ANALYTICAL

BIOCHEMISTRY

64,

A Rapid

367-371

(lY75)

Method

for

Ribonucleotide MOSE

Ribonucleoside homogenates

cessing

diphosphate

The of the reaction plate5. The

of multiple

Reductase

ROSSI AND BENITA

by following

deoxyribonucleotide. radioactivity PEI-cellulose

Determination

the

Activity

DE PETROCELLIS

reductahe activity i? determined conversion

enzymatic products method

of cytosine

reaction separated is rapid

of

ribonucleotide

in centrifuged to cytosine

i\ measured hy monitoring by thin layer chromatography and permit\ the Gmultaneous

the on pro-

samples.

The enzyme ribonucleotide reductase participates in the biosynthesis of DNA by reducing ribonucleosides diphosphate to the corresponding deoxyribonucleotides. Ribonucleotide reductase is the key enzyme for the biosynthesis of DNA precursors since animal cells synthesize (1.r tm\w only ribonucleotides. In addition, in Esc~hc~richitr coli and in rat hepatoma the enzyme is subject to complex regulation by the end products t 1.3) and thus the reduction of ribonucleotides is a rate controlling step in the pathway leading to the biosynthesis of DNA. The amount of ribonucleotide reductase activity in the cells is low, and sensitive methods are required for its measurement. The method of Reichard c,t rrl. ( I-3). consisting in the separation by chromatography on Dowex-50 column of the complex nucleotide mixture arising from the reaction, has been generally employed. This method is very sensitive and reproducible but requires for each sample a Dowex column and a quite long elution period to separate ribonucleotides from deoxyribonucleotides. This paper describes a chromatographic technique that, using labeled CDP, allows the determination of ribonucleotide reductase activity on a large number of samples in a short time. The separations are carried out by anion exchange chromatography on thin layers of PEI-Cellulose. This technique has been utilized for quantitative determinations of ribonucleotide reductase activity during embryonic development of Parrtc,ctt~IY)~L~.s li\.idtts sea urchin embryos (4).

368

ROSSI

AND

MATERIALS

DE

PETROCELLIS

AND

METHODS

Thin layers of PEI-cellulose and of DEAE were obtained from Machery-Nagel Co. and Merck; [2-‘-‘C]cytidine diphosphate from New England Nuclear; unlabeled nucleotides from Sigma Chemical Co. Other reagents were of analytical grade. The ribonucleotide reductase source was embryos of P. lit~idus 5-6 hr after fertilization. The embryos washed with 0.63 M NaCl were suspended in a buffer containing 0.1 M Tris-HCI and 1 mM DTT at pH 8.5, and disrupted by sonication using the microtip of the Branson J-17 A sonitier ( 10 set four times at 50% power). The homogenate was then centrifuged for 15 min at 27,OOOg and the supernatant assayed for the enzyme. The standard incubation mixture, derived from that described by Moore (5). contained 8.7 mM K-phosphate buffer pH 7.0, 4.4 mM ATP, 3.6 mM Mg-acetate, 8.7 mM NaF, 0.05 mM Fe”+ chloride, 6.0 mM DTT, 0.156 mM [‘YII]CDP (Sp act = 75,000-80,000 cpm/mpmole); 115 ~1 of incubation mixture contained l-3 mg of enzyme protein. Incubation was at 30°C for 30 min. The reaction was terminated by the addition of 150 ~1 of 2 M PCA: the denatured proteins were removed by centrifugation at 37,000~ for IO min and 10 (~1 of 0.1 M carrier dCMP was added to 150 ~1 of supernatant. The solution was then put in a boiling water bath for 10 min, to hydrolyse di- and triphosphates to monophosphates, cooled and neutralized by addition of 10 N KOH. In these conditions complete hydrolysis of nucleosides diphosphate and triphosphate to monophosphate was achieved and no radioactivity was found in the spots corresponding to them. After 15 min at 0°C the KClO, precipitate was removed by centrifugation and 10 ~1 of each neutral and protein-free solution were applied (on a line 2.5 cm long) on a PEI plate. The lines were spaced 1.5 cm from each other. The chromatograms were developed in one direction. at room temperature (18-20°C) using 4% H:,BO,-3 M LiCl (3: 1 v/v) (6). The time required for migration was 2 hr. The plates were prewashed with distilled water and dried overnight at room temperature. Spots were detected with a Mineralight shortwave lamp, cut out of the plate and the radioactivity monitored in a scintillation counter by adding 20 ml of toluene-liquiflor as scintillator fluid. A zero time control, consisting of an incubation mixture where the reaction had been stopped immediately after the addition of the enzyme, was prepared for each sample. All the radioactive determinations were corrected for the amount of radioactivity of the zero time control (- 200 cpm). To separate the three ribonucleotides CMP, CDP. and CTP from the corresponding deoxyribonucleotides dCMP. dCDP. and dCTP. the mixture was applied as a single spot on a DEAE plate and the chromat-

RIBONUCLEOTIDE

REDUCTASE

ASSAY

369

ogram was developed in two directions (7). Mono-, di-, and triphosphates were separated in the first direction, using 0.01 M HCI. In the second direction, ribonucleotides were separated from deoxyribonucleotides using, as solvent, 95% ethanol-l M ammonium acetate saturated with tetraborate ( I : 1 v/v). Protein was determined by the procedure of Lowry ct al. (8). RESULTS

AND

DISCUSSION

Figure 1 shows a typical separation on PEI plate of dCMP from CMP carried out spotting IO ~1 of assay mixture. dCR, CR, C. and dUMP (not reported in the figure), that can be the degradation products of CMP and dCMP, migrate more than dCMP almost with the same R,. On the contrary CDP, CTP, dCDP, and dCTP migrate less than CMP. However since dCMP migrates more than the corresponding ribonucleotide, it is possible to exclude contamination due to the substrate. Control of the extent of the hydrolysis step is possible by measuring the radioactivity corresponding to the different spots. Some radioactivity, in addition to that in the dCMP spot, is found associated with dUMP and never has been more than 10% of that found in the dCMP spot. This might

I-I