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227

RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE

Ribonucleoside

Diphosphate co//)

Reductase

(Escherichia

B y LARS T H E L A N D E R , B R I T T - M A R I E SJOBERG, a n d S T A F F A N ERIKSSON NDP + thioredoxin-(SHh

Mg2+

~ dNDP + thioredoxin-S2

Deoxyribonucleotides are synthesized by a direct reduction of the corresponding ribonucleotides. The reaction is catalyzed by ribonucleotide reductase which in this way is involved in the control of DNA synthesis. The enzyme has been purified to homogeneity and studied extensively in Escherichia coli and Lactobacillus leichmannii. 1 The preparation of ribonucleotide reductase from T4-infected E. coli by affinity chromatography z and the partial purification of the enzyme from Novikoff hepatoma z in rats were described in this series previously. An E. coli strain (KK546) 4 which is lysogenic for a defective lambda carrying the genes of ribonucleotide reductase overproduces the enzyme upon induction. The preparation of large amounts of homogeneous enzyme rests on the use of this source containing 10% of the total protein as ribonucleotide reductase. Assay Methods Principle. Ribonucleotide reductase from E. coli consists of two nonidentical subunits, proteins B1 and B2, which have no biological activity when assayed separately. In the presence of Mg z+ they combine to form the active enzyme. Therefore, one subunit is always assayed in the presence of an excess of the o t h e r ) In the reduction of ribonucleotides, stoichiometric amounts of oxidized thioredoxin are formed. This low-molecular-weight protein is reduced by N A D P H in a reaction catalyzed by a flavoprotein, thioredoxin reductase. 1 Thioredoxin-S2 + NADPH --~ thioredoxin-(SH)2 + NADP ÷

By combining the two reactions ribonucleotide reductase activity can be measured by spectrophotometric determination of N A D P H oxidation) 1 H. P. C. Hogenkamp and G, N. Sando, Struct. Bonding (Berlin) 20, 23f (1974). 2 O. Berglund and F. Eckstein, this series, Vol. 34B, p. 253. 3 E. C. Moore, Vol. 12A, p. 155. 4 S. Eriksson, B.-M. Sj6berg, S. Hahne, and O. Karlstrrm, J. Biol. Chem. 252, 6132 (1977). N. C. Brown, Z. N. Canellakis, B. Lundin, P. Reichard, and L. Thelander, Eur. J. Biochem. 9, 561 (1969). METHODS

IN ENZYMOLOGY,

VOL. LI

Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181951-

228

DEOXYNUCLEOTIDE SYNTHESIS

[30]

The spectrophotometric assay cannot be used with crude extracts due to unspecific oxidation of NADPH. Instead ribonucleotide reductase activity in crude extracts is determined by measurements of the formation of [3H]dCDP from [3H]CDp.5

A. Spectrophotometric Assay Procedure. The enzyme was incubated at 25 ° in a mixture containing 200 nmoles ATP, 1.6/zmoles MgCI2, 80 nmoles NADPH, 5/xmoles N-2hydroxyethylpiperazine-N'-2-ethanesulphonic acid buffer (pH 7.6), 300 pmoles thioredoxin, 40 pmoles thioredoxin reductase, 10 nmoles EDTA, and 65 nmoles dithiothreitol in a final volume of 0.13 ml. The reaction was started by the addition of 75 nmoles CDP, and the oxidation of NADPH was monitored at 340 nm with a Zeiss automatic recording spectrophotometer equipped with microcuvettes. The best determinations of activity were obtained when 1-2.5 units of one subunit were assayed in the presence of a 5-fold excess of the other subunit. Before addition of CDP the background oxidation of NADPH was recorded for some minutes, and this background was subtracted from the N A D P H oxidation observed after addition of CDP. Comments. Dithiothreitol stabilizes the B1 subunit of ribonucleotide reductase, 6 and therefore it is included in the incubation mixture in a final concentration of 0.5 mM. At higher concentrations dithiothreitol starts to chemically reduce oxidized thioredoxin and. competes with N A D P H as hydrogen donor. 5 EDTA is included in the stock solutions of thioredoxin and thioredoxin reductase to protect these proteins. B. [OH] CDP Assay

Procedure. The same incubation mixture was used as in the spectrophotometric assay but [3H]CDP (specific activity 2 x 106 cprn//zmole) replaced the CDP and the reaction was started by the addition of enzyme. Thioredoxin reductase can be replaced by 10 mM dithiothreitol. 5 After 10 min at 25 ° the reaction was stopped by the addition of 0.5 ml of 1 M perchloric acid and 0.5/zmole of dCMP carrier. Precipitated protein was carefully removed by centrifugation, and then the nucleotides were hydrolyzed to monophosphates by heating at 100 ° for 10 min. Potassium hydroxide (4 M) was added to neutralize the pH using phenol red as indicator, the potassium perchlorate was precipitated on ice for 10 min and then removed by centrifugation, and the sample was chromatographed on a 1 × 3.5 cm Dowex column (Dowex 50W-X8, 200--400 6 L. Thelander, J. Biol. Chem. 248, 4591 (1973).

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RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE

229

mesh, H +, in Biorad Econo columns 1 × 10 cm with polypropylene funnels). On elution with 0.2 M acetic acid, CMP eluted in the first 55 ml and dCMP in the following 25 ml. The chromatographic procedure was finished in 2 hr. Aliquots of 1 ml from the dCMP eluate were analyzed for radioactivity using Instagel (Packard) and a liquid scintillation counter. The amount of dCMP carrier in the dCMP eluate was determined by reading the absorbance at 280 nm. Usually recoveries were around 60-70%. The values from the radioactivity measurements were finally corrected for the recoveries of the dCMP carrier.

Definition of Unit and Specific Activity. One unit of ribonucleotide reductase activity is defined as the amount of proteins B1 or B2 which catalyzes the formation of 1 nmole of dCDP per minute at 25 ° under standard conditions in the presence of an excess of the complementary subunit. This definition of a unit is one-tenth of earlier used values. 5 The specific activity of a preparation is defined as units of enzyme activity per milligram of protein.

Purification Procedure

Reagents Strain: E. coli KK546 (F- thr leu thi thy tlr tonA lac supE44 nrdA nalA malA/hdnrd-1 ~b515 519 CI857 $7 xis6) Culture medium (in grams/liter): Bacto-tryptone (10), yeast extract (5), NaC1 (10), glucose (1); pH is adjusted to 7.0 before autoclaving. Prior to use 2 ml of sterile 0.5 M MgSO, are added. Buffer A: 50 mM Tris-Cl (pH 7.6)-20% glycerol-15 mM MgC12 Dithiothreitol (A-grade, Calbiochem) Streptomycin sulfate (Novo Industri A/S) dATP-Sepharose (prepared according to Berglund and Eckstein 2) Immuno adsorbent columns* consisting of anti B1 or anti B2 yglobulin covalently bound to Sepharose 4B Collodion bags, type SM 13200 (Sartorius-Membranfilter, 34 GSttingen, West Germany) Ultrogel AcA 34 (LKB-Beckman) Growth and Disintegration of Bacteria. A 250-liter culture of E. coli KK546 was grown with moderate aeration 0 2 5 liters/min) at 33 °. At a density of 5 x l0 a cells/ml the temperature was raised to 43 ° in less than 5 min by circulation of steam through the cooling jacket of the tank. After 20 min at 43 ° the culture was cooled to 37 ° by circulation of tap water through the cooling jackets. The culture was grown at 37 ° for 2 hr and then rapidly cooled to l0 ° by circulation of ice water. The cells (1.35

230

DEOXYNUCLEOTIDESYNTHESIS

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kg) were harvested through centrifugation, rapidly frozen, and stored at - 2 0 °. Immediately prior to the preparation of the crude extract the ceils were disintegrated at - 2 0 - - 2 5 ° in a bacterial press. 5

Crude Extract. Frozen disintegrated cells were extracted in a loosely fitting glass homogenizer with 3 volumes of buffer A containing 4 mM DTT for 10-15 rain until the frozen cells were thawed. Insoluble cell debris was removed by centrifugation at 35,000 g for 40 min. Streptomycin Precipitation. The supernatant solution was precipitated by addition of 0.2 volume of a 5% neutralized solution of streptomycin. The precipitant was slowly added (15 rain) with continuous stirring, the resulting suspension was stirred for an additional 15 rain, and the precipitate was removed by centrifugation. Ammonium Sulfate Fractionation. The supernant solution was precipitated by the addition of solid ammonium sulfate to give 60% saturation (0.39 g/ml solution). The suspension was stirred for 60 rain, and the precipitate was collected by centrifugation and dissolved in 1/4 of the original volume in buffer A containing 4 mM DTT. Ammonium sulfate was removed by passing this solution through a column of Sephadex G-25 equilibrated with the same buffer. Affinity Chromatography on dATP-Sepharose. The desalted protein fraction was adsorbed to a column of dATP-Sepharose 2 (2000 enzyme units/ml), which had previously been equilibrated with buffer A containing 4 mM DTT. The column was washed free of nonspecifically bound proteins with 8-9 column volumes of the same buffer. Most of the protein B2 activity in the extract was retarded on the column but 1020% of the activity was found in the wash fraction. The dithiothreitol concentration was then increased to 10 mM. This improved the stability of protein B1 so that the column could be left overnight. Two different elution procedures were then used. Alternative I, which yielded a complex of proteins B1 and B2, was used when separation of the two subunits from each other was unnecessary. Alternative II was used to obtain protein B1 and protein B2 in separate fractions from which either subunit could subsequently be further purified. Elution Alternative I. The column was eluted with 3-4 volumes of buffer A containing 10 mM DTT and 10 mM ATP. The resulting eluate contained a mixture of the two subunits with 4.5 mg of protein B! per mg of protein B2, i.e., approximately 2 molecules of protein B1 per molecule of protein B2. There was less than 15% contaminating proteins in the mixture of

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RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE

231

proteins B 1 and B2 as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme prepared by this very rapid procedure can be used as helper enzyme when other components of the ribonucleotide reductase system are to be assayed, i.e., the hydrogen donor system: thioredoxin, thioredoxin reductase,1 or glutaredoxin. 7 Elution Alternative H. Elution of protein B2 was started with 6 volumes of 10 mM Tris-C1 (pH 7.6)-20% glycerol-10 mM DTT. The eluate contained 50% of the applied protein B2 and 2-3% of contaminating protein B 1. Some contaminating protein was then desorbed by a second wash of the column with 4 volumes of 0.1 M Tris-C1 (pH 7.6)-0.2 M NaC1-20% glycerol-10 mM DTT. The column was again equilibrated with troffer A containing 10 mM DTT, and protein B1 was eluted with 3-4 volumes of buffer A containing 10 mM DTT and l0 mM ATP. Approximately 80% of the protein B1 and 10% of the protein B2 activity eluted in this ATP pool. Either subunit was 70-80% pure at this stage, and the elution procedure is schematically depicted in Fig. 1. Chromatography on Anti-B1 and Anti-B2 T-Globulin Sepharose Col-

1 1.5

,x I

Fnt

i 1.0

wash

Protun B2 ~

I

" \l Second ~1~ wash

tL5

BUtCh volume (ml) Fro. 1. Affinity c h r o m a t o g r a p h y on d A T P - S e p h a r o s e , elution alternative II. A desalted protein fraction (607 m g in 34 ml) w a s applied to 15 ml of d A T P - S e p h a r o s e and c h r o m a t o g r a p h e d as described in the text. A b s o r b a n c e at 280 n m , - (the a b s o r b a n c e at 290 n m w a s m e a s u r e d in the A T P eluate and t h e n u s e d to calculate the a b s o r b a n c e at 280 n m after subtraction of the A T P absorption, A28o ~ 2 × A290). Protein B2 activity, r~; protein B 1 activity, []. r A. H o l m g r e n , Proc. Natl. Acad. Sci. U.S.A. 73, 2275 (1976).

232

DEOXYNUCLEOTIDE SYNTHESIS

[30]

umns. Antisera against proteins B 1 and B2 were prepared from rabbit as described by Berglund 8 and precipitated with ammonium sulfate to 40% saturation. The dissolved and desalted y-globulin fraction had a protein concentration of 8 mg/ml. This fraction was coupled to CNBr-activated Sepharose 4B by the method described by Cuatrecasas. 9 The coupled Sepharose had an antigen-binding capacity of approximately 0.05 mg/ml. The protein B1 preparation from elution alternative II was next exposed to anti-B2 antibodies, and the protein B2 preparation was exposed to anti-B1 antibodies by immediately passing the eluates through the antibody-y-globulin Sepharose columns. The anti-B2 column was equilibrated with buffer A containing 10 mM DTT before use and eluted with 2 volumes of buffer A containing 10 mM DTT and 0.2 M NaCI. The anti-B1 column was equilibrated with 50 mM Tris-C1 (pH 7.6)-20% glycerol-0.2 M NaC1 and eluted with 2 volumes of the same buffer. By this procedure most of the contaminating subunit was removed. The columns were regenerated by washing with 3 column volumes of 6 M guanidine hydrochloride which removed the bound antigen. Concentration by Ultradialysis. Both eluates were concentrated by ultradialysis in collodion bags against buffer A containing 10 mM DTT (protein B1) or 50 mM Tris-Cl (pH 7.6)-20% glycerol-0.2 M NaC1 (protein B2) to a concentration of approximately 40 mg protein per ml. After the concentrated protein fraction had been removed, the empty dialysis bag was left in the buffer for 1 hr. Approximately 0.1 ml of buffer diffused back into the bag and was added to the previous fraction. This gave a recovery of 80-90% of both protein and activity and up to 200-fold concentration. Chromatography on Ultrogel AcA 34. Each concentrate from the 3,_ globulin Sepharose chromatography was further purified on Ultrogel AcA 34. This gel allows high enough flow rates for the chromatography to be completed within 20 hr. PROTEIN B1. The column was equilibrated and eluted with buffer A containing 10 mM DTT. The enzyme activity emerged from the column in a peak that coincided with the major protein peak. Protein B2 activity eluted slightly ahead of the main protein B 1 peak. This position in the chromatogram corresponds to the molecular weight of the complex between the two subunits. The fractions with constant specific activity were pooled and concentrated by ultradialysis. The pooled protein B1 contained no ATP as judged by the absorbance ratio of 260/280 nm. 80. Berglund, J. Biol. Chem. 250, 7450 (1975). P. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970).

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233

RIBONUCLEOSIDE DIPHOSPHATE R E D U C T A S E

0 0

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Ribonucleoside diphosphate reductase (Escherichia coli).

[30] [30] 227 RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE Ribonucleoside Diphosphate co//) Reductase (Escherichia B y LARS T H E L A N D E R , B R I...
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