RESUMEN
Protein structures are dynamic and can explore a large conformational landscape1,2. Only some of these structural substates are important for protein function (such as ligand binding, catalysis and regulation)3-5. How evolution shapes the structural ensemble to optimize a specific function is poorly understood3,4. One of the constraints on the evolution of proteins is the stability of the folded 'native' state. Despite this, 44% of the human proteome contains intrinsically disordered peptide segments greater than 30 residues in length6, the majority of which have no known function7-9. Here we show that the entropic force produced by an intrinsically disordered carboxy terminus (ID-tail) shifts the conformational ensemble of human UDP-α-D-glucose-6-dehydrogenase (UGDH) towards a substate with a high affinity for an allosteric inhibitor. The function of the ID-tail does not depend on its sequence or chemical composition. Instead, the affinity enhancement can be accurately predicted based on the length of the intrinsically disordered segment, and is consistent with the entropic force generated by an unstructured peptide attached to the protein surface10-13. Our data show that the unfolded state of the ID-tail rectifies the dynamics and structure of UGDH to favour inhibitor binding. Because this entropic rectifier does not have any sequence or structural constraints, it is an easily acquired adaptation. This model implies that evolution selects for disordered segments to tune the energy landscape of proteins, which may explain the persistence of intrinsic disorder in the proteome.
Asunto(s)
Entropía , Evolución Molecular , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Uridina Difosfato Glucosa Deshidrogenasa/química , Uridina Difosfato Glucosa Deshidrogenasa/metabolismo , Regulación Alostérica/efectos de los fármacos , Secuencia de Aminoácidos , Humanos , Proteínas Intrínsecamente Desordenadas/antagonistas & inhibidores , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Pliegue de Proteína , Desplegamiento Proteico , Proteoma/química , Proteoma/metabolismo , Especificidad por Sustrato , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidoresRESUMEN
UDP-glucose 6-dehydrogenase (UGDH) produces UDP-α-D-glucuronic acid, the precursors for glycosaminoglycans (GAGs) and proteoglycans of the extracellular matrix. Elevated GAG formation has been implicated in a variety of human diseases, including glioblastoma (GBM). In our previous study, we found that Krüppel-like factor 4 (KLF4) promotes GBM cell migration by binding to methylated DNA, mainly methylated CpGs (mCpG) and transactivating gene expression. We identified UDGH as one of the downstream targets of KLF4-mCpG binding activity. In this study, we show that KLF4 upregulates UGDH expression in a mCpG-dependent manner, and UGDH is required for KLF4-induced cell migration in vitro. UGDH knockdown decreases GAG abundance in GBM cells, as well as cell proliferation and migration in vitro. In intracranial xenografts, reduced UGDH inhibits tumor growth and migration, accompanied by a decrease in the expression of extracellular matrix proteins such as tenascin C, brevican. Our studies demonstrate a novel DNA methylation-dependent UGDH upregulation by KLF4. Developing UGDH antagonists to decrease the synthesis of extracellular matrix components will be a useful strategy for GBM therapy.
Asunto(s)
Neoplasias Encefálicas/enzimología , Glioblastoma/enzimología , Factores de Transcripción de Tipo Kruppel/metabolismo , Regulación hacia Arriba , Uridina Difosfato Glucosa Deshidrogenasa/genética , Animales , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Islas de CpG , Metilación de ADN , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Glioblastoma/genética , Glicosaminoglicanos/metabolismo , Humanos , Factor 4 Similar a Kruppel , Ratones , Trasplante de Neoplasias , Proteoglicanos/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidoresRESUMEN
6-Thiopurine (6TP) is an actively prescribed drug in the treatment of various diseases ranging from Crohn's disease and other inflammatory diseases to acute lymphocytic leukemia and non-Hodgkin's leukemia. While 6TP has beneficial therapeutic uses, severe toxicities are also reported with its use, such as jaundice and liver toxicity. While numerous investigations into the mode in which toxicity originates has been undertaken. None have investigated the effects of inhibition towards UDP-Glucose Dehydrogenase (UDPGDH), an oxidative enzyme responsible for UDP-glucuronic acid (UDPGA) formation or UDP-Glucuronosyl transferase (UGT1A1), which is responsible for the conjugation of bilirubin with UDPGA for excretion. Failure to excrete bilirubin leads to jaundice and liver toxicity. We proposed that either 6TP or its primary oxidative excretion metabolites inhibit one or both of these enzymes, resulting in the observed toxicity from 6TP administration. Inhibition analysis of these purines revealed that 6-thiopurine has weak to no inhibition towards UDPGDH with a Ki of 288⯵M with regard to varying UDP-glucose, but 6-thiouric (primary end metabolite, fully oxidized at carbon 2 and 8, and highly retained by the body) has a near six-fold increased inhibition towards UDPGDH with a Ki of 7⯵M. Inhibition was also observed by 6-thioxanthine (oxidized at carbon 2) and 8-OH-6TP with Ki values of 54 and 14⯵M, respectively. Neither 6-thiopurine or its excretion metabolites were shown to inhibit UGT1A1. Our results show that the C2 and C8 positions of 6TP are pivotal in said inhibition towards UDPGDH and have no effect upon UGT1A1, and that blocking C8 could lead to new analogs with reduced, if not eliminated jaundice and liver toxicities.
Asunto(s)
Bilirrubina/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas , Mercaptopurina/metabolismo , Mercaptopurina/toxicidad , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Uridina Difosfato Glucosa Deshidrogenasa/metabolismo , Antimetabolitos/metabolismo , Antimetabolitos/toxicidad , Hígado/efectos de los fármacos , Hígado/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismoRESUMEN
OBJECTIVE: The purpose of this study was to investigate the in vitro inhibitory effects of the edible microalga Aphanizomenon flos-aquae (AFA) on human UDP-α-d-glucose 6-dehydrogenase (UGDH) activity, a cytosolic enzyme involved both in tumor progression and in phytochemical bioavailability. METHODS: Both the hydrophilic and ethanolic AFA extracts as well as the constitutive active principles phycocyanin (PC), phycocyanobilin (PCB) and mycosporine-like amino acids (MAAs) were tested. RESULTS: Among AFA components, PCB presented the strongest inhibitory effect on UGDH activity, acting as a competitive inhibitor with respect to UDP-glucose and a non-competitive inhibitor with respect to NAD(+). In preliminary experiments, AFA PCB was also effective in reducing the colony formation capacity of PC-3 prostate cancer cells and FTC-133 thyroid cancer cells. CONCLUSIONS: Overall, these findings confirmed that AFA and its active principles are natural compounds with high biological activity. Further studies evaluating the effects of AFA PCB in reducing tumor cell growth and phytochemical glucuronidation are encouraged.
Asunto(s)
Aphanizomenon/química , Inhibidores Enzimáticos/farmacología , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , MasculinoRESUMEN
UDP-glucose dehydrogenases (EC 1.1.1.22) are responsible for the conversion of UDP-glucose to UDP-glucuronic acid, a key precursor in the biosynthesis of glycoconjugates. Herein we report the discovery and characterization of a UDPglucose dehydrogenase (GbUGD) from Granulibacter bethesdensis, a bacterium originally isolated from the lymph nodes of patients with chronic granulomatous disease (CGD). The recombinant form of the protein was expressed in high yield and the purified enzyme showed highest activity at 37°C/pH 9.0 and was strongly inhibited by Zn(2+) ions, sodium dodecyl sulfate (SDS) and urea. UDP-xylose, an allosteric feedback inhibitor, reduced significantly the activity of the enzyme. High activities were observed using the co-substrates UDP-glucose and NAD+, whereas no activity could be detected using other nucleotide sugars or NADP(+) as potential alternative substrates. The high activity combined with the simple purification procedure used make GbUGD a valuable new alternative biocatalyst for the synthesis of UDP-glucuronic acid or the development of NAD+ regeneration systems.
Asunto(s)
Acetobacteraceae/enzimología , Uridina Difosfato Glucosa Deshidrogenasa/química , Uridina Difosfato Glucosa Deshidrogenasa/metabolismo , Acetobacteraceae/genética , Clonación Molecular , Detergentes/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Concentración de Iones de Hidrógeno , Cinética , Metales/farmacología , Desnaturalización Proteica/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Uridina Difosfato Glucosa Deshidrogenasa/genéticaRESUMEN
Human UDP-α-d-glucose-6-dehydrogenase (hUGDH) displays hysteresis because of a slow isomerization from an inactive state (E*) to an active state (E). Here we show that the structure of E* constrains hUGDH in a conformation that favors feedback inhibition at physiological pH. The feedback inhibitor UDP-α-d-xylose (UDP-Xyl) competes with the substrate UDP-α-d-glucose for the active site. Upon binding, UDP-Xyl triggers an allosteric switch that changes the structure and affinity of the intersubunit interface to form a stable but inactive horseshoe-shaped hexamer. Using sedimentation velocity studies and a new crystal structure, we show that E* represents a stable conformational intermediate between the active and feedback-inhibited conformations. Because the allosteric switch occludes the cofactor and substrate binding sites in the inactive hexamer, the intermediate conformation observed in the crystal structure is consistent with the E* transient observed in relaxation studies. Steady-state analysis shows that the E* conformation enhances the affinity of hUGDH for the allosteric inhibitor UDP-Xyl by 8.6-fold (Ki = 810 nM). We present a model in which the constrained quaternary structure permits a small effector molecule to leverage a disproportionately large allosteric response.
Asunto(s)
Uridina Difosfato Glucosa Deshidrogenasa/química , Regulación Alostérica , Unión Competitiva , Dominio Catalítico , Cristalografía por Rayos X , Estabilidad de Enzimas , Retroalimentación Fisiológica , Humanos , Concentración de Iones de Hidrógeno , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Ligandos , Modelos Moleculares , Conformación Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Uridina Difosfato Glucosa Deshidrogenasa/metabolismo , Uridina Difosfato Xilosa/metabolismo , Uridina Difosfato Xilosa/farmacologíaRESUMEN
INTRODUCTION: The objective of this study was to investigate the possible role of UDP-glucose dehydrogenase (UGDH) in osteoarthritis (OA) and uncover whether, furthermore how interleukin-1beta (IL-1ß) affects UGDH gene expression. METHODS: UGDH specific siRNAs were applied to determine the role of UGDH in proteoglycan (PG) synthesis in human articular chondrocytes. Protein levels of UGDH and Sp1 in human and rat OA cartilage were detected. Then, human primary chondrocytes were treated with IL-1ß to find out whether and how IL-1ß could regulate the gene expression of UGDH and its trans-regulators, that is Sp1, Sp3 and c-Krox. Finally, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and stress-activated protein kinase/c-Jun N-terminal kinase (SAP/JNK) inhibitor SP600125 were used to pick out the pathway that mediated the IL-1ß-modulated PGs synthesis and gene expression of UGDH, Sp1, Sp3 and c-Krox. RESULTS: UGDH specific siRNAs markedly inhibited UGDH mRNA and protein expression, and thus led to an obvious suppression of PGs synthesis in human articular chondrocytes. UGDH protein level in human and rat OA cartilage were much lower than the corresponding controls and negatively correlated to the degree of OA. Decrease in Sp1 protein level was also observed in human and rat OA cartilage respectively. Meanwhile, IL-1ß suppressed UGDH gene expression in human articular chondrocytes in the late phase, which also modulated gene expression of Sp1, Sp3 and c-Krox and increased both Sp3/Sp1 and c-Krox/Sp1 ratio. Moreover, the inhibition of SAP/JNK and p38 MAPK pathways both resulted in an obvious attenuation of the IL-1ß-induced suppression on the UGDH gene expression. CONCLUSIONS: UGDH is essential in the PGs synthesis of articular chondrocytes, while the suppressed expression of UGDH might probably be involved in advanced OA, partly due to the modulation of p38 MAPK and SAP/JNK pathways and its trans-regulators by IL-1ß.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Osteoartritis/metabolismo , Proteoglicanos/biosíntesis , Uridina Difosfato Glucosa Deshidrogenasa/biosíntesis , Anciano , Animales , Cartílago Articular/patología , Células Cultivadas , Condrocitos/patología , Femenino , Humanos , Persona de Mediana Edad , Osteoartritis/patología , Proteoglicanos/antagonistas & inhibidores , ARN Interferente Pequeño/farmacología , Ratas , Ratas Wistar , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidoresRESUMEN
UDP-glucose dehydrogenase (Ugd) generates UDP-glucuronic acid, an important precursor for the production of many hexuronic acid-containing bacterial surface glycostructures. In Escherichia coli K-12, Ugd is important for biosynthesis of the environmentally regulated exopolysaccharide known as colanic acid, whereas in other E. coli isolates, the same enzyme is required for production of the constitutive group 1 capsular polysaccharides, which act as virulence determinants. Recent studies have implicated tyrosine phosphorylation in the activation of Ugd from E. coli K-12, although it is not known if this is a feature shared by bacterial Ugd proteins. The activities of Ugd from E. coli K-12 and from the group 1 capsule prototype (serotype K30) were compared. Surprisingly, for both enzymes, site-directed Tyr â Phe mutants affecting the previously proposed phosphorylation site retained similar kinetic properties to the wild-type protein. Purified Ugd from E. coli K-12 had significant levels of NAD substrate inhibition, which could be alleviated by the addition of ATP and several other nucleotide triphosphates. Mutations in a previously identified UDP-glucuronic acid allosteric binding site decreased the binding affinity of the nucleotide triphosphate. Ugd from E. coli serotype K30 was not inhibited by NAD, but its activity still increased in the presence of ATP.
Asunto(s)
Adenosina Trifosfato , Escherichia coli K12/enzimología , Proteínas de Escherichia coli , NAD , Uridina Difosfato Glucosa Deshidrogenasa , Factores de Virulencia , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Escherichia coli K12/genética , Escherichia coli K12/patogenicidad , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Mutagénesis Sitio-Dirigida , Mutación Missense , NAD/química , NAD/genética , NAD/metabolismo , Polisacáridos/biosíntesis , Polisacáridos/química , Polisacáridos/genética , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Uridina Difosfato Glucosa Deshidrogenasa/química , Uridina Difosfato Glucosa Deshidrogenasa/genética , Uridina Difosfato Glucosa Deshidrogenasa/metabolismo , Factores de Virulencia/química , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
UDP-α-D-xylose (UDX) acts as a feedback inhibitor of human UDP-α-D-glucose 6-dehydrogenase (hUGDH) by activating an unusual allosteric switch, the Thr131 loop. UDX binding induces the Thr131 loop to translate ~5 Å through the protein core, changing packing interactions and rotating a helix (α6(136-144)) to favor the formation of an inactive hexameric complex. But how does to conformational change occur given the steric packing constraints of the protein core? To answer this question, we deleted Val132 from the Thr131 loop to approximate an intermediate state in the allosteric transition. The 2.3 Å resolution crystal structure of the deletion construct (Δ132) reveals an open conformation that relaxes steric constraints and facilitates repacking of the protein core. Sedimentation velocity studies show that the open conformation stabilizes the Δ132 construct as a hexamer with point group symmetry 32, similar to that of the active complex. In contrast, the UDX-inhibited enzyme forms a lower-symmetry, horseshoe-shaped hexameric complex. We show that the Δ132 and UDX-inhibited structures have similar hexamer-building interfaces, suggesting that the hinge-bending motion represents a path for the allosteric transition between the different hexameric states. On the basis of (i) main chain flexibility and (ii) a model of the conformational change, we propose that hinge bending can occur as a concerted motion between adjacent subunits in the high-symmetry hexamer. We combine these results in a structurally detailed model for allosteric feedback inhibition and substrate--product exchange during the catalytic cycle.
Asunto(s)
Uridina Difosfato Glucosa Deshidrogenasa/química , Regulación Alostérica/genética , Catálisis , Cristalografía por Rayos X , Retroalimentación Fisiológica , Eliminación de Gen , Humanos , Modelos Moleculares , Conformación Proteica , Especificidad por Sustrato/genética , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Uridina Difosfato Glucosa Deshidrogenasa/genéticaRESUMEN
4-Methylumbelliferone (4-MU) is described as a selective inhibitor of hyaluronan (HA) production. It is thought that 4-MU depletes UDP-glucuronic acid (UDP-GlcUA) substrate for HA synthesis and also suppresses HA-synthase expression. The possibility that 4-MU exerts at least some of its actions via regulation of UDP-glucose dehydrogenase (UGDH), a key enzyme required for both HA and sulphated-glycosaminoglycan (sGAG) production, remains unexplored. We therefore examined the effects of 4-MU on basal and retroviral UGDH-driven HA and sGAG release in cells derived from chick articular cartilage and its influence upon UGDH protein and mRNA expression and HA and sGAG production. We found that 4-MU: i) suppressed UGDH mRNA and protein expression and chondrogenic matrix accumulation in chick limb bud micromass culture, ii) significantly reduced both HA and sGAG production and iii) more selectively reversed the potentiating effects of UGDH overexpression on the production of HA than sGAG. Understanding how GAG synthesis is controlled and the mechanism of 4-MU action may inform its future clinical success.
Asunto(s)
Condrogénesis/efectos de los fármacos , Matriz Extracelular/metabolismo , Ácido Hialurónico/antagonistas & inhibidores , Himecromona/análogos & derivados , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Animales , Células Cultivadas , Embrión de Pollo , Medios de Cultivo Condicionados , Glicosaminoglicanos/antagonistas & inhibidores , Glicosaminoglicanos/metabolismo , Ácido Hialurónico/metabolismo , Himecromona/farmacología , Esbozos de los Miembros/citología , Terapia Molecular Dirigida , Transcripción Genética , Uridina Difosfato Glucosa Deshidrogenasa/biosíntesis , Uridina Difosfato Glucosa Deshidrogenasa/genéticaRESUMEN
Biosynthesis of the glycosaminoglycan precursor UDP-α-D-glucuronic acid occurs through a 2-fold oxidation of UDP-α-D-glucose that is catalysed by UGDH (UDP-α-D-glucose 6-dehydrogenase). Structure-function relationships for UGDH and proposals for the enzymatic reaction mechanism are reviewed in the present paper, and structure-based sequence comparison is used for subclassification of UGDH family members. The eukaryotic group of enzymes (UGDH-II) utilize an extended C-terminal domain for the formation of complex homohexameric assemblies. The comparably simpler oligomerization behaviour of the prokaryotic group of enzymes (UGDH-I), in which dimeric forms prevail, is traced back to the lack of relevant intersubunit contacts and trimmings within the C-terminal region. The active site of UGDH contains a highly conserved cysteine residue, which plays a key role in covalent catalysis. Elevated glycosaminoglycan formation is implicated in a variety of human diseases, including the progression of tumours. The inhibition of synthesis of UDP-α-D-glucuronic acid using UGDH antagonists might therefore be a useful strategy for therapy.
Asunto(s)
Uridina Difosfato Glucosa Deshidrogenasa/química , Uridina Difosfato Glucosa Deshidrogenasa/metabolismo , Animales , Inhibidores Enzimáticos/uso terapéutico , Humanos , Ácido Hialurónico/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Uridina Difosfato Glucosa Deshidrogenasa/genética , Uridina Difosfato Ácido Glucurónico/metabolismoRESUMEN
We have examined polyphenols as potential inhibitors of UDP-glucose dehydrogenase (UGDH) activity. Gallic acid and quercetin decreased specific activities of UGDH and inhibited the proliferation of MCF-7 human breast cancer cells. Western blot analysis showed that gallic acid and quercetin did not affect UGDH protein expression, suggesting that UGDH activity is inhibited by polyphenols at the post-translational level. Kinetics studies using human UGDH revealed that gallic acid was a non-competitive inhibitor with respect to UDP-glucose and NAD+. In contrast, quercetin showed a competitive inhibition and a mixed-type inhibition with respect to UDP-glucose and NAD+, respectively. These results indicate that gallic acid and quercetin are effective inhibitors of UGDH that exert strong antiproliferative activity in breast cancer cells.
Asunto(s)
Neoplasias de la Mama/enzimología , Inhibidores Enzimáticos/farmacología , Ácido Gálico/farmacología , Quercetina/farmacología , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , NAD/farmacología , Uridina Difosfato Glucosa/farmacologíaRESUMEN
The effects of piperine, a major ingredient of black pepper, on UDP-glucose dehydrogenase (UDP-GDH) and glucuronidation potentials of rat and guinea pig liver and intestine were studied. Piperine caused a concentration-related strong inhibition of UDP-GDH (50% at 10 microM) reversibly and equipotently, in both tissues. Partially purified rat liver UDP-GDH was used to obtain the kinetic values at pH optima of 9.4 and 8.6. At pH 9.4: KmUDP-glucose = 15 microM, Vmax = 5.2 nmol NADH/min/mg protein, Ki = 6 microM. With NAD, a Ki of 16 microM was obtained. At pH 8.6: Km = 35 microM, Vmax = 7.5 nmol, Ki = 15 microM. In all of these cases, piperine caused non-competitive inhibition. Data from structure-activity comparisons of piperine analogs indicated that the presence of conjugated double bonds in the side chain of the molecule is a factor in piperine inhibition. However, the UDP-glucuronic acid (UDPGA) contents were decreased less effectively by piperine in isolated rat hepatocytes compared with enterocytes of guinea pig small intestine. Piperine at 50 microM caused a marginal decrease of UDPGA in hepatocytes when the rate of glucuronidation of 3-hydroxybenzo[a]pyrene (3-OH-BP) decreased by about 40%. The decrease obtained at 10 microM piperine in intestinal cells was comparable to that obtained at 50-100 microM in hepatocytes. UDP-glucuronosyltransferase (UGT) activities towards 3-OH-BP (UGT1A1) and 4-OH-biphenyl (UGT2B1) were also determined. Piperine did not affect the rate of glucuronidation of 4-OH-biphenyl in rat liver, whereas that of 3-OH-BP was impaired significantly. In guinea pig small intestine, both these activities were inhibited significantly requiring less than 25 microM piperine to produce a more than 50% inhibition of UGT(s). The results suggested that (i) piperine is a potent inhibitor of UDP-GDH, (ii) inhibition is offered exclusively by the conjugated double bonds of the molecule, and (iii) piperine exerts stronger effects on intestinal glucuronidation than in rat liver.
Asunto(s)
Alcaloides , Glucuronidasa/metabolismo , Intestino Delgado/enzimología , Hígado/enzimología , Piperidinas/farmacología , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Animales , Benzodioxoles , Células Cultivadas , Cobayas , Cinética , Masculino , Microsomas Hepáticos/enzimología , Alcamidas Poliinsaturadas , Ratas , Relación Estructura-Actividad , Uridina Difosfato Glucosa Deshidrogenasa/análisisRESUMEN
The enzymatic basis for inhibition of drug glucuronidation during ethanol oxidation was investigated in isolated rat hepatocytes. The intracellular rate of glucuronidation was varied independently by controlling the steady-state O2 concentration and the concentrations of UDP-glucose and UDP-glucuronic acid were measured in the absence and presence of 20 mM ethanol. Ethanol caused substantial inhibition of the glucuronidation rate which corresponded to a significant decrease in UDP-glucuronic acid concentration but not in UDP-glucose concentration. A plot of glucuronidation rate as a function of cellular UDP-glucuronic acid concentration yielded a single curve for incubations with or without ethanol; a similar plot of glucuronidation against UDP-glucose concentration gave separate curves for the two incubation conditions. These results clearly define the UDP-glucose dehydrogenase reaction as the site of inhibition during ethanol oxidation.
Asunto(s)
Deshidrogenasas de Carbohidratos/antagonistas & inhibidores , Etanol/metabolismo , Hígado/enzimología , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Acetaminofén/análogos & derivados , Acetaminofén/aislamiento & purificación , Acetaminofén/metabolismo , Aerobiosis , Animales , Cinética , Masculino , Consumo de Oxígeno , RatasAsunto(s)
Deshidrogenasas de Carbohidratos/antagonistas & inhibidores , Fibroblastos/enzimología , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Azúcares de Uridina Difosfato/farmacología , Uridina Difosfato Xilosa/farmacología , Células Cultivadas , Histocitoquímica , HumanosRESUMEN
Half-of-the-sites reactivity of the catalytic site thiol groups of UDPglucose dehydrogenase (UDPglucose:NAD+ 6-oxidoreductase, EC 1.1.1.22) can be ascribed either to the induction of conformational asymmetry following derivatization of one half of the subunits or to intrinsic conformational differences in the subunits of the native enzyme. If the half-sites reactivity behavior is due to induction effects, the magnitude of the induction could be expected to depend on the nature of the covalent modification. On the other hand, if the half-sites reactivity behavior is due to pre-existing asymmetry and there is no communication between catalytic centers, the properties of unmodified sub-units should be independent of the nature of the covalent derivative introduced on the modified subunits. According to the induced asymmetry hypothesis, the catalytic activity of half-sites modified enzyme might be different for different covalent modifications, whereas for the rigid pre-existing asymmetry hypothesis the catalytic activity of half-sites modified enzyme should be the same regardless of the modifying group. During the course of catalytic site thiol group modification by a number of thiol specific reagents, the loss of enzyme activity was equivalent to the degree of modification for most of the reagents employed. However, with iodoacetate and 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid, half-sites modification of UDPglucose dehydrogenase reduced catalytic activity by 58 and 78%, respectively, of the initial activity. These observations are consistent with a model in which there is communication between catalytic sites. Electron microscopy shows that the six subunits of UDPglucose dehydrogenase are arranged as a hexagonal planar ensemble.
Asunto(s)
Deshidrogenasas de Carbohidratos/antagonistas & inhibidores , Hígado/enzimología , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Animales , Sitios de Unión/efectos de los fármacos , Bovinos , Yodoacetamida/análogos & derivados , Yodoacetamida/farmacología , Yodoacetatos/farmacología , Microscopía Electrónica , Naftalenosulfonatos/farmacología , Conformación Proteica/efectos de los fármacosRESUMEN
The influence of incubation with D-penicillamine on pure enzyme preparations and on enzymatic activities of serum and skin homogenates was investigated. Three of the nine enzymatic activities studied underwent significant changes. Such effects of D-penicillamine must be taken into consideration if therapeutic or unwanted actions of this drug are to be fully understood; they are elicited by concentrations reached under conditions used for human therapy.
Asunto(s)
Enzimas/metabolismo , Penicilamina/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Fosfatasa Alcalina/antagonistas & inhibidores , Animales , Glucosafosfato Deshidrogenasa/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Humanos , Ratas , Piel/enzimología , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidoresAsunto(s)
Deshidrogenasas de Carbohidratos , Hígado/enzimología , Uridina Difosfato Glucosa Deshidrogenasa , Aminoácidos/análisis , Animales , Sitios de Unión , Deshidrogenasas de Carbohidratos/antagonistas & inhibidores , Bovinos , Ratas , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidoresAsunto(s)
Oxidorreductasas de Alcohol/efectos de la radiación , Uridina Difosfato Glucosa Deshidrogenasa/efectos de la radiación , Animales , Catálisis , Cisteamina/farmacología , Densitometría , Relación Dosis-Respuesta en la Radiación , Electroforesis en Gel de Poliacrilamida , Rayos gamma , Hígado/enzimología , Ratas , Espectrofotometría , Uridina Difosfato Glucosa/farmacología , Uridina Difosfato Glucosa Deshidrogenasa/análisis , Uridina Difosfato Glucosa Deshidrogenasa/antagonistas & inhibidores , Uridina Difosfato Xilosa/farmacologíaRESUMEN
During the transition from primary wall formation to secondary thickening there is a marked shift in the synthesis of pectin, hemicellulose and cellulose. The activities of the enzymes [UDP-D-galactose 4-epimerase (EC 5.1.3.2)8 UDP-l-arabinose 4-epimerase (EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D--glucuronate decarboxylase (EC 4.1.1.35)] were measured in cambial cells, differentiating xylem cells and differentiated xylem cells isolated from sycamore and poplar trees, and phloem cells from poplar. At the final stage of the differentiation of cambium to xylem there was a decrease in activity of the enzymes directly involved in producing the soluble precursors of pectin (DUP-D-galactose 4-epimerase and UDP-L-arabinose 4-epimerase and an increase in those producing the precursors of hemicellulose (UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase). These results strongly suggest ahat the changes were correlated with the differences observed in the chemical composition of the wall during development. The changes found in the catalytic activity of the enzymes of nucleoside diphosphate sugar interconversion exert a coarse control over the synthesis of pectin and hemicelluloses. The tissues at all stages of development contained the necessary enzyme activities to produce all the precursors of pectin and hemicellulose, even at the final stage of differentiation when no pectin was formed.