Ribonucleotide reductase assay

Jenh C-H, Geyer PK, Johnson LF (1985) Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse line. Mol Cell Biol 5 2527-2532 Jong AY, Yu K, Zhou B, Frgala T, Reynolds CP, Yen Y (1998) A simple and sensitive ribonucleotide reductase assay. J Biomed Sci 5 62-68... 

All eukaryotic ribonucleotide reductases assayed so far in vitro are inhibited by hydroxyurea, and a wide variety of eukaryotic cells and organisms is found susceptible to DNA synthesis inhibition by the drug . Nevertheless, a clearcut dependency like in E. coli cannot be stated as investigation of the eukaryotic enzymes is lagging and because hydroxyurea exerts manifold cellular effects, some without obvious relation to the inhibition of ribonucleotide reduction (cf. Table 9). 

Ribonucleotide reductase activity was assayed based on CDP reduction, using a modified method of Jong et al. (1998), with the [ CICDP reduction product determined as radioactivity incorporated into DNA in a series of two coupled reactions, catalyzed by nucleoside diphosphate kinase and DNA polymerase (Klenow fragment). A 40 pi reaction mixture contained 50 mM Hepes pH 7.2, 10 mM dithiothreitol. 

Additional support for the Mn specificity of native B. ammoniagenes ribonucleotide reductase is provided by its inhibition by cyanide (Fig. 5). The iron- and B 12-enzymes are not affected by cyanide which forms cyano and cyanoaquo complexes with Mn" but not with Fe or Fe" " " under typical enzyme assay conditions at ambient temperature. We thus believe that manganese in the Brevibacterium enzyme system is the functional, catalytic counterpart of iron found in E. coli and eukaryotic ribonucleotide reductases. The nature of Mn -protein interaction remains to be studied after further purification. 

It has so far been impossible to isolate ribonucleotide reductases from homogenates of higher plants such as the germinating wheat embryo, or bean root tips and cell cultures which all contain only minute amounts of enzyme As these activities are clearly growth-correlated like in other organisms and standard assay conditions are required, there is no evidence for a plant-specific, diverse type of reductase. 

The topology of the radical site pocket of calf thymus and mouse fibroblast ribonucleotide reductase was recently probed with a series of hydroxamate inhibitors of increasing bulkiness and will be discussed in the following section. Other mammalian sources from which ribonucleotide reductases have been isolated and more or less purified include rat Novikoff hepatoma and regenerating rat liver" rabbit bone mar-row 5,66) Ejjj-iich ascites tumor cells of mice and cultured human lymphoblast cells Some of their properties are described in Table 2. Many more animal and human cells were assayed for enzyme activity, frequently in mutant cell lines, to test for cell cycle dependence, mechanisms of metabolic regulation, drug resistance, and correlation with tumor growth rates. Representative studies of this kind, which rapidly expand in number, are summarized in Table 3. 

Whether or not pyrimidine and purine ribonucleotides are reduced by a single enzyme is a matter of some controversy. The monomeric reductase of L. leichmannii and the enzyme of E. coll where substrates bind exclusively to Bas well as calf thymus and human reductase consist of only one holoenzyme species capable to reduce all the ribonucleotides. In contrast rat liver ribonucleotide reductase during purification dissociated into one protein fraction specific for CDP reduction and one reducing the three other diphosphates from which a specific UDP reductase activity could then be obtained Other reports of differential ADP and CDP reductase activities in rodent enzyme preparations are difficult to assess. In reductase assays one can easily encounter slow reduction rates for one particular ribonucleotide when not all conditions like pH, ionic strength, effector nucleotide and dithiol concentrations, and stoichiometry of subunits, are at an optimum. Therefore differential activities not necessarily indicate different enzyme species. 

The activity of the proteinaceous inhibitors isolated from animal cells is inversely correlated with the proliferation rate of their source. Only recently was such an inhibitor characterized as complex mixture of enzymes like phosphatases and kinases that act on the substrate CDP and on the effector ATP The assumption is justified that this class of substances merely interferes with the assay and does not define specific cellular inhibitors of ribonucleotide reductase. Nevertheless it must be realized that ribonucleotide reductase activity is well coordinated with cell proliferation and is strictly controlled within the cell cycle. Hence a regulatory role of inhibitors besides the control of gene expression and fine tuning by allosteric effectors is a priori conceivable. 

The initiation or arrest of enzyme synthesis responsible for the peak of ribonucleotide reductase is discussed below. Other factors which may modulate the activity in vivo or when measured in crude homogenates include allosteric control by the endogenous deoxyribonucleotides, the action of late S phase-specific inhibitors like the one found in Achlya, or the redox status of thioredoxin and glutaredoxin however the cell cycle dependence of these reactions is little known. It is therefore desirable to assay ribonucleotide reductase in preparations which have been subjected to at least one precipitation and dialysis step. While reliable cell cycle-dependent activity data are thus obtained, the absolute figures are frequently an order of magnitude too low to account for the cell s need of ribonucleotide reduction for DNA synthesis. This unsatisfactory condition is most likely a problem of quinary enzyme structure (see below) but is not felt to invalidate the accumulated evidence for tight correlation of ribonucleotide reduction and the cell cycle as a whole. 

The isolated reductase (above) was used in assays to direct further fractionation experiments which culminated in the isolation of the physiological reducing system this turned out to be a previously unreci ized hydrogen transport system. This system, which connects ribonucleotide reductase to the NADPH-NADP+ system, was found to be a two-component system con.sisting of a small sulfhydryl protein, thioredoxin, and a flavoprotein, thioredoxin reductase. Thioredoxin is the reductant which specifically interacts with the ribonucleotide reductase. In the presence of catalytic amounts of thioredoxin, the thioredoxin reductase will link NADPH with the reduction of ribonucleoside diphosphates as follows ... 

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