BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95126

ON THE RELATIVE FUNCTIONING OF THE PATHWAYS FOR FORMATION

OF THYMIDINE NUCLEOTIDES IN THE REGENERATING LIVER AND

SPLEEN OF THE RAT

M O N N A C R O N E " AND S. I T Z H A K I

Department o/ Radiotherapeuties, University o/ Cambridge, Cambridge (Great Britain) (Received May 28th, I964)

SUMMARY

Experiments were carried out to give information on the relative functioning, in regenerating rat liver and in spleen, of the two possible pathways for the bio- synthesis of thymidine nucleotides, namely: Pathway I: uridine monophosphate-+ deoxyuridine monophosphate--> thymidine monophosphate, and Pathway II: uridine monophosphate -+ cytidine monophosphate -~ deoxycytidine monophosphate -+ deoxyuridine monophosphate -+ thymidine monophosphate.

The pyrimidines of ribonucleic acid and deoxyribonucleic acid were labelled by a single injection of [6-14C]orotic acid in the control rats. In the experimental animals, in addition to the labelled orotic acid, unlabelled eytidine was injected in divided doses before and after the orotic acid injection. It was noted that in the latter series the unlabelled cytidine caused a great decrease in the specific activities of cytidylic acid isolated from ribonucleic acid and of deoxycytidine from deoxyribonucleic acid, of regenerating liver and spleen, reflecting dilutions of the metabolic pools of cytidine nucleotides. In contrast, there was a comparatively small drop in the specific activity of deoxyribonucleic acid-thymidine. It is concluded that in both tissues, ttlymidine monophosphate is formed from uridine monophosphate to a greater extent through Pathway I without the intermediate formation of cytosine nucleotides stipulated by Pathway II.

INTRODUCTION

The enzymic patterns concerning the synthesis of thymidine nucleotides in various tissues point to two possible pathways 1 for the formation of TMP from UMP via deUMP:

* P re sen t address : F i n sen Labora to ry , F i n s e n In s t i t u t e , Copenhagen , D e n m a r k .

Biochim. Biophys. Acta, 95 (1965) 7-13

PATHWAYS FOR FORMATION OF THYMIDINE NUCLEOTIDES 9

Pathway I: UMP -+ deUMP ~ TMP, and Pathway II: UMP ~ CMP ~ deCMP ~ deUMP ~ TMP. For instance, the enzyme deoxycytidylate deaminase which would be expected to play an important role in Pathway II has been found in several mammalian tissues especially those with highly active DNA synthesis 1-s. It should be pointed out that the conversions of the nucleotides are depicted above as taking place at the mono- phosphate level for reasons of simplification only. Results from several laboratories have indicated that a number of these reactions actually occur at a higher phosphate level, e.g. the formation of cytidine nucleotides from uridine nucleotides 4-e and the reduction of uracil and cytosine ribonucleotides to their deoxyribose analogues 7-9.

The work described here was carried out to give information on the relative functioning of the two pathways in vivo. Unlabelled cytidine was administered simultaneously with E6-1*Clorotic acid into rats in order to dilute the metabolic pools of cytidine and deoxycytidine nucleotides. Consequently, the changes in the specific activities of these compounds were reflected in similar changes in the specific activities of the pyrimidines of RNA and DNA. If Pathway I is the predominant one, little change in the specific activity of DNA-thymidine would be expected. However if Pathway II is the most active of the two, a marked decrease in the specific activity of DNA-thymidine should occur. This report deals with experiments on rat rege- nerating liver and spleen. A preliminary report of this work has been published 1°.

MATERIALS

E6-14CJOrotic acid was obtained from the Radiochemical Centre, Amersham. Two samples were used: the first, having a specific activity of IO. I #C/#mole, was used for Expts. 1-3 and the second, having a specific activity of II.6/~C/#mole, was used for Expts. 4-11. Cytidine was purchased from Sigma Chemical Co., DNAase I (EC 3.1.4.5) (I × crystallized) from Worthington Biochemical Corporation, and snake venom (Crotalus adamanteus) from Ross Allen's Reptile Institute, Silver Springs, Fla.

METHODS

Animal experimental procedures

Partial hepactectomy was carried out by the method of HIGGINS AND AN- DERSON 11 on male albino rats weighing 200-220 g. Twenty-three hours after the operation, each animal received IO/~C [6-14Clorotic acid by a single intraperitoneal injection. In the experiments on spleen, non-operated rats weighing 13o-14o g were each given a single intraperitoneal injection of Ioo#C E6-14Clorotic acid.

In each series of experiments, the animals were divided into two groups. In the experimental group each rat received nnlabelled cytidine dissolved in 0. 9 % NaC1, given intraperitoneally in 3 equal doses in the following schedule: the first dose 30 rain before, the second at the same time as, and the third 30 rain after the injection of the labelled orotic acid (in Expts. 1- 5 and 9-11, the last injection of cytidine was given subcutaneously). The total amount of cytidine administered into

Biochim. Biophys. dcta, 95 (1965) 7-13

I 0 M. CRONE, S. ITZHAKI

each rat in the different experiments is specified in each case (Tables I and II) . In the control group, the rats were injected with o. 9 % NaC1 on a similar schedule. The animals were killed I h after the injection of the labelled orotic acid. The re- generating liver or spleen was removed, cleaned and frozen quickly in liquid N~. The organs from two animals treated similarly were pooled for each experiment.

Extraction o/nucleic acids and their subsequent degradation

The frozen tissue was homogenized in 0.6 N HC104. The acid-insoluble residue was collected by centrifugation and washed successively with o.6 N HCIO 4, then twice each with o.o6 N HCI04, 95 % ethanol and absolute ethanol, and finally with ether. These procedures were carried out at o-4 °. The lipids were then removed by repeated extractionswith methanol-chloroform (I : I, v/v) on a boiling-water bath. The residue was extracted 3 or 4 times with IO % NaC1 at ioo ° for 3o rain each time, care being taken to keep the suspension at pH 6.5-7 during the whole period of extraction. To the combined extracts 2.5 volumes of ethanol were added and the sodium nucleate precipitate was collected after cooling.

The sodium nucleates were fractionated into ribonucleotides and DNA by alkaline hydrolysis (o.3 N KOH at 37 ° for 18 h) and subsequent addition of HC104 (ref. 12) to a final concentration of o. 5 N to precipitate the DNA. After centrifugation, the supernatant containing the ribonucleotides was heated in a boiling-water bath for I h*, cooled, neutralized with KOH and concentrated to a small volume. Cytidylic and uridylic acids were then separated by paper electrophoresis la on \Vhatman paper No. 3 MM in o.o5 M formate buffer (pH 3.5) and eluted from their paper bands with 0.2 N formic acid.

The DNA was degraded by DNAase and snake venom to nucleosides which were separated by paper chromatography ~vVhatman paper No. 3 MM) using n- butanol-o.6 N NH4OI-I (6:1, v/v) as solvent 14. Thymidine and deoxycytidine were then eluted from their bands with o.2 N formic acid.

Determination o/ specific activities

The concentrations of the pyrimidine compounds in the eluates were determined spectrophotometrically using the following emM values 1~ at pH 2: uridylic acid, 9.9 at 262 m#; cytidylic acid, 13.o at 279 m#; thymidine, 9.65 at 267 m#; deoxycy- tidine 13.2 at 28o m#. Appropriate blank paper eluates were also taken. For the determination of radioactivity, aliquots of the eluates were plated on alnminium planchets and counted as "infinitely thin" layers (less than o.2 mg/cm 2) using a windowless gas-flow counter to a standard error of less than I °/o. Two or three samples were plated and counted for each eluate and the mean values are given in the tables.

* This t r e a t m e n t hyd ro lyzed the pu r ine nuc leo t ides l eav ing the py r imid ine nuc leo t ides in tac t . Th i s s tep was found useful in t he s epa ra t i on of cyt idyl ic a n d ur idyl ic acids by pape r e lec t rophores is s ince the pape r could t h e n be loaded w i t h more ma te r i a l t h a n if the four nuc leot ides were still i n t ac t .

Biochim. Biophys. Acta, 95 (1965) 7 - I3

P A T H W A Y S FOR FORMATION OF T H Y M I D I N E N U C L E O T I D E S I I

RESU L T S A N D DISCUSSION

Regenerating liver

The experiments were carried out on this tissue at a time after partial he- patectomy when DNA synthesis was at its most active stage is. The administration of unlabelled cytidine to the operated rats simultaneously with [6-14C]orotic acid (according to the schedule under Methods)resulted in decreases in the specific ac- tivities of cytidylic acid and deoxycytidine isolated from the nucleic acids of the re- generating liver, as compared with the values obtained in the control animals injected with the labelled precursor only (Table I ),reflecting dilutions in the corresponding so-

T A B L E I

CHANGES IN THE SPECIFIC ACTIVITIES OF THE PYRIMIDINES OF NUCLEIC ACIDS OF REGENERATING

LIVER FOLLOWING THE INJECTION OF [6-14C]OROTIC ACID AND UNLABELLED CYTIDINE INTO PAR-

TIALLY~ HEPATECTOM IZED RATS

Expt. Total amount No. o/ unlabelled

cytidine infected (mmoles)

Specific activity (counts~rain per i, mole)

RNA DNA

Uridylic Cytidylic Deoxycytidine Thymidine acid acid

I - - 57 ooo 20 650 5050 9320 2 o.18 63 4oo 8ooo 2o9 ° IO 15o 3 1.2 57 5 ° 0 343 68 598o

4 - - 45 850 13 300 561o 12 200 5 o.18 51 IOO 8950 2720 78oo

6 - - 47 4 ° 0 I6 200 454 ° 8000 7 0.3 41 5 ° o 635 175 4620 8 o.6 37 9 ° 0 62 5 ° 2850

luble nucleotide pools. The total amounts of cytidine injected (in divided doses ) varied from o.18 to 1.2 mmoles and the drop in the specific activities was much more pronounced in the animals receiving the larger doses. However, the specific-activity values of thymidine showed moderate decreases only, while no significant changes occurred in those of the uridylic acid. Thus, during these rather short-interval experiments, the uridine nucleotide pools were not appreciably diluted by the de- amination of cytidine nucleotides.

Under these circumstances, therefore, the observed dilution of the thymidine of DNA could be explained only by taking into consideration the functioning of Pathway II. I t should be noted, however, that in all experiments the values of specific activity of thymidine did not show decreases comparable to those occurring in the corresponding values for deoxycytidine even when higher doses of cytidine were given. These results are thus consistent with the assumption that both of the two pathways function in regenerating liver. However, they eliminate Pathway n as the predominant one since they indicate that there is an appreciable formation of thymidine nucleotides directly from uridine nucleotides through deUMP, i.e. via Pathway I, and without the intermediate formation of cytosine nueleotides.

Biochim. Biophys. Acta, 95 (1965) 7 -13

IS M. CRONE, 8. ITZHAKI

This conclusion is similar to that of HECHT AND POTTER 17 who studied the incor- poration of EI4C~cytidine into the DNA of regenerating liver.

Spleen

As in the case of regenerating liver, a marked reduction in the specific activity of the isolated cytidylic acid and deoxycytidine was found in the spleen after injection of unlabelled cytidine together with the labelled orotic acid (Table II). The specific activity of uridylic acid of RNA was unchanged after the smaller dose of cytidine

T A B L E I I

CHANGES IN THE SPECIFIC ACTIVITIES OF THE P Y R I M I D I N E S OF NUCLEIC ACIDS OF SPLEEN FOLLOWING THE I N J E C T I O N OF [6-14C]oROTIC ACID AND UNLABELL E D C Y T I D I N E INTO RATS

Expt. Total amount No. o/ unlabelled

cytidine in#cted (mmoles)

Specific activity (counts/rain per #mole)

RNA DNA

Uridylic Cytidylic Deoxycytidine Thymidine acid acid

9 - - 73 ° 0 755 33 ° 2 7 6 IO o . 1 2 5 7 7 o o 173 lO 3 585 I I o . 8 3 3 6 2 o 25 15 292

(Expt. IO), but with the larger dose (Expt. I I ) some dilution of uridine nucleotides seemed to occur, resulting in a decrease in the specific activity of uridylic acid. On the other hand, even in this experiment where the incorporation of 14C into the cytosine of both RNA and DNA was greatly reduced, the specific activity of thy- midine remained similar to that of the control. Thus, from this point of view these results resemble those of the liver and they indicate that in the spleen also, thymidine nucleotides are synthesized mainly through Pathway I.

The incorporation of the labelled orotic acid into the pyrimidines of nucleic acids is much lower in the spleen than in the regenerating liver despite the larger amount of radioactivity used for the spleen experiments (IOO/uC per rat in the spleen series and IO #C in the liver series). Not only the formation of uridine nu- cleotides from orotic acid, but also their further conversion, seems to be very low in the spleen compared with the corresponding processes in the regenerating liver. A possible explanation for this observation is that orotic acid is not a very effective precursor for the synthesis of RNA and DNA pyrimidines in the spleen; this in- dicates that the spleen may use other preformed precursors as well for this purpose. It is interesting in this connection that in a study of the distribution of radioactivity from E14C~orotic acid injected into tumour-bearing rats, HURLBERT AND POTTER 18 found that a relatively high percentage of the isotope was located in the liver at various times after the injection.

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PATHWAYS FOR FORMATION OF THYMIDINE NUCLEOTIDES 13

ACKNOWLEDGEMENTS

We wish to thank Professor J. S. MITCHELL, F. R. S., for his hospitality during the stay of one of us (M.C.) in this Department. This work was aided by grants from the British Empire Cancer Campaign.

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