RESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) play an important function in various biochemical processes as they generate reducing power of the cell. Thus, metabolic reprogramming of reduced nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis is reported to be a vital step in cancer progression as well as in combinational therapeutic approaches. In this study, N-benzoylindoles 9a--9d, which form the main framework of many natural indole derivatives such as indomethacin and N-benzoylindoylbarbituric acid, were synthesized through three easy and effective steps as an in vitro inhibitor effect of G6PD and 6PGD. The N-benzoylindoles inhibited the enzymatic activity with IC50 in the range of 3.391505 µM for G6PD and 2.19-990 µM for 6PGD.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Eritrocitos/enzimología , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Indoles/farmacología , Modelos Moleculares , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/metabolismo , Sitios de Unión , Unión Competitiva , Espectroscopía de Resonancia Magnética con Carbono-13 , Cromatografía de Afinidad , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/síntesis química , Activadores de Enzimas/química , Activadores de Enzimas/metabolismo , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Glucosafosfato Deshidrogenasa/metabolismo , Indoles/síntesis química , Indoles/química , Indoles/metabolismo , Cinética , Estructura Molecular , NADP/química , NADP/metabolismo , Fosfogluconato Deshidrogenasa/química , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Fosfogluconato Deshidrogenasa/metabolismo , Espectroscopía de Protones por Resonancia Magnética , Ratas , Homología Estructural de Proteína , Temperatura de TransiciónRESUMEN
G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2', 5'-ADP Sepharose 4B affinity chromatography. The purified enzymes showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the enzymes were estimated to be 134 kDa for G6PD, 107 kDa for 6PGD and 121 kDa for GR by Sephadex G-150 gel filtration chromatography, and the subunit molecular weights was respectively found to be 66, 52 and 63 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, optimum temperature, KM and Vmax values for substrates were determined. Product inhibition studies were also performed. The enzymes were inhibited by levofloxacin, furosemide, ceftazidime, cefuroxime and gentamicin as in vitro with IC50 values in the range of 0.07-30.13 mM. In vivo studies demonstrated that lung GR was inhibited by furosemide and lung 6PGD was inhibited by levofloxacin.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Glutatión Reductasa/aislamiento & purificación , Pulmón/enzimología , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Animales , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Glucosafosfato Deshidrogenasa/metabolismo , Glutatión Reductasa/antagonistas & inhibidores , Glutatión Reductasa/metabolismo , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive.
Asunto(s)
Digoxina/química , Dopamina/química , Inhibidores Enzimáticos/química , Furosemida/química , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Glutatión Reductasa/aislamiento & purificación , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Animales , Unión Competitiva , Pruebas de Enzimas , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Glucosafosfato Deshidrogenasa/química , Glutatión Reductasa/antagonistas & inhibidores , Glutatión Reductasa/química , Concentración de Iones de Hidrógeno , Cinética , Masculino , Peso Molecular , Miocardio/química , Miocardio/enzimología , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/química , Unión Proteica , Ratas , Ratas Sprague-Dawley , Especificidad por Sustrato , TemperaturaRESUMEN
Considerable information exists on the physiological correlates of life history adaptation, while molecular data on this topic are rapidly accumulating. However, much less is known about the enzymological basis of life history adaptation in outbred populations. In the present study, we compared developmental profiles of fat body specific activity, kinetic constants of homogeneously purified and unpurified enzyme, and fat body enzyme concentration of the pentose-shunt enzyme, 6-phosphogluconate dehydrogenase (6PGDH, E.C.1.1.1.44) between the dispersing [long-winged, LW(f)] and flightless [short-winged, SW] genotypes of the cricket Gryllus firmus. Neither kcat nor the Michaelis constant for 6-phosphogluconate differed between 6PGDH from LW(f) versus SW morphs for either homogeneously purified or unpurified enzyme. Purified enzyme from the LW(f) morph exhibited reduced KM for NADP(+), but this was not observed for multiple KM(NADP+) estimates for unpurified enzyme. A polyclonal antibody was generated against 6PGDH which was used to develop a chemiluminescence assay to quantify 6PGDH concentration in fat body homogenates. Elevated enzyme concentration accounted for all of the elevated 6PGDH specific activity in the LW(f) morph during the juvenile and adult stages. Finally, activity of another pentose-shunt enzyme, glucose-6-phosphate dehydrogenase, strongly covaried with 6PGDH activity suggesting that variation in 6PGDH activity gives rise to variation in pentose shunt flux. This is one of the first life-history studies and one of the few studies of intraspecific enzyme adaptation to identify the relative importance of evolutionary change in enzyme concentration vs. kinetic constants to adaptive variation in enzyme activity in an outbred population.
Asunto(s)
Gryllidae/enzimología , Proteínas de Insectos/metabolismo , Fosfogluconato Deshidrogenasa/metabolismo , Alas de Animales/enzimología , Adaptación Fisiológica , Animales , Cuerpo Adiposo/enzimología , Proteínas de Insectos/inmunología , Proteínas de Insectos/aislamiento & purificación , Isoenzimas , Cinética , Mediciones Luminiscentes/métodos , Especificidad de Órganos , Fosfogluconato Deshidrogenasa/análisis , Fosfogluconato Deshidrogenasa/inmunología , Fosfogluconato Deshidrogenasa/aislamiento & purificación , ConejosRESUMEN
6-Phosphogluconate dehydrogenase (6PGD), the third enzyme in the pentose phosphate pathway, was recently identified as a novel target in human lung cancer. In this report, we present an expression and purification scheme of recombinant human 6PGD from Escherichia coli. Using a DE3 derivative strain expressing tRNAs for seven rare codons in E. coli called Rosetta2 (DE3), a large quantity of soluble human 6PGD can be expressed with an N-terminal histidine tag and purified by a one-step purification procedure to near homogeneity without denaturants or refolding. Three to seven milligrams of purified protein could be obtained from 100 ml of culture. This recombinant human 6PGD follows classic Michaelis-Menton saturation kinetics with respect to both substrates NADP(+) and 6-phosphogluconate. The respective k(cat) and K(m) were comparable to those of 6PGDs purified from mammalian tissues. Using this purified 6PGD enzyme, we devised an endpoint colorimetric assay suitable for high-throughput screening for human 6PGD inhibitors.
Asunto(s)
Histidina/genética , Histidina/aislamiento & purificación , Fosfogluconato Deshidrogenasa/genética , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Gluconatos/metabolismo , Histidina/química , Humanos , Cinética , Datos de Secuencia Molecular , Fosfogluconato Deshidrogenasa/química , Fosfogluconato Deshidrogenasa/metabolismo , Plásmidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , SolubilidadRESUMEN
6-Phosphogluconate dehydrogenase (6-PGD, E.C.: 1.1.1.44) was purified and characterized from the hepatopancreas of grass carp (Ctenopharyngodon idella) for the first time. Grass carp represents the second largest aquaculture industry in the world after silver carp, constituting 14.7% of the world aquaculture production, with an average annual increase of 14% in China, mainly as a source of food. The purification procedure involved a single 2', 5'-ADP-Sepharose 4B affinity chromatographic step by using different elution buffers. The enzyme was purified 309-fold with a specific activity of 5.259 U/mg protein and yield of 68%. The purity and subunit molecular weights of the 6-PGD were checked on SDS-PAGE and purified enzyme showed a single band on the gel. The subunit molecular mass was 57 kDa, with an optimum pH, temperature and ionic strength at 7.96, 50 degrees C and 100 mM Tris-HCl, respectively. The Km values of 6-PGA and NADP+ were 0.019 and 0.0052 mM, respectively, while Vm of 6-PGA and NADP+ was 0.69 U/ml. Dissociation constants (Ki) for 6-PGA and NADP+ were 2.05 and 0.12 mM, respectively. NADPH inhibited the enzyme in a competitive manner and its Ki value was 0.032 mM. The Cu2+, Zn2+, Cd2+ and Al3+ showed inhibitory effects on the enzyme with IC50 values of 0.293, 0.099, 0.045 and 1.526 mM, respectively. All tested metals inhibited the enzyme in a competitive manner, indicating that these metals might be toxic even at low concentrations for the 6-PGD. As the fish is one of valuable foodstuff of animal sources for human consumption, under certain environmental conditions, metal ions accumulated in fish up to a lethal concentration may be harmful for human health. Therefore, it is impending to reduce the concentration of metal ions in contaminated lakes and rivers for fishery and also for human health.
Asunto(s)
Carpas , Hepatopáncreas/enzimología , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Fosfogluconato Deshidrogenasa/metabolismo , Animales , Concentración de Iones de Hidrógeno , Cinética , Fosfatos/metabolismo , TemperaturaRESUMEN
Haemophilus parasuis (H. parasuis) is a swine pathogen responsible for the Glässer's disease. In order to understand the pathogenesis of the H. parasuis infection, the gnd gene encoding a cell surface protein, 6-phosphogluconate-dehydrogenase (6PGD) of H. parasuis was inducibly expressed in Escherichia coli BL21 with a hexahistidyl N-terminus to permit its purification. Western blotting using the r6PGD-specific antiserum showed that the 6PGD protein is on the cell surface of H. parasuis. The characterization of 6PGD in H. parasuis pathogenesis involved as an adhesion and its immunogenicity in mice was further investigated. The adherence assay with H. parasuis and swine alveolar epithelial cells (SJPLC) pre-incubated with (His)(6)6PGD and non-incubated SJPLC showed a noticeable reduction in the adhesion of H. parasuis in the (His)(6)6PGD pre-incubated SJPLC compared to the non-incubated SJPLC. Further, the r6PGD protein induces the production of IL-8 and IL-6 by SJPLC. Furthermore, immunization with the r6PGD protein can provide the protective efficacy by 75% following intraperitoneal administration of a 5×LD(50) dose of H. parasuis SH0165, and elicited a good protective immune response, which demonstrated the importance of 6PGD to bacterial pathogenesis. Identification and characterization of the role of H. parasuis 6PGD in adhesion and immunogenicity will allow us to use this protein to develop new antimicrobial therapies and/or vaccines.
Asunto(s)
Haemophilus parasuis/enzimología , Fosfogluconato Deshidrogenasa/genética , Animales , Western Blotting , Pared Celular/enzimología , Pared Celular/genética , Clonación Molecular , ADN Bacteriano/genética , Femenino , Genes Bacterianos/genética , Haemophilus parasuis/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Fosfogluconato Deshidrogenasa/biosíntesis , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Alveolos Pulmonares/microbiología , Mucosa Respiratoria/microbiología , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Porcinos/microbiología , Enfermedades de los Porcinos/microbiologíaRESUMEN
The enzymes of glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and glutathione reductase (GR) were purified from rat kidney in one chromatographic step consisting of the use of the 2',5'-ADP Sepharose 4B by using different elution buffers. This purification procedure was accomplished with the preparation of the homogenate and affinity chromatography on 2',5'-ADP Sepharose 4B. The purity and subunit molecular weights of the enzymes were checked on SDS-PAGE and purified enzymes showed a single band on the gel. The native molecular weights of the enzymes were found with Sephadex G-150 gel filtration chromatography. Using this procedure, G6PG, having the specific activity of 32 EU/mg protein, was purified 531-fold with a yield of 88%; 6PGD, having the specific activity of 25 EU/mg protein, was purified 494-fold with a yield of 73%; and GR, having the specific activity of 33 EU/mg protein, was purified 477-fold with a yield of 76%. Their native molecular masses were estimated to be 144 kDa for G6PD, 110 kDa for 6PGD, and 121 kDa for GR and the subunit molecular weights were found to be 68, 56, and 61 kDa, respectively. A new modified method to purify G6PD, 6PGD, and GR, namely one chromatographic step using the 2',5'-ADP Sepharose 4B, is described for the first time in this study. This procedure has several advantages for purification of enzymes, such as, rapid purification, produces high yield, and uses less chemical materials.
Asunto(s)
Cromatografía de Afinidad/métodos , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Glutatión Reductasa/aislamiento & purificación , Riñón/enzimología , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Sefarosa/análogos & derivados , Animales , Tampones (Química) , Electroforesis en Gel de Poliacrilamida , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/metabolismo , Glutatión Reductasa/química , Glutatión Reductasa/metabolismo , Riñón/química , Peso Molecular , Fosfogluconato Deshidrogenasa/química , Fosfogluconato Deshidrogenasa/metabolismo , Ratas , Ratas Sprague-Dawley , Sefarosa/químicaRESUMEN
The inhibitory effects of some drugs on 6-phosphogluconate dehydrogenase from human erythrocytes have been investigated. For this purpose, initially, erythrocyte 6-phosphogluconate dehydrogenase was purified 3364 times in a yield of 58% by using ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity gel. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. This method was utilized for all kinetic studies. Many commonly used drugs were investigated in this study. Some drugs (ketotifen (K(i): 8.3 +/- 1.7 microM), dacarbazine (K(i): 10.1 +/- 0.7 microM), meloxicam (K(i): 50.9 +/- 13.2 microM), furosemide (K(i): 127 +/- 37.8 microM), methotrexate (K(i): 136.7 +/- 25.3 microM), metochloropramide hydrochloride (K(i): 2.1113 +/- 0.6979 mM), ritodrine hydrochloride (K(i): 6.0353 +/- 1.2783 mM), and gadopentetic acid (K(i): 73.4 +/- 21.9 mM)) inhibited enzyme activity in vitro. K(i) constants for the enzyme were found by means of Lineweaver-Burk graphs. All drugs showed non-competitive inhibition. In addition, IC(50) values of the drugs were determined by plotting activity percent vs [I].
Asunto(s)
Eritrocitos/enzimología , Fosfogluconato Deshidrogenasa/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Cinética , Preparaciones Farmacéuticas , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Análisis EspectralRESUMEN
Inhibitory effects of some drugs were investigated on human erythrocyte 6-phosphogluconate dehydrogenase obtained with a 6552-fold purification in a yield of 78% using 2', 5'-ADP Separose 4B affinity gel. Which on SDS polyacrylamide gel electrophoresis showed a single band. Larnoxicam, metronidazole, imipenem, ornidazole, vancomycin, clindamycin, and amoxicillin exhibited inhibitory effects on the enzyme in vitro with IC50 values of 0.17, 0.23, 0.43, 21.79, 46.39, 117.43 and 287.35 mM, and the Ki constants 0.40 +/- 0.04, 0.57 +/- 0.06, 0.77 +/- 0.11, 42.40 +/- 2.89, 65.60 +/- 4.03, 130.22 +/- 9.21, and 287.58 +/- 10.56 mM, respectively. While vancomycin, clindamycin and amoxicillin showed competitive inhibition the other drugs displayed noncompetitive inhibition.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Eritrocitos/efectos de los fármacos , Eritrocitos/enzimología , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/metabolismo , Humanos , Fosfogluconato Deshidrogenasa/aislamiento & purificaciónRESUMEN
6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg. On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed. The pH optimum was 7.35. Using Arrhenius plots, Ea, DeltaH, Q10 and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4 degrees C, respectively. The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 595 +/- 213 microM for 6PG and 53.03+/-1.99 microM for NADP. 1/Vm versus 1/6PG and 1/NADP plots gave a Vm value of 8.91+/-1.92 U/mg protein. NADPH is the competitive inhibitor with a Ki of 31.91+/-1.31 microM. The relatively small Ki for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD and 6PGD.
Asunto(s)
Intestino Delgado/enzimología , Fosfogluconato Deshidrogenasa/metabolismo , Animales , Activación Enzimática , Femenino , Concentración de Iones de Hidrógeno , Cinética , Peso Molecular , Fosfogluconato Deshidrogenasa/química , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Ratas , Temperatura , TermodinámicaRESUMEN
6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44; 6PGD) was purified from chicken liver; some kinetic and characteristic properties of the enzyme were investigated. The purification procedure consisted of four steps: preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Thanks to the four consecutive procedures, product having a specific activity of 61 U (mg proteins)(-1), was purified 344-fold with a yield of 5.57%. Optimum pH, stable pH, optimum temperature, and KM and Vmax values for NADP+ and 6PG substrates were determined for the enzyme. Molecular weight of the enzyme was also determined by Sephadex G-200 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Ki values and inhibition types were estimated by means of Lineweaver-Burk graphs obtained for NADPH and CO2 products.
Asunto(s)
Fraccionamiento Químico/métodos , Cromatografía de Afinidad/métodos , Fosfogluconato Deshidrogenasa/química , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Animales , Pollos , Dextranos , Activación Enzimática , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Especificidad por Sustrato , TemperaturaRESUMEN
The in vitro and in vivo effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase were investigated. Human erythrocyte 6-phosphogluconate dehydrogenase was purified with ammonium sulphate precipitation, 2',5' ADP-Sepharose 4B affinity and gel filtration chromatography. Some antibiotics (netilmicin sulphate, cefepime, amikacin, isepamycin, chloramphenicol, ceftazidim, teicoplanin, ampicillin, ofloxacin, levofloxacin, cefotaxime, penicillin G, gentamicin sulphate, ciprofloxacin) inhibited enzyme activity in vitro but others (cefozin, decefin, streptomycin, combisid, and meronem) were devoid of inhibitory effects. For the drugs having low IC50 values (netilmicin sulphate and cefepime), in vivo studies were performed in rats. Netilmicin sulphate at 15-mg/kg inhibited enzyme activity significantly (p < 0.001) 1 h, 2 h, and 3 h after dosing and cefepime at 200-mg/kg very significantly (p < 0.001) inhibited the enzyme 1 h and 2 h after dosing. Netilmicin sulphate and cefepime inhibited rat erythrocyte 6-phosphogluconate dehydrogenase both in vivo and in-vitro.
Asunto(s)
Antibacterianos/farmacología , Eritrocitos/efectos de los fármacos , Eritrocitos/enzimología , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Humanos , Fosfogluconato Deshidrogenasa/química , Fosfogluconato Deshidrogenasa/aislamiento & purificación , RatasRESUMEN
In this study, 6-phosphogluconate dehydrogenase (E.C.1.1.44; 6PGD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps that are preparation of homogenate ammonium sulfate fractionation and on DEAE-Sephadex A50 ion exchange. The enzyme was obtained with a yield of 49% and had a specific activity of 18.3 U (mg proteins)(-1) (Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry, 2nd Ed.; Worth Publishers Inc.: N.Y., 2000, 558-560). The overall purification was about 339-fold. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method at 340 mn. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 97.5 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a subunit molecular weight of 24.1 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found as 8.0, 8.0, and 50 degrees C, respectively. In addition, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk plots.
Asunto(s)
Petroselinum/química , Fosfogluconato Deshidrogenasa/química , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Hojas de la Planta/química , Activación Enzimática , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Peso Molecular , Petroselinum/enzimología , Fosfogluconato Deshidrogenasa/biosíntesis , Hojas de la Planta/enzimología , TemperaturaRESUMEN
Previous attempts to purify chloroplast 6-phosphogluconate dehydrogenase (cp6PGDH), a key enzyme of the oxidative pentose phosphate pathway, have been unsuccessful due to rapid activity loss. An efficient purification protocol was developed and the enzyme from spinach leaves was purified 1000-fold to apparent homogeneity with a specific activity of 60 U.mg-1. The enzyme is a homodimer with subunits of 50 kDa. Antibodies raised against the purified cp6PGDH detected a 53-kDa protein from a crude extract, indicating alterations during purification. Purified cp6PGDH was microsequenced and the corresponding spinach cDNA was cloned using PCR techniques and degenerate primers. The cDNA for cytosolic 6PGDH from spinach was cloned for comparison. Phylogenetic analysis in the context of available homologues from eukaryotes and eubacteria revealed that animal and fungal cytosolic 6PGDH sequences are more similar to their homologues from gamma-proteobacteria, whereas plant 6PGDH is more similar to its cyanobacterial homologues. The ancestral gene for higher plant 6PGDH was acquired from the antecedent of plastids through endosymbiosis and gene transfer to the nucleus. A subsequent gene duplication gave rise to higher plant cytosolic 6PGDH, which assumed the function of its pre-existing cytosolic homologue through endosymbiotic gene replacement. The protein phylogeny of both 6PGDH and of the first enzyme of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase, indicate a surprisingly close relationship between the plant and Trypanosoma brucei lineages, suggesting that T. brucei (a relative of Euglena gracilis) may be secondarily nonphotosynthetic.
Asunto(s)
Cloroplastos/enzimología , Cloroplastos/genética , Fosfogluconato Deshidrogenasa/genética , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Spinacia oleracea/enzimología , Spinacia oleracea/genética , Secuencia de Aminoácidos , Animales , Archaea/enzimología , Archaea/genética , Secuencia de Bases , Clonación Molecular , Cianobacterias/enzimología , Cianobacterias/genética , Citosol/enzimología , Cartilla de ADN/genética , Genes Bacterianos , Genes de Plantas , Isoenzimas/genética , Datos de Secuencia Molecular , Filogenia , Plastidios/enzimología , Plastidios/genética , Homología de Secuencia de Aminoácido , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genéticaRESUMEN
The glucose-6-phosphate (Glc6P) and 6-phosphogluconate (6PG) dehydrogenases of the amino-acid-producing bacterium Corynebacterium glutamicum were purified to homogeneity and kinetically characterized. The Glc6P dehydrogenase was a heteromultimeric complex, which consists of Zwf and OpcA subunits. The product inhibition pattern of the Glc6P dehydrogenase was consistent with an ordered bi-bi mechanism. The 6PG dehydrogenase was found to operate according to a Theorell-Chance ordered bi-ter mechanism. Both enzymes were inhibited by NADPH and the 6PG dehydrogenase additionally by ATP, fructose 1,6-bisphosphate (Fru1,6P2), D-glyceraldehyde 3-phosphate (Gra3P), erythrose 4-phosphate and ribulose 5-phosphate (Rib5P). The inhibition by NADPH was considered to be most important, with inhibition constants of around 25 microM for both enzymes. Intracellular metabolite concentrations were determined in two isogenic strains of C. glutamicum with plasmid-encoded NAD- and NADP-dependent glutamate dehydrogenases. NADP+ and NADPH levels were between 130 microM and 290 microM, which is very much higher than the respective Km and Ki values. The Glc6P concentration was around 500 microM in both strains. The in vivo fluxes through the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified enzymes determined in vitro were in agreement with the same fluxes determined by NMR after 13C-labelling. From the derived kinetic model thus validated, it is concluded that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH and NADP+ concentrations and the specific enzyme activities of both dehydrogenases.
Asunto(s)
Corynebacterium/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Vía de Pentosa Fosfato , Fosfogluconato Deshidrogenasa/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Isótopos de Carbono , Fructosadifosfatos/metabolismo , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Glutamato Deshidrogenasa/genética , Glutamato Deshidrogenasa/metabolismo , Gliceraldehído 3-Fosfato/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutación , NAD/metabolismo , NADP/metabolismo , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Ribulosafosfatos/metabolismo , Fosfatos de Azúcar/metabolismoRESUMEN
The mRNA encoding the 51-kDa subunit of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver was reverse-transcribed and amplified. The resulting cDNA was reamplified in N-terminal and C-terminal segments and spliced to generate a full-length clone, and an internal cDNA fragment was also amplified. The full-length clone containing the complete coding sequence of the 6PGDH cDNA was sequenced and found to contain two mutations and two deletions in the internal region and two mutations outside of the internal region, an A to G point mutation at position 1407 that resulted in the amino acid change Gln 445 to Arg and a silent mutation at position 1426. The internal clone was sequenced and shown to be free of any mutations; therefore the internal piece was used to replace the same region in the full-length clone to correct the mutations in this region. The mutation at position 1407 which was outside of the internal region was corrected using site-directed mutagenesis. The cDNA with the correct codon was then subcloned into the bacterial expression vector pQE-30 and overproduced in Escherichia coli strain M15. A protein with a subunit molecular weight of 51,000 was expressed at a level of about 4.5% of the total soluble protein in M15 as judged by SDS/PAGE. Cloning into pQE-30 adds six histidines and a short linker to the N-terminus of the enzyme. The recombinant 6PGDH with His-tag was purified using the Ni-NTA affinity column supplied by Qiagen. The purification procedure resulted in a homogeneous protein by SDS/PAGE with 22.4-fold purification with an overall yield of 61%. The recombinant enzyme exhibits kinetic parameters within error identical to those measured for native sheep liver enzyme.
Asunto(s)
Hígado/enzimología , Fosfogluconato Deshidrogenasa/biosíntesis , Proteínas Recombinantes/biosíntesis , Animales , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Mutagénesis Sitio-Dirigida , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Fosfogluconato Deshidrogenasa/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , OvinosRESUMEN
The Rotofor cell is a preparative isoelectric focusing (IEF) apparatus, in which IEF is performed entirely in free solution. Electrofocusing in Rotofor cell has been described as well-suited for use at any stage of a purification scheme. However, it has some important limitations in resolving complex mixtures of proteins. This paper describes the advantages and disadvantages of using the Rotofor cell in purification protocols.
Asunto(s)
Proteínas/aislamiento & purificación , Animales , Química Encefálica , Electroforesis en Gel de Poliacrilamida , Focalización Isoeléctrica/instrumentación , Peso Molecular , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Ratas , Superóxido Dismutasa/aislamiento & purificaciónRESUMEN
The mechanism of action of 6-phosphogluconate dehydrogenase with the alternative substrate 2-deoxy 6-phosphogluconate was investigated using enzymes from sheep liver, human erythrocytes and Trypanosoma brucei. The three enzymes oxidize 2-deoxy 6-phosphogluconate, but only the sheep liver enzyme releases the intermediate 2-deoxy,3-keto 6-phosphogluconate. Kinetic comparison showed that an increase in the rate of NADP+ reduction at high pH is due to increased release of the intermediate, rather than an increase in the overall reaction rate. 2-Deoxy,3-keto 6-phosphogluconate is decarboxylated by the erythrocyte and trypanosome enzymes but not the liver one in the absence of either NADPH or 6-phosphogluconate, which act as activators. The pH dependence of decarboxylation and the degree of activation suggest that 6-phosphogluconate is the activator which operates under normal assay conditions, while NADPH acts mainly by increasing the binding of the intermediate. The data suggest that the activity of 6PGDH is subjected to a two-way regulation: NADPH, which regulates the pentose phosphate pathway, inhibits the enzyme, while 6-phosphogluconate, levels of which rise when NADPH inhibition is removed, acts as an activator ensuring that 6-phosphogluconate is rapidly removed.
Asunto(s)
Fosfogluconato Deshidrogenasa/metabolismo , Regulación Alostérica , Animales , Eritrocitos/enzimología , Escherichia coli/enzimología , Gluconatos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Hígado/enzimología , NADP/fisiología , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Ovinos , Trypanosoma brucei brucei/enzimologíaRESUMEN
6-Phosphogluconate dehydrogenase is one of the seven enzymes involved in the pentose phosphate pathway. Crystals of a mammalian and a protozoan enzyme have been obtained previously and structures determined. It is reported here that a bacterial 6-phosphogluconate dehydrogenase, from Lactococcus lactis, has been purified and used in crystallization trials. Large prisms suitable for a detailed structural analysis have been obtained and characterized as orthorhombic, space group F222, with a = 70.4, b = 105.7, c = 474.6 A. Diffraction has been observed to 2.2 A resolution using synchrotron radiation. Structural analysis, in combination with ongoing biochemical characterization, will assist the elucidation of the structure-activity relationships of this enzyme.