galtol(2 mM)+Gal(1 mM) concentrations usually measured in patients with galactokinase deficiency galactosaemia} on Mg2+-ATPase activity in suckling rat brain frontal cortex, hippocampus or hypothalamus homogenates. Gal-1-P significantly (p<0.001) enhanced enzyme activity in all the brain areas measured, whereas Galtol and Gal failed to cause any effect in the same regions. Mix A remarkably (p<0.001) stimulated Mg2+-ATPase in the studied areas. On the contrary, mix B had no effect. The supplementation of antioxidant l-cysteine (Cys) or reduced Glutathione (GSH) in mix A failed to reverse to normal the activated enzyme in frontal cortex and hypothalamus, while they significantly reduced Mg2+-ATPase activation in hippocampus. CONCLUSIONS: (a) Gal-1-P enormously activated Mg2+-ATPase in all the studied brain regions, (b) Mix A, also, excessively activated the enzyme in the same areas, (c) the production of free radicals may be implicated with the enzyme activation and (d) Cys or GSH significantly decreased the activated hippocampal Mg2+-ATPase.
Assuntos
Antioxidantes/farmacologia , Encéfalo/enzimologia , ATPase de Ca(2+) e Mg(2+)/biossíntese , Cisteína/farmacologia , Glutationa/farmacologia , Animais , Animais Lactentes , Encéfalo/efeitos dos fármacos , Combinação de Medicamentos , Ativação Enzimática , Feminino , Galactitol/farmacologia , Galactose/farmacologia , Galactosemias , Galactosefosfatos/farmacologia , Masculino , Ratos , Ratos WistarRESUMO
Previously we reported that stable transfection of human UDP-glucose pyrophosphorylase (hUGP2) rescued galactose-1-phosphate uridyltransferase (GALT)-deficient yeast from "galactose toxicity." Here we test in human cell lines the hypothesis that galactose toxicity was caused by excess accumulation of galactose-1-phosphate (Gal-1-P), inhibition of hUGP2, and UDP-hexose deficiency. We found that SV40-transformed fibroblasts derived from a galactosemic patient accumulated Gal-1-P from 1.2+/-0.4 to 5.2+/-0.5 mM and stopped growing when transferred from 0.1% glucose to 0.1% galactose. Control fibroblasts accumulated little Gal-1-P and continued to grow. The GALT-deficient cells had 157+/-10 micromoles UDP-glucose/100 g protein and 25+/-5 micromoles UDP-galactose/100 g protein when grown in 0.1% glucose. The control cells had 236+/-25 micromoles UDP- glucose/100 g protein and 82+/-10 micromoles UDP-galactose/100 g protein when grown in identical medium. When we transfected the GALT-deficient cells with either the hUGP2 or GALT gene, their UDP-glucose content increased to 305+/-28 micromoles/100 g protein (hUGP2-transfected) and 210+/-13 micromoles/100 g protein (GALT-transfected), respectively. Similarly, UDP-galactose content increased to 75+/-12 micromoles/100 g protein (hUGP2-transfected) and 55+/-9 micromoles/100 g protein (GALT-transfected), respectively. Though the GALT-transfected cells grew in 0.1% galactose with little accumulation of Gal-1-P (0.2+/-0.02 mM), the hUGP2-transfected cells grew but accumulated some Gal-1-P (3.1+/-0.4 mM). We found that 2.5 mM Gal-1-P increased the apparent KM of purified hUGP2 for glucose-1-phosphate from 19.7 microM to 169 microM, without changes in apparent Vmax. The Ki of the reaction was 0.47 mM. Gal-1-P also inhibited UDP-N-acetylglucosamine pyrophosphorylase, which catalyzes the formation of UDP-N-acetylglucosamine. We conclude that intracellular concentrations of Gal-1-P found in classic galactosemia inhibit UDP-hexose pyrophosphorylases and reduce the intracellular concentrations of UDP-hexoses. Reduced Sambucus nigra agglutinin binding to glycoproteins isolated from cells with increased Gal-1-P is consistent with the resultant inhibition of glycoprotein glycosylation.
Assuntos
Fibroblastos/metabolismo , Galactosefosfatos/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/deficiência , Uridina Difosfato Glucose/deficiência , Linhagem Celular Transformada , Transformação Celular Viral , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Galactose/farmacologia , Galactosefosfatos/farmacologia , Glicosilação , Humanos , Cinética , Fenótipo , Vírus 40 dos Símios/genética , Transfecção , UTP-Glucose-1-Fosfato Uridililtransferase/genética , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/genética , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismoRESUMO
Binding characteristics of two types of ligands for human neo-C-reactive protein (neo-CRP), which is a conformationally altered but physiologically relevant form of CRP, were studied fluorometrically by probing CRP immobilized on a polystyrene surface with europium-labeled ligands. Two Eu-ligands used were bovine serum albumin derivatives that contain on average 40 residues of ligand structures, one derivative containing phosphorylcholine (PC) and the other lactosyl residues. The PC-containing ligands required the presence of calcium for binding, whereas galactose-containing derivatives bound in the absence of calcium. The optimal pH for the PC-dependent binding was broad (pH 6-8), whereas the best binding pH for the galactose-dependent binding was around 6. The carbohydrate-mediated binding is rather nonspecific: the binding site prefers galactose configuration, but other hexoses can be accommodated. The two best monosaccharide inhibitors at this site were galactose-6-phosphate and galacturonic acid, suggesting the importance of having a negatively charged group at C-6 position of galactose. In fact, the phosphate-binding site is common to both PC and sugar phosphates, and the choline- and the sugar-binding sites are probably located on either side of the phosphate-binding site. Binding characteristics of Eu-labeled PC-BSA to neo-CRP are quite similar to that found for native CRP in solution phase [Lee et al. (2002) J. Biol. Chem., 277, 225-232], whereas binding of sugar phosphates by neo-CRP shows considerably less stringent requirements compared to native CRP. For instance, galactose-alpha1-phosphate was not inhibitory at all in the native CRP binding assay, whereas it was a good inhibitor in the neo-CRP assay.
Assuntos
Proteína C-Reativa/metabolismo , Galactose/metabolismo , Lactose/metabolismo , Ligantes , Fosforilcolina/análogos & derivados , Fosforilcolina/metabolismo , Sítios de Ligação , Proteína C-Reativa/química , Cálcio/farmacologia , Epitopos/análise , Galactosefosfatos/farmacologia , Ácidos Hexurônicos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Ácido Pentético/metabolismo , Fosfatos/metabolismo , Ligação Proteica , Soroalbumina Bovina/metabolismoRESUMO
To evaluate the effect of galactose metabolic disorders on the brain Na+,K+-ATPase in suckling rats. Separate preincubations of various concentrations (1-16 mM) of the compounds galactose-1-phosphate (Gal-1-P) and galactitol (galtol) with whole brain homogenates at 37 degrees C for 1 h resulted in a dose dependent inhibition of the enzyme whereas the pure enzyme (from porcine cerebral cortex) was stimulated. Glucose-1-phosphate (Glu-1-P) or galactose (Gal) stimulated both rat brain Na+,K+-ATPase and pure enzyme. A mixture of Gal-1-P (2 mM), galtol (2 mM) and Gal (4 mM), concentrations commonly found in untreated patients with classical galactosemia, caused a 35% (p < 0.001) rat brain enzyme inhibition. Additionally, incubation of a mixture of galtol (2 mM) and Gal (1 mM), which is usually observed in galactokinase deficient patients, resulted in a 25% (p < 0.001) brain enzyme inactivation. It is suggested that: a) The indirect inhibition of the brain Na+,K+-ATPase by Gal-1-P should be due to the presence of the epimer Gal and phosphate and that the pure enzyme direct activation by Gal-1-P and Glu-1-P to the presence of phosphate only. b) The observed brain Na+,K+-ATPase inhibitions in the presence of toxic concentrations of Gal-1-P and/or galtol could modulate the neural excitability, the metabolic energy production and the catecholaminergic and serotoninergic system.
Assuntos
Encéfalo/enzimologia , Erros Inatos do Metabolismo dos Carboidratos/metabolismo , Galactose/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Animais Lactentes , Erros Inatos do Metabolismo dos Carboidratos/enzimologia , Galactitol/farmacologia , Galactose/análogos & derivados , Galactose/farmacologia , Galactosefosfatos/farmacologia , Cinética , RatosRESUMO
Galactokinase (EC 2.7.1.6) catalyses the first step in the catabolism of galactose. Yeast galactokinase, Gal1p, and the closely related but catalytically inactive Gal3p, also function as ligand sensors in the GAL genetic switch. In the presence of galactose and ATP (the substrates of the reaction catalysed by Gal1p) Gal1p or Gal3p can bind to Gal80p, a transcriptional repressor. This relieves the inhibition of a transcriptional activator, Gal4p, and permits expression of the GAL genes. In order to learn more about the mechanism of ligand sensing by Gal3p and Gal1p, we studied the kinetics of the reaction catalysed by Gal1p. Galactose-1-phosphate, a product of the reaction, is a mixed inhibitor both with respect to galactose and to ATP suggesting that the reaction proceeds via a compulsory, ordered, ternary complex mechanism. There is little variation in either the turnover number or the specificity constants in the pH range 6.0-9.5, implying that no catalytic base is required in the reaction. These data are discussed both in the context of galactokinase enzymology and their implications for the mechanism of transcriptional induction.
Assuntos
Galactoquinase/genética , Galactoquinase/metabolismo , Ativação Transcricional/genética , Trifosfato de Adenosina/metabolismo , Ligação Competitiva , Inibidores Enzimáticos/farmacologia , Galactose/metabolismo , Galactosefosfatos/biossíntese , Galactosefosfatos/farmacologia , Concentração de Íons de Hidrogênio , Isótopos , Cinética , Ligantes , Ligação Proteica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Galactosaemia is an inborn error of metabolism characterized by irreversible damage to neural tissue. To evaluate whether galactose metabolic disorders, (e.g. classical galactosaemia, galactokinase deficiency galactosaemia), is implicated for alterations of brain Mg2+-ATPase activity, various concentrations (1-16 mM) of galactose, galactose-1-phosphate, galactitol, glucose-1-phosphate or glucose were preincubated with whole brain homogenates of suckling rats at 37 degrees for 1 hr. Mg2+-ATPase activities were determined according to Bowler & Tirri's (1974). Galactose-1-phosphate or glucose-1-phosphate excessively activated the brain Mg2+-ATPase in a concentration-dependent way. Additionally, galactitol, galactose or glucose stimulated the enzyme up to 35-45% (P < 0.001) at concentrations >4 mM. A mixture of galactose-1-phosphate (2 mM), glactitol (2 mM) and galactose (4 mM), concentrations commonly found in blood and brain of untreated patients with classical galactosaemia, resulted in a 500% enzyme activation (P < 0.001) as compared to control. Moreover, a mixture of galactitol (2 mM) and galactose (1 mM), concentrations measured in patients with galactokinase deficiency, caused an enzyme stimulation (35%, P < 0.001). These findings suggest: a) The great Mg2+-ATPase activation by galactose-1-phosphate or glucose-1-phosphate may be due to the epimer of galactose and the presence of phosphorus. b) The brain Mg2+-ATPase stimulation by galactose and its derivatives could be toxic by modulating the Mg2+ concentration, the ATP availability, the activity of other ATP- and Mg2+-dependent enzymes as well as the rates of protein synthesis and cell growth.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , ATPase de Ca(2+) e Mg(2+)/metabolismo , Galactose/metabolismo , Animais , Animais Lactentes , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Feminino , Galactitol/metabolismo , Galactitol/farmacologia , Galactose/farmacologia , Galactosefosfatos/metabolismo , Galactosefosfatos/farmacologia , Glucose/metabolismo , Glucose/farmacologia , Glucofosfatos/metabolismo , Glucofosfatos/farmacologia , Técnicas In Vitro , Masculino , Ratos , Ratos WistarRESUMO
To evaluate whether in classical galactosemia galactose (Gal), galactose-1-phosphate (Gal-1-P) and galactitol (Galtol) affect brain acetylcholinesterase (AChE) activity, various concentrations (1-16 mM) of these compounds were preincubated with brain homogenates of suckling rats as well as with pure eel Electroforus electricus AChE at 37 degrees C for 1 h. Initially, Galtol (up to 2.0 mM) increased (25%) AChE activity which decreased. thereafter, reaching the control value in high Galtol concentrations. Gal-1-P decreased gradually the enzyme activity reaching a plateau (38%), when incubated with 8-16 mM. However, when the usually found 2 mM of Galtol and 2 mM of Gal-1-P, concentrations in galactosemia were added in the incubation mixture simultaneously, brain AChE was stimulated (16%). Galtol or Gal-1-P modulated brain AChE as well as enzyme activity of E.electricus in the same way. Gal, Glucose (Glu) and glucose-1-phosphate (Glu-1-P) had no effect on AChE activity. It is suggested that Galtol as well as Gal-1-P can affect acetylcholine degradation acting directly on AChE molecule. Consequently the direct action of these substances on the enzyme might explain the brain cholinergic dysfunction in untreated galactosemia patients.
Assuntos
Acetilcolinesterase/metabolismo , Encéfalo/enzimologia , Galactosemias/enzimologia , Animais , Feminino , Galactitol/farmacologia , Galactose/farmacologia , Galactosefosfatos/farmacologia , Cinética , Masculino , Ratos , Ratos WistarRESUMO
A new methodology for chemical differentiation of one amino and four hydroxyl groups of D-galactosamine derivatives and its application for the synthesis of D-galactosamine-4-phosphate analogs of lipid A are described. Preliminary examination of biological activity revealed that the synthetic monosaccharides show mitogenic activity.
Assuntos
Galactosamina/análogos & derivados , Galactosefosfatos/síntese química , Lipídeo A/análogos & derivados , Mitógenos/síntese química , Galactosamina/síntese química , Galactosamina/química , Galactosamina/farmacologia , Galactosefosfatos/química , Galactosefosfatos/farmacologia , Lipídeo A/farmacologia , Mitógenos/farmacologiaRESUMO
The syntheses of the poly-phosphorylated galactosides 6, 8, 10, 13, 16, and 20, isolated as sodium salts, have been performed. The non-phosphorylated disaccharide 17 and trisaccharide 21 have been prepared via glycosylation of the 2-(trimethylsilyl)ethyl galactosides 3 and 2, respectively, and subsequent complete deprotection. Preliminary insulin-like activity of the phosphorylated derivatives is reported.
Assuntos
Galactose/análogos & derivados , Galactosefosfatos/farmacologia , Galactosídeos/farmacologia , Insulina/metabolismo , Proteínas Quinases/efeitos dos fármacos , Células Cultivadas , Fibroblastos/citologia , Galactosefosfatos/síntese química , Galactosídeos/síntese química , HumanosRESUMO
The Escherichia coli uhp T gene encodes an active transport system for sugar phosphates. When the uhp T gene was carried on a multicopy plasmid, amplified levels of transport activity occurred, and growth of these strains was inhibited upon the addition of various sugar phosphates. Two different mechanisms for this growth inhibition were distinguished. Exposure to glucose-6-phosphate, fructose-6-phosphate or mannose-6-phosphate, which enter directly into the glycolytic pathway, resulted in cessation of growth and substantial loss of viability. Cell killing was correlated with the production of the toxic metabolite, methylglyoxal. In contrast, addition of 2-deoxyglucose-6-phosphate, galactose-6-phosphate, glucosamine-6-phosphate or arabinose-5-phosphate, which do not directly enter the glycolytic pathway, resulted in growth inhibition without engendering methylglyoxal production or cell death. Inhibition of growth could result from excessive accumulation of organophosphates in the cell or depletion of inorganic phosphate pools as a result of the sugar-P/Pi exchange process catalysed by UhpT. The phosphate-dependent uptake of glycerol-3-phosphate by the GlpT antiporter was strongly inhibited under conditions of elevated sugar-phosphate transport. There are thus two separate toxic effects of elevated sugar-phosphate transport, one of which was lethal and related to increased flux through glycolysis. It is likely that the control of uhpT transcription by catabolite repression exists to limit the level of UhpT transport activity and thereby prevent the toxic events that result from elevated uptake of its substrates.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Proteínas de Transporte de Monossacarídeos , Fosfatos Açúcares/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Divisão Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Frutosefosfatos/farmacologia , Galactosefosfatos/farmacologia , Glucose-6-Fosfato , Glucofosfatos/farmacologia , Glicerofosfatos/metabolismo , Glicólise , Manosefosfatos/farmacologia , Pentosefosfatos/farmacologia , Aldeído Pirúvico/metabolismoRESUMO
Porcine submaxillary beta-galactoside alpha(1----2)-fucosyltransferase is known to transfer a fucosyl residue from guanosine 5'-diphosphofucose (GDP-fucose) to the 2-OH group of beta-D-galactopyranosides with inversion of configuration at the fucopyranosyl anomeric carbon. A bisubstrate analog (1) of the postulated transition-state for this reaction, which has O-2 of phenyl beta-D-galactopyranoside attached to the terminal phosphorous of GDP through a flexible ethylene bridge, has been chemically synthesized and evaluated as an inhibitor of this enzyme. Compound 1 was found to be a competitive inhibitor with respect to both GDP-fucose and phenyl beta-D-galactopyranoside for both the membrane-bound and soluble forms of the fucosyltransferase. It was also a competitive inhibitor with respect to the alternate acceptor beta DGal(1----3)beta DGlcNAcO(CH2)8-COOMe. The Ki values were in the range 2.3-16 microM. Compound 1 is the first example of a bisubstrate analog inhibitor for a glycosyltransferase which binds to both the acceptor and donor recognition sites of the enzyme. The potential of a bisubstrate analog strategy for the production of specific glycosyltransferase inhibitors is discussed.
Assuntos
Fucosiltransferases/antagonistas & inibidores , Galactosefosfatos/farmacologia , Nucleotídeos de Guanina/farmacologia , Guanosina Monofosfato/farmacologia , Hexosefosfatos/farmacologia , Hexosiltransferases/antagonistas & inibidores , Animais , Sítios de Ligação , Ligação Competitiva , Galactosefosfatos/metabolismo , Galactosídeos/metabolismo , Glicosilação , Guanosina Difosfato/metabolismo , Guanosina Difosfato Fucose/metabolismo , Guanosina Monofosfato/metabolismo , Cinética , Estrutura Molecular , Conformação Proteica , Glândula Submandibular/enzimologia , Suínos , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
The increase of intracellular cyclic AMP levels suppresses the cytotoxic activities of lymphocytes and monosaccharides interfere with cell to cell and cell to cytokine interactions, and these effects on natural killer (NK)/antibody-dependent cell-mediated cytotoxicity (ADCC) and lymphokine activated killer (LAK) activities were examined. Dibutyryl cyclic AMP markedly suppressed NK, IL-2-augmented NK and ADCC activities in a dose-dependent manner but not previously induced LAK activity. Induction of LAK was inhibited. Dibutyryl cyclic GMP had no effect. Addition of mannose 6-phosphate and galactose 6-phosphate strongly inhibited NK and ADCC activities, but not LAK activity. These results suggest that the lytic mechanism for NK and ADCC activity is different from that of LAK activity.