RESUMEN
While the application of enzymes to synthetic and industrial problems continues to grow, the major development today is focused on multi-enzymatic cascades. Such systems are particularly attractive, because many commercially available enzymes operate under relatively similar operating conditions. This opens the possibility of one-pot operation with multiple enzymes in a single reactor. In this paper the concept of modules is introduced whereby groups of enzymes are combined in modules, each operating in a single reactor, but with the option of various operating strategies to avoid any complications of nonproductive interactions between the enzymes, substrates or products in a given reactor. In this paper the selection of modules is illustrated using the synthesis of the bulk chemical, gluconic acid, from lignocellulosic waste.
Asunto(s)
Catalasa/química , Celulasas/química , Gluconatos/síntesis química , Glucosa Oxidasa/química , Lignina/química , Modelos Estadísticos , beta-Glucosidasa/química , Biocatálisis , Catalasa/metabolismo , Celulasas/metabolismo , Simulación por Computador , Fermentación , Gluconatos/química , Gluconatos/metabolismo , Glucosa/química , Glucosa/metabolismo , Glucosa Oxidasa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Lactonas/química , Lactonas/metabolismo , Lignina/metabolismo , Ingeniería Metabólica/métodos , Temperatura , Residuos , beta-Glucosidasa/metabolismoRESUMEN
The effects of glucose starvation on glycogen synthase (GS) activity and protein expression were investigated. Fibroblasts were cultured in medium supplemented with either glucose or pyruvate. Pyruvate-cultured cells exhibited UDP-glucose contents that amounted to approximately 10% of those in cells cultured with glucose. GS activity, protein and mRNA amounts in pyruvate-cultured cells were decreased to approximately 35, 60, and 60%, respectively, of values in glucose-cultured cells. Incubation of extracts from glucose-cultured cells with radioactive UDP-glucose resulted in substantial binding of ligand to immunoprecipitated GS. However, binding in immunoprecipitates from pyruvate-cultured cells was decreased to approximately 25% of values in glucose-cultured cells. These data indicate that glucose starvation and the subsequent depletion of UDP-glucose result in: (1) inactivation of GS, owing to a decrease in its ability to bind UDP-glucose, and (2) decreased amount of GS protein, owing to a decrease in the levels of GS mRNA.
Asunto(s)
Fibroblastos/metabolismo , Glucosa/deficiencia , Glucógeno Sintasa/metabolismo , Uridina Difosfato Glucosa/deficiencia , Animales , Células Cultivadas , Cricetinae , Cricetulus , Activación Enzimática , Inhibidores Enzimáticos , Pulmón/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Inanición/metabolismoRESUMEN
A low level of UDP-Glc occurs in cells exposed to hypoxia or glucose starvation. This work reveals that a 65% reduction in the cellular UDP-Glc level causes up-regulation of the mitochondrial chaperone GRP75 and the endoplasmic reticulum (ER) resident chaperones GRP58, ERp72, GRP78, GRP94, GRP170, and calreticulin. Conditions that cause misfolding of proteins within the ER activate the transcription factors ATF6alpha/beta and induce translation of the transcription factors XBP-1/TREB5 and ATF4/CREB2. These transcription factors induce the overexpression of ER chaperones and CHOP/GADD153. However, the 65% decrease in the cellular UDP-Glc level does not cause activation of ATF6alpha, splicing of XBP-1/TREB5, induction of ATF4/CREB2, or expression of CHOP/GADD153. The activity of the promoters of the ER chaperones is increased in UDP-Glc-deficient cells, but the activity of the CHOP/GADD153 promoter is not affected, in comparison with their respective activities in cells having compensated for the UDP-Glc deficiency. The results demonstrate that the unfolded protein response remains functionally intact in cells with a 65% decrease in the cellular UDP-Glc level and provide evidence that this decrease is a stress signal in mammalian cells, which triggers the coordinate overexpression of mitochondrial and ER chaperones, independently of the ER stress elements.
Asunto(s)
Retículo Endoplásmico/metabolismo , Glucosa/metabolismo , Oxígeno/metabolismo , Uridina Difosfato Glucosa/deficiencia , Factor de Transcripción Activador 6 , Adenosina Trifosfato/química , Alelos , Empalme Alternativo , Animales , Western Blotting , Proteínas Potenciadoras de Unión a CCAAT/genética , Calreticulina/biosíntesis , Proteínas Portadoras/biosíntesis , Línea Celular , Supervivencia Celular , Cricetinae , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel Bidimensional , Chaperón BiP del Retículo Endoplásmico , Fibroblastos/metabolismo , Genes Dominantes , Genes Reporteros , Glicoproteínas/biosíntesis , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/biosíntesis , Hipoxia , Isomerasas/biosíntesis , Luciferasas/metabolismo , Espectroscopía de Resonancia Magnética , Glicoproteínas de Membrana/biosíntesis , Proteínas de la Membrana/biosíntesis , Mitocondrias/metabolismo , Chaperonas Moleculares/biosíntesis , Mutación , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Transducción de Señal , Factor de Transcripción CHOP , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Tunicamicina/farmacología , Regulación hacia ArribaRESUMEN
The regulatory role of UDP-glucose in glycogen biogenesis was investigated in fibroblasts containing a point mutation in the UDP-glucose pyrophosphorylase gene and, consequently, chronically low UDP-glucose levels (Qc). Comparisons were made with cells having the intact gene and restored UDP-glucose levels (G3). Glycogen was always very low in Qc cells. [(14)C]Glucose incorporation into glycogen was decreased and unaffected by insulin in Qc cells, whereas insulin stimulated glucose incorporation by approximately 50% in G3 cells. Glycogen synthase (GS) activity measured in vitro was virtually absent and the amount of enzyme in Qc cells was decreased by about 50%. The difference in GS activity between cells persisted even when G3 cells were devoid of glycogen. Incubation of G3 cell extracts with either exogenous UDP-glucose or glycogen resulted in increases in GS activity. Incubation of Qc cell extracts with exogenous UDP-glucose had no effect on GS activity; however, incubation with glycogen fully restored enzyme activity. Incubation of G3 cell extracts with radioactive UDP-glucose resulted in substantial binding of ligand to immunoprecipitated GS, whereas no binding was detected in Qc immunoprecipitates. Incubation of Qc cell extracts with exogenous glycogen fully restored UDP-glucose binding in the immunoprecipitate. These data suggest that chronically low UDP-glucose levels in cells result in inactivation of GS, owing to loss of the ability of GS to bind UDP-glucose.