RESUMEN
In all living organisms, deoxyribonucleotides, the DNA precursors, are produced by reduction of the corresponding ribonucleotides catalyzed by ribonucleotide reductase. In mammals as in Escherichia coli, the enzyme consists of two proteins. Protein R1 is the proper reductase as it contains, in the substrate binding site, the reducing active cysteine pair. Protein R2 provides a catalytically essential organic radical. Here we report the cloning, expression, purification and characterization of protein R1 from Arabidopsis thaliana. Expression in E. coli was made possible by coexpression of tRNAArg4 which is required for the utilization of AGA and AGG as codons for arginines. Protein R1 shows extensive similarities with protein R1 from mammals: (a) it shows 69% amino-acid sequence identity to human and mouse R1 protein; (b) it is active during CDP reduction by dithiothreitol, in the presence of protein R2 [Sauge-Merle, S., Laulhère, J.-P., Coves, J., Ménage, S., Le Pape, L. & Fontecave, M. (1997) J. Biol. Inorg. Chem. 2, 586-594]; (c) activity is stimulated by thioredoxin and ATP and is inhibited by dATP, showing that as in the mammalian enzyme, the plant ribonucleotide reductase seems to be allosterically regulated by positive (ATP) and negative (dATP) effectors.
Asunto(s)
Arabidopsis/enzimología , Proteínas de Plantas/genética , Ribonucleótido Reductasas/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Proteínas Bacterianas/química , Clonación Molecular , Codón , Citidina Difosfato/metabolismo , ADN Complementario/genética , Nucleótidos de Desoxiadenina/farmacología , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Ratones , Datos de Secuencia Molecular , Oxidación-Reducción , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Lipid peroxidation in rat liver microsomes induced by asbestos fibres, crocidolite and chrysotile, is greatly increased in the presence of NADPH, leading to malondialdehyde levels comparable with those induced by CCl4, a very strong inducer of lipid peroxidation. This synergic effect only occurs during the first minutes and could be explained by an increase or a regeneration of the ferrous active sites of asbestos by NADPH, which in turn could rapidly be prevented by the adsorption of microsomal proteins on the surface of the fibres. It is not inhibited by superoxide dismutase, catalase and mannitol, indicating that oxygen radicals are not involved in the reaction. It is also not inhibited by desferrioxamine, indicating that it is not due to a release of free iron ions in solution from the fibres. Lipid peroxidation in NADPH-supplemented microsomes is also greatly increased upon addition of magnetite. This could be linked to the presence of ferrous ions in this solid iron oxide, since the ferric oxides haematite and goethite are completely inactive.