Purification and Properties of Ribonucleotide Reductase from Leukemic Mouse Spleen*

was observed in the specific activity of ribonucleotide reductase in the a the properties of the the

The reduction of CDP was stimulated by dCTP and ATP.
UDP reduction was stimulated by ATP. The reduction of ADP was stimulated by GTP and dGTP. The GDP reduction was stimulated by dTTP.
In the presence of ATP, the weak activators, dTTP and dATP, inhibit the CDP reduction.
With minor exceptions, the ribonucleotide reductase from murine leukemic spleen closely resembles that of Novikoff hepatoma.
Regulation of the enzymatic reduction of ribonucleotides to deoxyribonucleotides has been investigated in several microbial and animal systems.
The fact that deoxyribonucleotides are present only in very small amounts in cells (11) suggests that the reduction of ribonucleotides to deoxyribonucleotides would be a suitable locus for the regulation of deoxyribonucleic acid formation (5,12). In view of the probable importance of this reaction in cell proliferation, and the potential value of this reaction as a target site for antimetab- olites, it seemed pertinent to determine the levels of the enzyme and regulation of this system in the course of viral leukemogenesis. Changes in ribonucleotide reductase have been observed following the infection of bacteria (13,14) and animal cells with viruses.
A 3-to 5-fold increase in activity occurs upon polyoma virus infection of contact-inhibited mouse kidney cells (15). The ribonucleotide reductase level in cell-free extracts of Yaba Pox virus tumor is from 2-to IO-fold higher than the levels found in normal monkey tissues (16).
The present paper reports a striking increase in ribonucleotide reductase activity in the spleen of mice following infection with a murine leukemia virus and describes some properties of the partially purified enzyme. The results show that the reduction occurs mainly at the diphosphate level and requires a sulfhydryl compound as a hydrogen donor. Although the enzyme resembles the E. coli (17) and the Novikoff hepatoma (18) ribonucleotide reductases in many respects, several differences were found in the allosteric effecters regulating this system in the leukemic mouse spleen.

Preparation of Enzyme-Female
Swiss mice, weighing approximately 25 g, were inoculated intraperitoneally with 0.2 ml of a cell-free extract prepared from spleens of DBA/2 mice, which had been inoculated with Friend murine leukemia virus (19).
Control animals were injected with an equal volume of 0.15 M NaCl or with 1 mg of phenylhydrazine in 0.2 ml of 0.15 M NaCI.
The spleens from the virus-infected animals weighed up to 20 times more than spleens of normal controls.
The administration of phenylhydrazine, which causes a hemolytic anemia in the mouse, results in a 2-to a-fold increase in splenic weight.
Groups of six to 10 animals were killed at various time intervals.
The enzyme was purified from the spleen of animals killed 5 days postinoculation.
Ten spleens, weighing a total of 14 g, were pooled and homogenized in an equal volume of 0.02 M Tris-HCl buffer, pH 7.0, in a Potter-Elvehjem homogenizer fitted with a Teflon pestle.
The homogenate was centrifuged at 20,000 X g for 45 min.
The supernatant fluid was passed through a column (2.4 x 40 cm) of Sephadex G-25 to eliminate endogenous nucleotides and eluted with the same Tris HCI buffer.
Streptomycin sulfate was added to the eluate to a final concentration of 1% (w/v) and the precipitate was removed by centrifugation.
An ammonium sulfate precipitation step followed.
The precipitate formed after the addition of ammonium sulfate (1.95 g to 10 ml) was removed by centrifugation. An additional 0.92 g of ammonium sulfate was added to the supernatant fluid.
The precipitate, which represents a 25 to 40% saturated ammonium sulfate fraction, was dissolved in 1 ml of 0.02 M Tris-HCl buffer, pH 7.0. The solution was passed through a column, 1 X 30 cm, of Sephadex G-25 which was eluted with the same Tris buffer.
All procedures were performed at 4". The enzyme solution was used immediately or kept at -60" for up to 1 week.
Enzyme Assay Method-The assay method for ribonucleotide reductase in our laboratory was described previously (20). The assay mixture, in a final volume of 120 ";All substrates were tested at the 0.5 mu level according to thelassay system described in the text. nucleosides on the paper was determined with a liquid scintillation counter.
Protein was determined according to the procedure of Lowry et al. (21). Specific activity is expressed as millimicromoles of substrate reduced per mg of protein per 15 min.
Duplicate assays agreed within &15%. Chemicals-Nucleotides and nucleosides were purchased from Calbiochem and Sigma. Dithiothreitol was purchased from Calbiochem.
Bacterial alkaline phosphatase was obtained from Worthington.
All radioactive chemicals were purchased from Schwarz BioResearch.

RESULTS
The specific activity of ribonucleotide reductase in the spleen following infection with murine leukemia virus is shown in Fig. I. On the 2nd day after inoculation, the specific activity had risen to over 3 times that of the control.
Over the next 3 days, there was a sharp increase in the enzyme level to a peak specific activity approximately 13 times higher than the average for normal spleen.
Thereafter, a gradual decline occurred and, by Day 14, the moan specific activity had fallen to about 3 times that of the normal level. The specific activity in the spleen of the phenylhydrazine-treated animals was 1.5 to 3 times that of controls. The purification of ribonucleotide reductase from leukemic spleen is summarized in Table I. After ammonium sulfate precipitation, the enzyme became very labile, losing 50% of its activity when stored at 4" and 90% of its activity when kept at room temperature for 40 hours. Attempts to stabilize the enzyme with 2-mercaptoethanol (1O-4 M), dithiothreitol (10e4 M), ascorbic acid (low4 M), 10% glycerol, EDTA (low4 M), Mg++ (1O-4 M), Ca++ (5 X 10F3 M), or phenylmethyl sulfonyl fluoride were without effect. dCTP and ATP did not stabilize the enzyme for storage or purification.
Precipitation of the enzyme at pH 5.0 resulted in a 75% loss in specific activity. Treatment of the preparation with alumina C-r or calcium phosphate gel, chromatography on DEAE-cellulose, or further filtration through Sephadex resulted in either no further increase or a loss in specific activity.
A loss in activity with dialysis has been reported for the ribonucleotide reductase from Yaba Pox virus tumor and some normal monkey tissues (16).
Requirement of Reaction- Fig.  2 shows the effects of magnesium acetate, ferric chloride, dithiothreitol, and ATP on the reaction. The reduction showed a requirement for dithiothreitol and ATP. Higher concentrations of ATP were markedly inhibitory. Slight stimulation by Mg++ was observed. Higher concentrations of Mg++ or Fe+++ ion result in moderate inhibition.
General Properties of Enzyme-The ribonucleotide reductase from murine leukemic spleen was not stimulated by the addition of deoxyadenosyl 13rz in a concentration of 10h5 ELI. The optimal pH for the reduction of CDP (in potassium phosphate buffer) occurs between pH 6.8 and pH 7.4, as shown in Fig. 38.
A linear relationship between the concentration of enzyme and the reaction rate was observed (Fig. 3B).
When a time study of the reduction of CDP was carried out (Fig. 3C), it was found that the rate decreased after 10 min.
Cytidine Nucleotide SpeciJicity-A comparison of different cytidine nucleotides as substrates is shown in Table II. The highest reduction rate was obtained with CDP. The reaction proceeded at approximately two-thirds maximal rate when CMP or CTP was substituted for CDP. Since phosphatases and kinases were still present in the preparation, it seems likely that some conversion of the tri-and monophosphate to the nucleoside diphosphate may have occurred. Nucleotide Activation and Inhibition Effects on Reduction of CDP-L4 comparison of the effects of different nucleoside triphosphates on CDP reduction is shown in Fig. 4. The reduction of CDP was greatly influenced by the presence of different nucleotides.
The addition of dCTP and ATP resulted in marked stimulation of the reaction; dTTP and dATP were less effective than dCTP and ATP.
It is difficult to show any inhibitory effects in the absence of ATP because of the very low reduction rate.
The effects of nucleotides on CDP reduction in the presence of 2 InM ATP are shown in of dCTP stimulate the reduction of CDP, whereas dTTP, dATP, and larger amounts of dCTP inhibit the reaction. Reduction of UDP-The effects of nucleotides upon the reduction of UDP are shown in Fig. 6. ATP markedly stimulated the reaction.
Slight stimulation by dATP was also noted on UDP reduction.
The striking stimulatory effect which dCTP has on CDP reduction is lacking when UDP is the substrate. Reduction of ADP-As shown in Fig. 7, reduction of ADP proceeded at a much greater velocity in the presence of GTP or dGTP.
dTTP and dUTP showed a lesser stimulatory effect on ADP reduction.
Reduction of GDP-The addition of dTTP resulted in a striking increase in GDP reduction, as shown in Fig. 8. Optimal activity was observed at a dTTP concentration of 1 x lo+ RI. dUTP and dGTP showed lower stimulatory effects. ATP stimulated this reaction slightly.
Although not shown in the figure, in the presence of dTTP, the reduction of GDP was inhibited by dATP to the same extent as the reduction of CDP (Fig. 5).
Similar rates were obtained when the same purified enzyme preparation was tested with CDP, UDP, GDP, or ADP as substrate.
In the absence of ATP or other effecters, the barely detectable reaction rates ranged from 0.01 to 0.02 mpmole of ribonucleotide reduced per mg of protein per 15 min. With optimal effector concentrations, a 7-to 30-fold increase in reaction rate was observed (Figs. 4 to 7).
No differences were found between the enzymes purified from leukemic spleens, normal spleen, and spleens of control animals injected with phenylhydrazine. ATP and dCTP stimulated CDP reduction. dGTP and dTTP stimulated reduction of ADP and GDP.
The activities were too low to allow any conclusion concerning inhibition by negative effecters.

DISCUSSION
The marked increase in the specific activity of ribonucleotide reductase in a rapidly growing neoplastic tissue is consistent by guest on July 9, 2020 with the function of the enzyme in providing deoxyribonucleotide precursors for DNA synthesis.
The decline in specific activity after Day 6 was unexpected since the spleen continued to increase in size, doubling its weight over the next 4 days. It is conceivable that sufficient amounts of deoxyribonucleotides had accumulated to allow DNA synthesis to continue without a further increase in the enzyme.
Repression of further enzyme synthesis by a metabolite offers a possible explanation for the decrease in ribonucleotide reductase activity. The purification of ribonucleotide reductase from leukemic spleen, like that of the enzyme from Novikoff hepatoma cells, has been complicated by the enzyme's instability during storage or manipulation.
Two differences between the spleen enzyme and that from Novikoff hepatoma were noted during the purification procedure.
Gel filtration of the spleen enzyme through Sephadex G-25 results in a greater increase in specific activity than does dialysis or gel filtration of the Novikoff hepatoma enzyme.
Since there was a 5-fold increase in total activity of the spleen enzyme following this step, it seems probable that a significant quantity of endogenous nucleotides were removed; this would result in less dilution of the radioactivity in the ribonucleotide added to the assay and could account for the increase in specific and total activity.
The removal of an inhibitor by Sephadex filtration would offer an alternative explanation for this finding.
The second difference noted in the spleen ribonucleotide reductase was that the precipitation at pH 5.0 (18,22), a useful step in the course of the purification of the enzyme from E. coli or Novikoff hepatoma, resulted in a loss of specific activity of the spleen enzyme.  Reichard and Moore (7,8,24), the effects of dTTP and dATP are dependent upon the concentration of ATP.
The two deoxyribonucleotides can therefore function as either inhibitors or activators. dCTP markedly stimulated the reaction in the absence of ATP and also caused a slight stimulation if ATP was present in the reaction.
It is worth noting that dCTP acts as an allosteric stimulator on the CDP reduction in the murine leukemic spleen while it inhibits the Yaba tumor enzyme.
As has been observed in the other systems, dTTP stimulates the reduction of the purine nucleotides, and dTTP is the most potent stimulator of GDP reduction in the E. co& Novikoff hepatoma, and leukemic spleen system. dUTP and dGTP are almost as effective in stimulating the reaction. This relationship is reversed in ADP reduction, in which the greatest stimulation occurs with dGTP, and dTTP stimulation is considerably less than that observed with dGTP.
A marked stimulation of ADP reduction by GTP was also noted in this study.
The positive and negative influence of nucleotide effecters in the leukemic spleen ribonucleotide reductase follows the general pattern first described for the E. coli enzyme and even more closely resembles the regulatory controls observed in the Novikoff hepatoma enzyme. Some differences, however, exist between these neoplastic tissues.
For example, dCTP which is a weak stimulator of CDP reduction in the No&off hepatoma reductase has a marked effect on the spleen enzyme.
On the other hand, GTP, which stimulates CDP reduction in the hepatoma system, does not appear to affect the reaction of the spleen enzyme with this substrate.
It is possible that the quantitative variation in these effecters may stem from further interconversions or from differences in their rates of degradation by the relatively impure enzyme preparations. The effects of nucleotides on ribonucleotide reductase from murine leukemic spleen are summarized in Table III. Despite the increase in ribonucleotide reductase activity in the leukemic spleen, no differences in the regulation by nucleotides were found from enzyme purified from normal spleen. The lack of qualitative differences might favor an increase in synthesis de noao of ribonucleotide reductase by the host cells over the coding for a different enzyme by the murine leukemic virus, as an explanation for the elevated enzyme level.
Acknowledgment-We wish to express our thanks to Miss Rose Meyers for her skillful technical assistance. This work was initiated while one of the authors (R. Silber) was a Visiting Scientist in the Department of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California.