RESUMEN
Nucleotides are synthesized from de novo and salvage pathways. To characterize the uridine salvage pathway, two genes, UKL1 and UKL2, that tentatively encode uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) bifunctional enzymes were studied in Arabidopsis thaliana. T-DNA insertions in UKL1 and UKL2 reduced transcript expression and increased plant tolerance to toxic analogs 5-fluorouridine and 5-fluorouracil. Enzyme activity assays using purified recombinant proteins indicated that UKL1 and UKL2 have UK but not UPRT activity. Subcellular localization using a C-terminal enhanced yellow fluorescent protein fusion indicated that UKL1 and UKL2 localize to plastids. The ukl2 mutant shows reduced transient leaf starch during the day. External application of orotate rescued this phenotype in ukl2, indicating pyrimidine pools are limiting for starch synthesis in ukl2. Intermediates for lignin synthesis were upregulated, and there was increased lignin and reduced cellulose content in the ukl2 mutant. Levels of ATP, ADP, ADP-glucose, UTP, UDP, and UDP-glucose were altered in a light-dependent manner. Seed composition of the ukl1 and ukl2 mutants included lower oil and higher protein compared with the wild type. Unlike single gene mutants, the ukl1 ukl2 double mutant has severe developmental defects and reduced biomass accumulation, indicating these enzymes catalyze redundant reactions. These findings point to crucial roles played by uridine salvage for photoassimilate allocation and partitioning.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Plastidios/enzimología , Uridina Quinasa/metabolismo , Uridina/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Biomasa , Celulosa/metabolismo , Fluorouracilo/farmacología , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Luz , Lignina/metabolismo , Mutagénesis Insercional , Ácido Orótico/farmacología , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Fenotipo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Plastidios/efectos de los fármacos , Plastidios/genética , Plastidios/fisiología , Proteínas Recombinantes de Fusión , Semillas/efectos de los fármacos , Semillas/enzimología , Semillas/genética , Semillas/metabolismo , Almidón/metabolismo , Uracilo/farmacología , Uridina/análogos & derivados , Uridina/farmacología , Uridina Quinasa/genética , Uridina Quinasa/aislamiento & purificaciónRESUMEN
Nucleoside analogs are effective inhibitors of the hepatitis C virus (HCV) in the clinical setting. One such molecule, 2'-C-methyl-cytidine (2'-MeC), entered clinical development as NM283, a valine ester prodrug form of 2'-MeC possessing improved oral bioavailability. To be active against HCV, 2'-MeC must be converted to 2'-MeC triphosphate which inhibits NS5B, the HCV RNA-dependent RNA polymerase. Conversion of 2'-MeC to 2'-MeC monophosphate is the first step in 2'-MeC triphosphate production and is thought to be the rate-limiting step. Here we investigate which of three possible enzymes, deoxycytidine kinase (dCK), uridine-cytidine kinase 1 (UCK1), or uridine-cytidine kinase 2 (UCK2), mediate this first phosphorylation step. Purified recombinant enzymes UCK2 and dCK, but not UCK1, could phosphorylate 2'-MeC in vitro. However, siRNA knockdown experiments in three human cell lines (HeLa, Huh7 and HepG2) defined UCK2 and not dCK as the key kinase for the formation of 2'-MeC monophosphate in cultured human cells. These results underscore the importance of confirming enzymatic kinase data with appropriate cell-based assays. Finally, we present data suggesting that inefficient phosphorylation by UCK2 likely limits the antiviral activity of 2'-MeC against HCV. This paves the way for the use of a nucleotide prodrug approach to overcome this limitation.
Asunto(s)
Antivirales/metabolismo , Citidina Monofosfato/metabolismo , Citidina/análogos & derivados , Desoxicitidina Quinasa/metabolismo , Profármacos/metabolismo , Uridina Quinasa/metabolismo , Biotransformación , Línea Celular , Citidina/metabolismo , Citidina Monofosfato/análogos & derivados , Desoxicitidina Quinasa/genética , Desoxicitidina Quinasa/aislamiento & purificación , Silenciador del Gen , Hepacivirus/efectos de los fármacos , Humanos , ARN Interferente Pequeño/genética , Uridina Quinasa/genética , Uridina Quinasa/aislamiento & purificaciónRESUMEN
Enzyme capable of catalyzing the phosphorylation of thymidine and uridine was isolated from Y. pseudotuberculosis cells by fractionation with the use of ammonium sulfate, ion exchange and affinity chromatography. The degree of purification of thymidine- and uridine-kinase was approximately 350 times, and at all stages of isolation the activity of both nucleoside-kinases was detected in the same peaks. The purified enzyme was capable of the phosphorylation of thymidine and uridine at temperatures of 8-10 degrees C to 50 degrees C and exhibited the maximum enzymatic activity at pH 8-8.5 and 45 degrees C in the presence of 0.5-1.0 mM MgCl2 and 2 mM ATP. The enzyme was found to have no strict substrate specificity and transferred the phosphate group from ATP to radiolabeled thymidine, uridine and desoxycytidine with different effectiveness, but did not use thymidine-monophosphate as phosphate acceptor.
Asunto(s)
Timidina Quinasa/aislamiento & purificación , Uridina Quinasa/aislamiento & purificación , Yersinia pseudotuberculosis/enzimología , Adenosina Trifosfato , Sulfato de Amonio , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Concentración de Iones de Hidrógeno , Cloruro de Magnesio , Fosforilación , Temperatura , Timidina Quinasa/metabolismo , Uridina Quinasa/metabolismoRESUMEN
Uridine kinase from Ehrlich ascites tumor cells has been purified about 60,000-fold to apparent homogeneity and with an overall recovery of about 40%. This purification was achieved using phosphocellulose and adenosine 5'-triphosphate-agarose affinity chromatography. The subunit molecular mass as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 31,000 daltons. With two-dimensional electrophoresis, only one spot was observed, indicating the absence of isoenzymes. Multiple peaks of activity are routinely observed on ion exchange chromatography or gel filtration, for both crude preparations or homogeneous uridine kinase, in agreement with our earlier results that this enzyme exists as multiple interconvertible oligomeric forms (Payne, R. C., and Traut, T. W. (1982) J. Biol. Chem. 257, 12485-12488). The purified enzyme has a specific activity of 283 mumol/min/mg of protein at 22 degrees C. Initial velocity studies using uridine and ATP are consistent with a sequential mechanism. Km values for uridine, cytidine, and ATP are 40, 57, and 450 microM, respectively. CTP and UTP are competitive inhibitors with respect to ATP, with Ki values for CTP and UTP of 10 and 61 microM, respectively. The enzyme was active with several nucleoside analogs, the Km values being 69 microM (5-fluorouridine), 200 microM (3-deazauridine), and 340 microM (6-azauridine). The pure enzyme is very sensitive to freezing, but can be maintained at O degrees C for 8 weeks with only 20% loss of activity. For long-term storage, enzyme in 50% glycerol can be maintained at -20 degrees C for many months with no detectable loss of activity.
Asunto(s)
Carcinoma de Ehrlich/enzimología , Fosfotransferasas/aislamiento & purificación , Uridina Quinasa/aislamiento & purificación , Animales , Unión Competitiva , Electroforesis en Gel de Poliacrilamida , Cinética , Ratones , Peso Molecular , Ribonucleótidos/farmacología , Especificidad por Sustrato , Uridina Quinasa/metabolismoRESUMEN
Uridine kinase from mouse Ehrlich ascites cells can exist in a variety of different aggregation states, from monomer up to aggregates that may contain 32 or more subunits. With very crude enzyme preparations, uridine kinase activity is always associated with several different coexisting molecular weight species. Changes in the aggregation state are produced in the presence of normal effectors (orthophosphate, ATP and CTP) at physiological concentrations. With uridine kinase that has been purified 9,000-fold, enzyme activity is associated with only a single molecular weight species, but is still responsive to the same physiological effectors. In the presence of orthophosphate, uridine kinase has a molecular weight of 380,000 (appropriate for a dodecamer). In the presence of CTP, the enzyme dissociates with concomitant loss of activity. The dissociated enzyme can be reassociated to the native size. These results imply that alteration of the enzyme's quaternary structure by normal effectors constitutes a mechanism for regulating uridine kinase activity in vivo.
Asunto(s)
Carcinoma de Ehrlich/enzimología , Citidina Trifosfato/farmacología , Nucleótidos de Citosina/farmacología , Fosfotransferasas/metabolismo , Uridina Quinasa/metabolismo , Animales , Sustancias Macromoleculares , Ratones , Peso Molecular , Conformación Proteica , Uridina Quinasa/aislamiento & purificaciónRESUMEN
Uridine kinase (ATP: uridine-5-phosphotransferase, EC 2.7.1.48) was isolated from cytosol of rat Zajdela ascite hepatoma cells by fractionation with ammonium sulfate and gel-filtration on Sephadex G-200. The enzyme has a pH optimum of 7.2 - 7.8; Km for uridine is 4.8 . 10(-5) M, that for ATP - 1.9 . 10(-4) M. The optimal ratio of ATP of Mg2+ is 2.6. The enzyme activity is inhibited by end products of pyrimidine biosynthesis with Ki for CTP of 6.0 . 10(-4) M and for UTP of 1.2 . 10(-3) M. The Ki values for uridine competitive analogs, i. e. 6-azauridine, 5-bromuridine and 5-azacytidine are equal to 4.0 . 10(-4) M, 1.5 . 10(-3) M and 2.5 . 10(-3) M, respectively. Further purification of the enzyme on Sepharose 4B allowed to obtain the most active, although heterogeneous fractions purified 86-fold, with specific activity of 11.2 mkmole/hour per mg of protein. Using electrofocusing, uridine kinase was found to consist of two major and one minor active fractions with pH of 6.2, 6.7 and 6.35, respectively. Chromatography on DEAE-cellulose DE-32 resulted in two major active fractions of the enzyme, differing in thermal stability and inhibition by CTP. It may be concluded that Zajdela ascite hepatoma cells contain at least two isoforms of uridine kinase.
Asunto(s)
Neoplasias Hepáticas Experimentales/enzimología , Fosfotransferasas/metabolismo , Uridina Quinasa/metabolismo , Animales , Citosol/enzimología , Cinética , Masculino , Ratas , Ribonucleótidos/farmacología , Uridina Quinasa/aislamiento & purificaciónAsunto(s)
Óxido de Aluminio , Aluminio , Nucleósidos/aislamiento & purificación , Nucleótidos/aislamiento & purificación , Fosfotransferasas/aislamiento & purificación , Adenosina Quinasa/aislamiento & purificación , Animales , Tampones (Química) , Humanos , Hígado/enzimología , Ratones , Sarcoma 180/enzimología , Timidina Quinasa/aislamiento & purificación , Uridina Quinasa/aislamiento & purificaciónRESUMEN
UMPK 3 is a rare variant of the polymorphic enzyme of human red cells, uridine monophosphate kinase. This homozygote phenotype was detected among the Warao Indians of Venezuela. The UMPK 1 and UMPK 3 enzymes were partially purified following the method described by Tend et al. (1976). The biochemical and kinetic parameters of both variants were studied in crude hemolysates and in partially purified enzymes. A comparison was made with the results previously reported by Teng for UMPK 1 and UMPK 2, and it was concluded that UMPK 3 seems to resemble the other two allelic gene products in Km values for UMP, CMP, and ATP but differs from them in electrophoretic mobility, pH optimum, and thermal stability.
Asunto(s)
Alelos , Frecuencia de los Genes , Indígenas Sudamericanos , Fosfotransferasas/genética , Uridina Quinasa/genética , Electroforesis , Eritrocitos/enzimología , Humanos , Concentración de Iones de Hidrógeno , Cinética , Uridina Quinasa/aislamiento & purificación , Uridina Monofosfato/metabolismo , VenezuelaAsunto(s)
Fosfotransferasas , Uridina Quinasa , Animales , Azacitidina/uso terapéutico , Encéfalo/enzimología , Carcinoma de Ehrlich/enzimología , Embrión de Pollo , Resistencia a Medicamentos , Fluorouracilo/uso terapéutico , Isoenzimas , Cinética , Hígado/enzimología , ARN/biosíntesis , Ratas , Sarcoma Experimental/tratamiento farmacológico , Sarcoma Experimental/enzimología , Uracilo/análogos & derivados , Uracilo/uso terapéutico , Uridina Quinasa/aislamiento & purificación , Uridina Quinasa/fisiologíaAsunto(s)
Geobacillus stearothermophilus/enzimología , Neoplasias Hepáticas Experimentales/enzimología , Fosfotransferasas/aislamiento & purificación , Uridina Quinasa/aislamiento & purificación , Animales , Citidina , Retroalimentación , Cinética , Ratas , Especificidad de la Especie , Especificidad por Sustrato , Temperatura , Uridina Quinasa/metabolismoAsunto(s)
Sarcoma de Mastocitos/enzimología , Fosfotransferasas/aislamiento & purificación , Uridina Quinasa/aislamiento & purificación , Animales , Radioisótopos de Carbono , Citidina , Haplorrinos , Cinética , Neoplasias Experimentales/enzimología , Espectrofotometría Ultravioleta/métodos , Especificidad por Sustrato , Uridina Quinasa/metabolismoRESUMEN
Partially purified enzyme fraction from rat kidney possessing high uridine kinase and phosphomonoesterase activity was insolubilized by means of zinc precipitation without substantial loss of the activity. While uridine kinase in a soluble and Zn-precipitated form was inhibited by low concentrations (0.5-1.0 mM) of Zn2+-ions, phosphomonoesterase was fully active. In contrast to the soluble fraction, the two enzymes in zinc-precipitated and lyophilized preparations were stable on heating at 100 degrees C. Metal complexed proteins catalyze the dephosphorylation of 5'-UMP, 6-AzaUMP as well as of 2'(3')-UMP or 2,4-dinitrophenyl phosphate indicating thus the presence of several phosphomonoesterases in the complex.
Asunto(s)
Riñón/enzimología , Monoéster Fosfórico Hidrolasas , Fosfotransferasas , Uridina Quinasa , Zinc , Animales , Precipitación Química , Femenino , Concentración de Iones de Hidrógeno , Cinética , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas/aislamiento & purificación , Unión Proteica , Ratas , Uridina Quinasa/aislamiento & purificación , Uridina Quinasa/metabolismo , Zinc/farmacologíaRESUMEN
Using Sepharose 6B coupled to N4-(5-aminopentyl)-cytidine at least three different peaks of rat kidney uridine kinase activity have been found. Prior to affinity chromatography the enzyme was partially purified by ammonium sulfate precipitation. The elution was carried out with 0.15 M NaCl or with 0.012 M ATP. The importance of different forms of uridine kinase in eukaryotic cells for cancer chemotherapy with uridine and cytidine analogues is discussed.
Asunto(s)
Cromatografía de Afinidad , Citidina/análogos & derivados , Riñón/enzimología , Fosfotransferasas/aislamiento & purificación , Uridina Quinasa/aislamiento & purificación , Animales , Femenino , Neoplasias/tratamiento farmacológico , RatasRESUMEN
Partial purification of uridine--cytidine kinase (EC 2.7.1.48) from foetal rat liver by chromatography on DEAE-cellulose gives two active fractions. The first in order of elution was identified as a form specific for foetal liver. It was purified 300-fold. The second fraction was common to foetal, and adult rat liver and spleen and was purified 20-fold. The foetal fraction of the enzyme was found to be heat-sensitive and protected against inactivation by PO34- anions. The two isolated forms have different apparent Km for uridine, respectively 410 muM for the foetal form and 52 muM for the adult form.
Asunto(s)
Hígado/enzimología , Fosfotransferasas/metabolismo , Uridina Quinasa/metabolismo , Animales , Estabilidad de Medicamentos , Femenino , Feto , Calor , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Masculino , Embarazo , Ratas , Bazo/enzimología , Uridina Quinasa/aislamiento & purificaciónRESUMEN
Partially purified calf brain uridine kinase precipitated by bivalent metal cations has been compared with the soluble enzyme fraction regarding its stability in the presence of inactivating factors. The freeze-dried preparations of uridine kinase precipitaated by Pb2+ or Zn2+ ions, althouth enzymatically highly active, are insoluble in aqueous solutions. The activity of metal-insolubilized enzymes disappears during their preincubation in acidic media or in the presence of silver ions. Also trypsin, chymotrypsin and cathepsin B1 caused decreases in enzyme activity. However, fractions which have been precipitated by metal ions and freeze-dried are stable at high temperatures, whereas the activity of soluble uridine kinase is completely lost. Both unheated metal-ion precipitated uridine kinase preparations and those heated at 100 degrees C are equally sensitive to the feedback inhibition by CTP.