RESUMEN
The single-copy genes encoding putative polyphosphate-glucose phosphotransferases (PPGK, EC 2.7.1.63) from two nitrogen-fixing Cyanobacteria, Nostoc sp. PCC7120 and Nostoc punctiforme PCC73102, were cloned and functionally characterized. In contrast to their actinobacterial counterparts, the cyanobacterial PPGKs have shown the ability to phosphorylate glucose using strictly inorganic polyphosphates (polyP) as phosphoryl donors. This has proven to be an economically attractive reagent in contrast to the more costly ATP. Cyanobacterial PPGKs had a higher affinity for medium-long-sized polyP (greater than ten phosphoryl residues). Thus, longer polyP resulted in higher catalytic efficiency. Also in contrast to most their homologs in Actinobacteria, both cyanobacterial PPGKs exhibited a modest but significant polyP-mannokinase activity as well. Specific activities were in the range of 180-230 and 2-3 µmol min(-1) mg(-1) with glucose and mannose as substrates, respectively. No polyP-fructokinase activity was detected. Cyanobacterial PPGKs required a divalent metal cofactor and exhibited alkaline pH optima (approx. 9.0) and a remarkable thermostability (optimum temperature, 45 °C). The preference for Mg(2+) was noted with an affinity constant of 1.3 mM. Both recombinant PPGKs are homodimers with a subunit molecular mass of ca. 27 kDa. Based on database searches and experimental data from Southern blots and activity assays, closely related PPGK homologs appear to be widespread among unicellular and filamentous mostly nitrogen-fixing Cyanobacteria. Overall, these findings indicate that polyP may be metabolized in these photosynthetic prokaryotes to yield glucose (or mannose) 6-phosphate. They also provide evidence for a novel group-specific subfamily of strictly polyP-dependent gluco(manno)kinases with ancestral features and high biotechnological potential, capable of efficiently using polyP as an alternative and cheap source of energy-rich phosphate instead of costly ATP. Finally, these results could shed new light on the evolutionary origin of sugar kinases.
Asunto(s)
Glucoquinasa/aislamiento & purificación , Glucoquinasa/metabolismo , Hexosas/metabolismo , Nostoc/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Polifosfatos/metabolismo , Clonación Molecular , Coenzimas/análisis , Glucoquinasa/química , Glucoquinasa/genética , Concentración de Iones de Hidrógeno , Peso Molecular , Nostoc/genética , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Multimerización de Proteína , TemperaturaRESUMEN
Glucokinase catalyzes the phosphorylation of glucose using ATP to yield glucose 6-phosphate. SgGlkA is a bacterial group III glucokinase from Streptomyces griseus that seems to play a regulatory role in carbon catabolite repression in this organism. SgGlkA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293â K. A crystal of SgGlkA in complex with glucose was obtained using a reservoir solution consisting of 0.9â M sodium/potassium tartrate, 0.2â M NaCl and 0.1â M imidazole pH 8.1 and diffracted X-rays to 1.84â Å resolution. The crystal of SgGlkA in complex with glucose belonged to space group P6(2)22 or P6(4)22, with unit-cell parameters a = b = 109.19, c = 141.18â Å. The crystal contained one molecule in the asymmetric unit.
Asunto(s)
Glucoquinasa/química , Glucosa/química , Streptomyces griseus/enzimología , Cristalización , Cristalografía por Rayos X , Glucoquinasa/aislamiento & purificación , Glucoquinasa/metabolismo , Glucosa/metabolismo , Unión ProteicaRESUMEN
Fungi contain several hexokinases, which are involved either in sugar phosphorylation or in carbon source sensing. Glucose and fructose phosphorylations appear to rely exclusively on glucokinase and hexokinase. Here, we characterized the catalytic glucokinase and hexokinase from the opportunistic human pathogen Aspergillus fumigatus and showed that both enzymes display different biochemical properties and play different roles during growth and development. Glucokinase efficiently activates glucose and mannose but activates fructose only to a minor extent. Hexokinase showed a high efficiency for fructose activation but also activated glucose and mannose. Transcript and activity determinations revealed high levels of glucokinase in resting conidia, whereas hexokinase was associated mainly with the mycelium. Consequentially, a glucokinase mutant showed delayed germination at low glucose concentrations, whereas colony growth was not overly affected. The deletion of hexokinase had only a minor impact on germination but reduced colony growth, especially on sugar-containing media. Transcript determinations from infected mouse lungs revealed the expression of both genes, indicating a contribution to virulence. Interestingly, a double-deletion mutant showed impaired growth not only on sugars but also on nonfermentable nutrients, and growth on gluconeogenic carbon sources was strongly suppressed in the presence of glucose. Furthermore, the glkA hxkA deletion affected cell wall integrity, implying that both enzymes contribute to the cell wall composition. Additionally, the absence of either enzyme deregulated carbon catabolite repression since mutants displayed an induction of isocitrate lyase activity during growth on glucose-ethanol medium. Therefore, both enzymes seem to be required for balancing carbon flux in A. fumigatus and are indispensable for growth under all nutritional conditions.
Asunto(s)
Aspergillus fumigatus/enzimología , Aspergillus fumigatus/crecimiento & desarrollo , Biocatálisis , Glucoquinasa/genética , Hexoquinasa/genética , Esporas Fúngicas/enzimología , Animales , Aspergilosis/microbiología , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Biocatálisis/efectos de los fármacos , Carbono/farmacología , Pared Celular/efectos de los fármacos , Pared Celular/enzimología , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Glucoquinasa/antagonistas & inhibidores , Glucoquinasa/aislamiento & purificación , Hexoquinasa/antagonistas & inhibidores , Hexoquinasa/aislamiento & purificación , Humanos , Pulmón/enzimología , Pulmón/microbiología , Ratones , Micelio/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Especificidad por Sustrato/efectos de los fármacos , Fosfatos de Azúcar/farmacología , Trehalosa/análogos & derivados , Trehalosa/farmacologíaRESUMEN
Uridine 5'-diphospho N-acetylglucosamine (UDP-GlcNAc) is an important nucleotide sugar in the biochemistry of all living organisms, and it is an important substrate in the synthesis of oligosaccharides. In the present work, three bioactive enzymes, namely, glucokinase (YqgR), GlcNAc-phosphate mutase (Agm1), and N-acetylglucosamine-1-phosphate uridyltransferase (GlmU), were produced effectively as soluble form in recombinant Escherichia coli. These three enzymes and dried yeast together were used to construct a multistep enzymatic system, which could produce UDP-GlcNAc efficiently with N-acetylglucosamine (GlcNAc) as the substrate. After the optimization of various reaction conditions, 31.5 mMUDP-GlcNAc was produced from 50 mMGlcNAc and 50 mMUMP.
Asunto(s)
Bacillus subtilis/enzimología , Escherichia coli/genética , Microbiología Industrial/métodos , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Uridina Difosfato N-Acetilglucosamina/metabolismo , Bacillus subtilis/genética , Clonación Molecular , Glucoquinasa/genética , Glucoquinasa/aislamiento & purificación , Glucoquinasa/metabolismo , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/aislamiento & purificación , Transferasas Intramoleculares/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/aislamiento & purificación , Nucleotidiltransferasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The glucokinase V62M and G72R mutations are naturally occurring and known to associate with hyperglycemia in humans. Structurally, V62 and G72 residues are located in close proximity to the allosteric site where hypoglycemia-linked activating mutations are clustered. To address the mechanism by which these variants alter the physiological phenotype, we characterized the biochemical and biophysical properties of the enzymes. Recombinant proteins were purified without affinity tags, and their steady-state kinetics and glucose binding affinities were determined. Both enzymes showed reduced rates of turnover (k(cat)) and reduced glucose affinity (i.e., increased K(0.5) and K(D) values). Their thermal stability did not largely differ from that of wild-type glucokinase. However, V62M and G72R lost the stabilizing protein interactions with glucokinase regulatory protein, which may contribute to lower activity in vivo. Both mutants were subject to activation by small molecule activators. In conclusion, the decreased enzyme activities of V62M and G72R observed in this study are consistent with the hyperglycemic phenotype.
Asunto(s)
Diabetes Mellitus Tipo 2/enzimología , Glucoquinasa/química , Glucoquinasa/metabolismo , Mutación Missense , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Estabilidad de Enzimas , Variación Genética , Glucoquinasa/genética , Glucoquinasa/aislamiento & purificación , Glucosa/metabolismo , Humanos , CinéticaRESUMEN
Glucokinase genes, found in the genome databases of Trypanosoma cruzi and Leishmania major, were cloned and sequenced. Their expression in Escherichia coli resulted in the synthesis of soluble and active enzymes, TcGlcK and LmjGlcK, with a molecular mass of 43 kDa and 46 kDa, respectively. The enzymes were purified, and values of their kinetic parameters determined. The K(m) values for glucose were 1.0 mM for TcGlcK and 3.3 mM for LmjGlcK. For ATP, the K(m) values were 0.36 mM (TcGlcK) and 0.35 mM (LmjGlcK). A lower K(m) value for glucose (2.55 mM) was found when the (His)(6)-tag was removed from the recombinant LmjGlcK, whereas the TcGlcK retained the same value. The V(max)'s of the T. cruzi and L. major GlcKs were 36.3 and 30.9 U/mg of protein, respectively. No inhibition was exerted by glucose-6-phosphate. Similarly, no inhibition by inorganic pyrophosphate was found in contrast to previous observations made for the T. cruzi and L. mexicana hexokinases. Both trypanosomatid enzymes were only able to phosphorylate glucose indicating that they are true glucokinases. Gel-filtration chromatography showed that the GlcK of both trypanosomatids may occur as a monomer or dimer, dependent on the protein concentration. Both GlcK sequences have a type-1 peroxisome-targeting signal. Indeed, they were shown to be present inside glycosomes using three different methods. These glucokinases present highest, albeit still a moderate 24% sequence identity with their counterpart from Trichomonas vaginalis, which has been classified into group A of the hexokinase family. This group comprises mainly eubacterial and cyanobacterial glucokinases. Indeed, multiple sequence comparisons, as well as kinetic properties, strongly support the notion that these trypanosomatid enzymes belong to group A of the hexokinases, in which they, according to a phylogenetic analysis, form a separate cluster.
Asunto(s)
Glucoquinasa/genética , Glucoquinasa/metabolismo , Leishmania major/enzimología , Trypanosoma cruzi/enzimología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía en Gel , Clonación Molecular , Dimerización , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Glucoquinasa/química , Glucoquinasa/aislamiento & purificación , Glucosa/metabolismo , Glucosa-6-Fosfato/farmacología , Cinética , Leishmania major/genética , Datos de Secuencia Molecular , Peso Molecular , Peroxisomas/química , Fosfatos/farmacología , Filogenia , Señales de Clasificación de Proteína/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Trichomonas vaginalis/genética , Trypanosoma cruzi/genéticaRESUMEN
According to analysis of proteomic profiling for Thermoanaerobacter tencongensis, TTE0090 could be a novel gene of glucokianse (GLK), though no GLK gene was annotated in the genomic data. With the methods of cloning and expression in vitro, the recombinant TTE0090 was successfully expressed and purified. The recombinant TTE0090 exhibited the catalysis of GLK, even at high temperatures. Detection of expression levels and catalysis of TTE0090 in vivo was furthermore carried out at different temperatures. The expression of TTE0090 was attenuated during the culture temperature elevated; however, the specific activity was positively correlated to temperature raised. This leads a possibility that the metabolic capacity of glycolysis in T. tencongensis is relatively constant at different temperatures. All the results herein demonstrate that TTE0090 is a novel gene of GLK. The studies on TTE0090 and its protein product, thus, may deepen our understanding of the adaptation mechanism of thermophilic bacteria living in harsh environment.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Expresión Génica , Glucoquinasa/química , Glucoquinasa/genética , Thermoanaerobacter/enzimología , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Catálisis , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Glucoquinasa/aislamiento & purificación , Glucoquinasa/metabolismo , Cinética , Temperatura , Thermoanaerobacter/químicaRESUMEN
The presence of glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.2) activity in seven strains of oral streptococci is demonstrated. The glucokinase purified from Streptococcus mutans SL-1 cells is shown to be a highly specific enzyme, phosphorylating only glucose (eight sugars tested). The enzyme is a true glucokinase: formation of the product, shown here to be glucose 6-phosphate, is dependent on the presence of glucose, ATP, divalent metal ion and enzyme. The Km for glucose is 1.40 mM, the pH optimum for the enzyme is a broad plateu from pH 7.1 to 9.5 and the molecular weight is estimated to be 40 000. The finding of a glucokinase in oral streptococci indicates the existence of an intracellular mechanism of glucose phosphorylation. The implications of this observation are discussed.
Asunto(s)
Glucoquinasa/metabolismo , Streptococcus mutans/enzimología , Glucoquinasa/aislamiento & purificación , Cinética , Especificidad por SustratoRESUMEN
Glucokinase (ATP-D-glucose 6-phosphotransferase, EC 2.7.1.2) was purified 144-fold from extracts of sucrose-grown Streptococcus mutans OMZ70 (ATCC 33535) cells. Twenty compounds were tested as potential substrates; only glucose (Km = 0.61 mM) was phosphorylated. The reaction catalyzed by the purified enzyme was dependent on the presence of glucose, nucleoside triphosphate and metal ion; glucose 6-phosphate and ADP were the products. Of the seven nucleoside triphosphates tested, ATP (Km = 0.21 mM) was the most efficient phosphate donor in the enzyme-catalyzed formation of glucose 6-phosphate. Both Mn2+ (relative activity, 173%) and Co2+ (264%) were more efficient than Mg2+ (100%) in supporting the enzyme reaction. The enzyme exhibited a broad maximal activity in the pH range from 7.5 to 9.5. The apparent molecular weight of glucokinase, as determined by gel filtration, was 41 000. With glucose held constant at either saturating or subsaturating levels, ADP was a noncompetitive inhibitor of ATP (Ki = 0.67 mM). ADP was an uncompetitive inhibitor of glucose (Ki = 0.71 mM) when ATP was held constant at either a saturating or subsaturating concentration. Glucose 6-phosphate was a competitive inhibitor of glucose (Ki = 0.31 mM) at saturating ATP and exhibited noncompetitive or mixed inhibition at a subsaturating ATP concentration. Glucose 6-phosphate was not an inhibitor toward ATP at saturating glucose concentrations, but exhibited noncompetitive inhibition at subsaturating glucose concentrations. The kinetic data support the postulation of a sequential mechanism for the glucokinase reaction; they are consistent with an ordered mechanism in which glucose binds first and glucose 6-phosphate dissociates last. Furthermore, the data suggest the existence of more than one enzyme binding site for the substrates of the glucokinase reaction.
Asunto(s)
Glucoquinasa/aislamiento & purificación , Streptococcus mutans/enzimología , Glucoquinasa/metabolismo , Cinética , Peso Molecular , Especificidad por SustratoRESUMEN
Three glucose-phosphorylating enzymes having different specificities for glucose and fructose were separated from the cell-free extract of Candida tropicalis by means of ammonium sulfate fractionation and chromatography on DEAE-cellulose and Sephadex G-100. Two of them, which phosphorylated fructose 1.5 times faster than glucose, were designated as hexokinase I and II (ATP : D-hexose 6-phosphotransferase, EC 2.7.1.1.), and the other with very low or no fructose-phosphorylating activity, as glucokinase (ATP : D-glucose 6-phosphotransferase, EC 2.7.1.2). Km values for glucose with both hexokinase I and glucokinase were 0.3 mM, and that for fructose with hexokinase I was 2.2 mM. Time-course changes in the levels of these enzymes in C. tropicalis growing on glucose and on n-alkane revealed that hexokinase was induced specifically by the sugars, while glucokinase was a constitutive enzyme. Addition of cycloheximide to the culture medium prevented the increase in the hexose-phosphorylating activity and in the Fru/Glu ratio (the ratio of enzymatic phosphorylation of fructose to that of glucose) in the cells. Although Candida lipolytica also contained hexokinase and glucokinase, both enzymes seemed to be constitutive.
Asunto(s)
Candida/enzimología , Hexoquinasa/metabolismo , Acetatos/metabolismo , Alcanos/metabolismo , Cicloheximida/farmacología , Etanol/metabolismo , Fructosa/metabolismo , Glucoquinasa/aislamiento & purificación , Glucoquinasa/metabolismo , Glucosa/metabolismo , Hexoquinasa/aislamiento & purificación , CinéticaRESUMEN
1. Glucokinase (ATP : D-glucose 6-phosphotransferase, EC 2.7.1.2) was extracted from pea seeds and purified by fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose and Sephadex. 2. The relative rates of phosphorylation of glucose, mannose and fructose (final concentration 5 mM) were 100, 64 and 11. 3. The Km for glucose of pea-seed glucokinase was 70 muM and the Km for mannose was 0.5 mM. The Km for fructose was much higher (30 mM). 4. Mg2+ ions were essential for activity. Mn2+ could partially replace Mg2+. 5. Enzyme activity was not inhibited by glucose 6-phosphate. A number of other metabolites had no effect on glucokinase activity. 6. Pea-seed glucokinase was inhibited by relatively low concentrations of ADP.
Asunto(s)
Glucoquinasa/metabolismo , Semillas/enzimología , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Cobre/farmacología , Fructosa/metabolismo , Glucoquinasa/aislamiento & purificación , Glucosa/metabolismo , Glucofosfatos/farmacología , Concentración de Iones de Hidrógeno , Cinética , Magnesio/farmacología , Manosa/metabolismo , Mercurio/farmacologíaRESUMEN
Alloxan inactivated glucokinase in intact, isolated pancreatic islets incubated in vitro. Inactivation of glucokinase was antagonized by 30 mM glucose present during incubation of islets with alloxan. Glucokinase partially purified from transplantable insulinomas or rat liver was inactivated by alloxan with a half-maximal effect at 2-4 microM alloxan. Inactivation of purified glucokinase was antagonized by glucose, mannose, and 2-deoxyglucose in order of decreasing potency but not by 3-O-methylglucose. Glucose anomers at 6 and 14 mM were discriminated as protecting agents, with the alpha-anomer more effective than the beta-anomer. Glucokinase was not protected from alloxan inactivation by N-acetylglucosamine, indicating that the reactive site for alloxan is not the active site; therefore, glucose may protect glucokinase by inducing a conformational change. Glucokinase is thought to be the glucose sensor of the pancreatic beta-cell. The finding that glucokinase is inactivated by alloxan and protected by glucose with discrimination of its anomers similar to inhibition of glucose-stimulated insulin secretion by alloxan supports this hypothesis and appears to explain the mechanism for inhibition of hexose-stimulated insulin secretion by this agent and the unique role of glucose and mannose as protecting agents.
Asunto(s)
Aloxano/farmacología , Glucoquinasa/antagonistas & inhibidores , Islotes Pancreáticos/enzimología , Animales , Glucoquinasa/aislamiento & purificación , Técnicas In Vitro , Insulinoma/enzimología , Cinética , Hígado/enzimología , Masculino , Monosacáridos/farmacología , Especificidad de Órganos , Neoplasias Pancreáticas/enzimología , Unión Proteica , Ratas , Ratas EndogámicasRESUMEN
Glucokinase from rat liver or transplantable, radiation-induced insulinomas was partially purified by ion exchange chromatography using DEAE-Cibacron Blue F3GA agarose. Phosphorylation of alpha,beta-D-mannose by glucokinase occurred with cooperative rate dependence on mannose concentration (nH: 1.50). Half-maximal phosphorylation rate occurred at 14 mM alpha,beta-D-mannose. The alpha- and beta-anomers of mannose were phosphorylated with sigmoidal kinetics (nH: 1.57 and 1.42, respectively). The affinity of glucokinase for alpha-D-mannose is higher than for beta-D-mannose (S0.5: 12 mM versus 19 mM). The maximum phosphorylation rate is slightly higher, about 10%, with beta-D-mannose than with alpha-D-mannose. Islet glucokinase has previously been shown to be chromatographically and kinetically identical to glucokinase from insulinoma and liver; therefore, evidence that glucokinase from these two tissues phosphorylates mannose with cooperative rate dependence and differentiates mannose anomers supports the glucokinase-glucose sensor hypothesis.
Asunto(s)
Adenoma de Células de los Islotes Pancreáticos/enzimología , Glucoquinasa/metabolismo , Insulinoma/enzimología , Hígado/enzimología , Manosa/metabolismo , Animales , Glucoquinasa/aislamiento & purificación , Cinética , Masculino , Trasplante de Neoplasias , Fosforilación , Ratas , Ratas Endogámicas , Estereoisomerismo , Especificidad por SustratoRESUMEN
Using cultured islets as the experimental system, this study established dosage-response and time-dependency curves of the inductive glucose effect on glucose-stimulated insulin release, glucose usage, and glucokinase activity. Glucose-stimulated insulin release in islets cultured for 1, 2, or 7 days was increased as a function of glucose concentration in the culture medium and as a function of time. Glucose usage in the cultured islets showed a close relationship with glucose concentration in the culture medium at both 2 and 7 days of culture. Glucokinase activity increased in islets cultured for 1, 2, or 7 days as a function of increasing glucose concentrations in the culture medium and as a function of time. The V(max) of glucokinase in islets cultured for 7 days in medium containing 30 mM glucose was twice the value of freshly isolated islets and was almost fivefold higher than that in islets cultured for 7 days in 3 mM glucose. The glucose induction of glucose-stimulated insulin release, of glucose usage, and of glucokinase activity were tightly correlated. The biochemical mechanisms of glucose induction of islet glucokinase were further studied. Immunoblotting with an antibody against C-terminal peptide of glucokinase showed that densities of a 52,000-kD protein band from tissue extracts of islets cultured for 7 days in 3, 12, and 30 mM glucose were 25, 44, and 270% compared with that of extract from freshly isolated islets (100%). RNA blot analysis of glucokinase mRNA demonstrated virtually the same levels in fresh islets and islets after 7 days of culture in 3 or 30 mM glucose. The adaptive response of glucokinase to glucose appears therefore to be occurring at a translational or posttranslational site in cultured islets. These data greatly strengthen the concept that glucose is the regulator that induces the activity of glucokinase, which in turn determines the rate change of glucose usage as well as glucose-stimulated insulin release from beta-cells. Thus, the hypothesis that glucokinase is the glucose sensor of beta-cells is strengthened further.
Asunto(s)
Glucoquinasa/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Animales , Células Cultivadas , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Glucoquinasa/biosíntesis , Glucoquinasa/aislamiento & purificación , Hexoquinasa/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/ultraestructura , Cinética , Masculino , Microscopía Electrónica , Modelos Biológicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
We investigated the subcellular localization, mobility, and activity of glucokinase in MIN6 cells, a glucose-responsive insulin-secreting beta-cell line. Glucokinase is present in the cytoplasm and a vesicular/granule compartment that is partially colocalized with insulin granules. The granular staining of glucokinase is preserved after permeabilization of the cells with digitonin. There was no evidence for changes in distribution of glucokinase between the cytoplasm and the granule compartment during incubation of the cells with glucose. The rate of release of glucokinase and of phosphoglucoisomerase from digitonin-permeabilized cells was slower when cells were incubated at an elevated glucose concentration (S0.5 approximately 15 mmol/l). This effect of glucose was counteracted by competitive inhibitors of glucokinase (5-thioglucose and mannoheptulose) but was unaffected by fructose analogs and may be due to changes in cell shape or conformation of the cytoskeleton that are secondary to glucose metabolism. Based on the similar release of glucokinase and phosphoglucoisomerase, we found no evidence for specific binding of cytoplasmic digitonin-extractable glucokinase. The affinity of beta-cells for glucose is slightly lower than that in cell extracts and, unlike that in hepatocytes, is unaffected by fructose, tagatose, or a high-K+ medium, which is consistent with the lack of change in glucokinase distribution or release. We conclude that glucokinase is present in two locations, cytoplasm and the granular compartment, and that it does not translocate between them. This conclusion is consistent with the lack of adaptive changes in the glucose phosphorylation affinity. The glucokinase activity associated with the insulin granules may have a role in either direct or indirect coupling between glucose phosphorylation and insulin secretion.
Asunto(s)
Glucoquinasa/metabolismo , Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Fracciones Subcelulares/metabolismo , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Digitonina/farmacología , Glucoquinasa/aislamiento & purificación , Hexoquinasa/metabolismo , Técnicas Inmunológicas , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Cinética , Concentración Osmolar , Fosforilación , Coloración y Etiquetado , Fracciones Subcelulares/efectos de los fármacos , Distribución TisularRESUMEN
The high Km glucose phosphorylation enzyme glucokinase is believed to be the beta-cell glucose sensor, i.e., the site in glucose metabolism that determines the sensitivity and specificity of glucose-induced insulin secretion. We investigated the regulation of this enzyme by measuring glucokinase Vmax and protein levels in isolated islets from hyperinsulinemic rats. Rats were infused for 48 h with 2 ml/h of 20% glucose, 50% glucose, or 0.45% NaCl (control rats). At the end of the infusion, 20% glucose-infused rats were normoglycemic and hyperinsulinemic (2.3-fold rise in basal plasma insulin level). Their islets had a 2.3-fold increase in insulin secretion at 8.3 mM glucose (51 +/- 10% of capacity vs. 22 +/- 5% in NaCl rats, P < 0.03), a 75% increase in glucokinase Vmax and little if any increase in glucokinase protein level (111 +/- 3% of control). The rats infused with 50% glucose had marked hyperglycemia and higher basal plasma insulin levels. Their islets were maximally stimulated by 8.3 mM glucose in combination with a 270% increase in glucokinase Vmax and a 69 +/- 11% increase in glucokinase protein level. Hexokinase Vmax was also doubled. Thus, compensatory increases in beta-cell glucose phosphorylation are a key mechanism for adaptive hyperinsulinemia. Our results show two types of regulation for the beta-cell high Km phosphorylation enzyme, glucokinase. The content of glucokinase protein is controlled by the plasma glucose level. Variable catalytic activity of this protein was also observed in this study.
Asunto(s)
Glucoquinasa/metabolismo , Hiperinsulinismo/enzimología , Islotes Pancreáticos/enzimología , Animales , Glucemia/metabolismo , Catálisis , ADN/metabolismo , Glucoquinasa/aislamiento & purificación , Glucosa/farmacología , Hexoquinasa/metabolismo , Hiperinsulinismo/fisiopatología , Immunoblotting , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Cinética , Masculino , Ratas , Ratas Sprague-Dawley , Tolbutamida/farmacologíaRESUMEN
D-Glyceraldehyde irreversibly inhibited rat liver glucokinase in a concentration-dependent manner. The inactivation of glucokinase by glyceraldehyde was blocked by the presence of its substrates such as glucose and mannose. Glucokinase was highly sensitive to glyceraldehyde compared with some other glycolytic enzymes (from animal tissues) including hexokinase, glucose-6-phosphate isomerase, 6-phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase. The amino acid analysis of untreated and glyceraldehyde-treated glucokinase suggested that glyceraldehyde-induced inactivation of glucokinase is caused by glycation of Lys residues of the enzyme by the triose. Treatment of pancreatic islets with 6 mM glyceraldehyde for 1 h at 37 degrees C caused both inactivation of glucokinase and inhibition of glucose-induced insulin secretion. Another glucose-phosphorylating enzyme (hexokinase) in pancreatic islets, however, was little affected by glyceraldehyde. In addition, glyceraldehyde did not affect the insulin secretory responses of islets to nonglucose secretagogues such as glyceraldehyde and Leu. When pancreatic islets were cultured with a lower concentration (1 mM) of glyceraldehyde for a longer time (17 h) in the presence of 10 mM glucose to mimic the in vivo conditions, both glucokinase activity and glucose-induced insulin secretion were again decreased. This study demonstrates that glucose-induced insulin secretion is impaired by glyceraldehyde through the inactivation of glucokinase. The implication of this finding in the pathophysiology of type II diabetes is discussed.
Asunto(s)
Glucoquinasa/antagonistas & inhibidores , Glucosa/farmacología , Gliceraldehído/farmacología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Aminoácidos/análisis , Animales , Femenino , Glucoquinasa/aislamiento & purificación , Glucosa/antagonistas & inhibidores , Hexoquinasa/metabolismo , Técnicas In Vitro , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Cinética , Hígado/enzimología , Monosacáridos/farmacología , Ratas , Ratas Wistar , Fosfatos de Azúcar/farmacologíaRESUMEN
Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.
Asunto(s)
Cromatografía de Afinidad/métodos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Endopeptidasas/biosíntesis , Endopeptidasas/genética , Endopeptidasas/aislamiento & purificación , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucoquinasa/biosíntesis , Glucoquinasa/genética , Glucoquinasa/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Solubilidad , alfa-Amilasas/biosíntesis , alfa-Amilasas/genética , alfa-Amilasas/aislamiento & purificaciónRESUMEN
In the liver many metabolic pathways are preferentially localized in different zones of the acinus. It is assumed that this zonation allows an efficient adaptation to different states of nutrition, because alternative pathways can be regulated independently. It is reported that the rate limiting enzyme for the glycolytic pathway, glucokinase (EC 2.7.1.2), is predominantly located in the pericentral zone. The gene expression of glucokinase is induced to a maximum level after a carbohydrate-rich diet. In starved or diabetic rats glucokinase gene expression is barely detectable. In postnatal development glucokinase is induced to significant levels only from day 14 onwards. The distribution of the glucokinase protein in the rat liver lobule in the first 4 weeks of postnatal life was investigated by immunohistochemistry and compared to the distribution observed in adult rats. In adult rats considerably high levels of glucokinase are measureable as shown by immunoblotting utilizing a monospecific antibody and a photometric assay of glucokinase enzyme activity, respectively. Immunohistochemically the hepatic glucokinase protein is detected in the perivenous area. During postnatal development, the quantities of hepatic glucokinase protein and glucokinase enzyme activity start to increase significantly from day 15 onwards. Subsequently, glucokinase levels rise further until day 29. In contrast to the results obtained by immunoblotting, glucokinase is already detectable in some liver cells in sections from 6-day-old rats by immunohistochemistry. The liver lobule structure at this age is not completely developed, therefore it is not possible to definitely assign these cells to periportal or pericentral areas. At day 10 post partum the number of glucokinase expressing cells, which appear to be localized preferentially in the periportal zone, increases. In agreement with the immunoblotting, an immense increase in glucokinase activity was observed at day 14. The periportal zonation, clearly detectable at this time, remains stable until day 24. In sections from 29-day-old rats the periportal zonation begins to change into a more homogeneous pattern with a slight preference for periportal areas. The observed appearance of the periportal zonation of glucokinase during neonatal development is obviously in contrast to the perivenous expression of glucokinase in adult rats.
Asunto(s)
Glucoquinasa/metabolismo , Hígado/enzimología , Hígado/crecimiento & desarrollo , Envejecimiento/fisiología , Animales , Animales Recién Nacidos , Citosol/enzimología , Electroforesis en Gel de Poliacrilamida , Femenino , Glucoquinasa/aislamiento & purificación , Immunoblotting , Técnicas para Inmunoenzimas , Masculino , Ratas , Ratas Sprague-Dawley , OvinosRESUMEN
The particulate glucokinase of cat liver is shown to be microsomal. The activity is readily solubilized by glucose-6-phosphate, ATP, pyrophosphate, high salt concentrations and, to a lesser extent, ribonucleoside triphosphates. The solubilization by glucose-6-phosphate is inhibited by Pi. Solubilizations by ATP and glucose-6-phosphate differ in their sensitivity to temperature changes; they are relatively specific for glucokinase as compared to solubilization by detergent (Triton X 100). The enzyme can be bound again to previously eluted microsomal membranes. Treatment of membrane with trypsin, at 0 degrees C, destroys the ability to rebind the enzyme to the membrane. It is suggested that electrostatic forces are of considerable importance for the binding of glucokinase to a possible protein binding site in the membrane.