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Metabolic studies on the sugars of nucleic acids
554
BIOCHIMICA EF BIOPHYICAS ACTA
BBA 95076
M E T A B O L I C S T U D I E S ON T H E S U G A R S O F N U C L E I C A C I D S II. laC-LABELLING OF R I B O S E AND D E O X Y R I B O S E AND ITS COMPARISON W I T H 32p I N C O R P O R A T I O N I N T O R I B O N U C L E I C ACID AND D E O X Y R I B O N U C L E I C ACID OF RAT-THYMUS CELLS S. ITZHAKI AND ELIZABETH D. WHITTLE Department o/ Radiotherapeutics, University o[ Cambridge, Cambridge (Great Britain) (Received March 24th, 1964)
SUMMARY I. R a t - t h y m u s cells were incubated with [14Ce]glucose. Ribose and deoxyribose moieties bound to the different bases of nucleic acids were isolated and the incorporation of laC into these sugars was determined. 2. Among the ribose moieties, those bound to adenine and guanine had the highest specific activities; that of uracil-bound ribose was lower, while the specific activity of the cytosine-bound ribose was the lowest, amounting to only a few per cent of that of the purine-ribose. 3. In deoxyribonucleic acid, similarly, the specific activity of the purinedeoxyribose was much higher (about 20 times) than that of the pyrimidine-deoxyribose. 4. On the other hand, when 32p was used as a precursor, no such differences were observed in its uptake into the 5'-nucleotides derived from ribonucleic acid, nor in its uptake into the deoxyribonucleic acid nucleotides. 5- These results are discussed in relation to the comparative utilization of the purine and pyrimidine nucleosides as precursors in the formation of nucleic acids.
INTRODUCTION The biosynthesis of the ribose moiety of RNA has been studied in several animal tissues and in microorganisms with the aid of specifically labelled glucose 1-3. These studies have shown that there are two pathways for the formation of ribose, namely the hexosemonophosphate oxidative route and the transaldolase (EC 2.2.1.2)transketolase (EC 2.2.1.1) reactions. The two routes have been shown to function in most cases simultaneously, although in general and depending on type of tissue, one route or the other is predominant. As regards the origin of deoxyribose of DNA, there is now abundant evidence that deoxyribonucleotides are formed from their ribonncleotide analogues 4-0. In a previous paper 1°, the isolation of the pyrimidine-bound ribose and deoxyBiochim. Biophys..4cta, 87 (1964) 554-563
METABOLIC STUDIES ON NUCLEIC ACID SUGARS. II.
555
ribose of nucleic acids has been described. This paper describes the use of this technique for investigating the labelling of the sugar moieties attached to the purine and pyrimidine bases of nucleic acids using [14C61glucose. From these experiments, together with those from parallel experiments on 32p uptake, the renewal of these components in the different types of nucleotides derived from nucleic acids may be compared. The observations reported here, of which brief accounts have appeareda~, 12, show that, while the extent of incorporation of 14C into the sugar moiety of nucleic acids of rat-thymus cells depends on the type of base it is attached to, no such differences are observed in the corresponding incorporation of 32p. MATERIALS AND METHODS
Materials E14Cn~Glucose and [32pJorthophosphate (in isotonic solution, pH 7) were purchased from the Radiochemical Centre, Amersham. The lyophilized snake venom used was that of the eastern diamond rattlesnake (Crotalus adamanteus) obtained from Ross Allen's Reptile Institute, Silver Springs, Florida; phosphodiesterase (EC 3.1.4.1 ) was prepared from the venom according to the method of KOERNER AND SINSHEIMERis. DNAase (EC 3.1.4-5) (once crystallized) was obtained from Worthington Biochemical Corporation, Freehold, New Jersey. I)owex-5o (H+), Dowex-2 (HCOs-) and mixed Dowex (COs-treated) were washed and prepared for use as described previously 1°.
Chromatography and electrophoresis Paper chromatography was carried out by the descending technique using the following solvents. Solvent I: the upper (organic) layer of an equilibrated mixture of ethyl acetate-water-acetic acid (3:3:I,V/V) freshly prepared14; Solvent 2: n-butanol-o.6 N NH4OH (6:1, v/v)15; Solvent 3: isopropanol-conc. HC1 (specific gravity I.I9)-water (17o:41:39, v/v)16; Solvent 4: isopropanol-water (7:3, v/v) with N H 3 in the gas phaselT; Solvent 5: n-butanol-water (86:14, v/v) aS. Paper electrophoresis was done on Whatman paper No. 3MM, 17 cm wide, soaked in 0.o5 M formate buffer (pH 3.5) with an apparatus similar to that of MARKHAMAND SMITH19. Location of purine and pyrimidine compounds on paper was carried out by ultraviolet photography is. Ribose and deoxyribose were detected on paper by spraying with aniline hydrogen phthalate ~°.
Tissue and incubation Thymus glands were obtained from male albino rats weighing about 13o-15o g. Cell suspensions from the thymus were prepared and incubated for 3 h as described earlier 1°. In the experiments with laC, each ml of the incubation medium contained 5 mg of E14Cnlglucose of known specific activity. In the s*l~ experiments Es2Plorthophosphate was added to the medium which contained also non-labelled glucose (5 mg/ml). At the end of incubation the contents of 2 or 3 flasks were pooled and the cells collected by centrifugation for the extraction of nucleic acids; thus each experiment represents tissue from 12-16 thymus glands. Biochim. Biophys. Acta, 87 (1964) 554-563
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S. ITZHAKI, E. D. WHITTLE
Extraction o/ nucleic acids Of the two methods described below', one or the other (as specified) was used in each experiment except Nos. 1-3; the procedures used in the latter are described with the relevant results. For the isolation of the sugars, the salt method was found eventually to be the most satisfactory of all since it yields in a convenient form both the ribonucleotides for subsequent separation by paper electrophoresis, and the DNA for degradation and isolation of the individual nucleosides by paper chromatography. Salf method: The acid-soluble material and tipids were extracted from the cells as described previously 1°. The sodium nucleates were extracted from the dried defatted tissue with hot io o/' Jo NaC1 by essentially the method of DAVlI)SOX axe) SMELLIE 21, and I-~URLBERT AND POTTER 22. Prior to heating at IOO°, the suspension of the tissue residue in IO O/~oNaC1 was brought to pH 6.5-7 by addition of NaOH (British Drug Houses Universal Indicator, used internally). It was left at room temperature with occasional shaking and further addition of NaOH till the pH remained constant; the pH was then maintained at this value throughout the salt extraction procedure. The sodium nucleates were fractionated into RNA (as nucleotides) and DNA by hydrolysis with 0.3 N KOH for 18 h at 37 ° and subsequent acidification with HC10 4 (ref. 23). The ribonucleotide supernatant was neutralized with KOH and concentrated under reduced pressure to a volume of about I ml for subsequent separation of the individual nucleotides by paper electrophoresis. The DNA was recovered from the residue by dissolving in dilute KOH and re-precipitating it at pH 4 with ethanol. Phenol method: KIRBY'S24 method for extraction of RNA from animal tissues with phenol was used. In Expt. 9 where the DNA was also required, it was separated from the phenol layer (which contained the residue of proteins and DNA) as follows. The liquid phase was withdrawn, the residue was homogenized with ethanol, spun down, washed several times with ethanol and finally with ether. After extraction of lipids, the DNA was extracted with hot IO % NaC1, treated with 0.3 N KOH to remove any residual RNA, and recovered by acidification as in the salt method above.
Degradation o[ D N A to nucleosides This was carried out by the combined action of deoxyribonuclease and snake venom zS. The DNA was dissolved in 0.02 M MgSO4 (IO mg DNA in I ml solution) and by adding I N NH4OH (final p H 7.6). A few crystals of DNAase and a few drops of chloroform were added and the mixture incubated at 37 ° for 6 h. NH4OI-I to bring to p]-I 9 and 0.25 volume of o.I M glycine buffer (pIZ 9) were then added, followed by snake venom in the ratio of 1.25 mg to IO mg DNA. After further incubation for 5 h, the mixture was centrifuged and the supernatant containing the deoxyribonucleosides was collected. For the isolation of the individual nucleosides the supernatant was applied to Whatman No. 3MM and chromatographed in Solvent 2 for 48 h. The bands were eluted with water and the eluates from several chromatograms were combined for the isolation of deoxyribose.
Isolation o/ ribose The purine-bound ribose was obtained by hydrolysis of the purine nucleotides with I N I-IC1 at IOO° for i h; for the pyrimidine-ribose, cytidylic and uridylic acids
Biochim. Biophys. Acta, 87 (1964) 554-563
METABOLIC STUDIES ON NUCLEIC ACID SUGARS.
II.
557
were first treated with Br~ and NaOH, then hydrolysed with HC1. In each case the mixture was neutralized with Dowex-2 (HC03-) and then deionized with mixed Dowex (CO2-treated) and the ribose isolated by paper chromatography in Solvent I. Details have been given previously TM.
Isolation o/ deoxyribose To the purine deoxyribonucleoside solution, about 3 ml of a thick suspension of Dowex-5o (H +) were added. The mixture was then shaken for 40 rain ~ and filtered and the resin washed. The combined filtrate and washings were neutralized with Dowex-2 (t-ICO3-) and deionized with mixed Dowex (C02-treated). Deoxycytidine and thymidine were first treated with Br2 and NaOH, then hydrolysed in 0.05 N HC1 at IOO° for 15 min (ref. to) and the mixture was neutralized and deionized with resin as above. In each case deoxyribose was isolated by chromatography on W h a t m a n paper No. I in Solvent I for 15 h in a manner similar to that for ribose.
Isolation o/5'-nucleotides in 32p experiments After digestion of the DNA with DNAase (as a b o v e ) t h e solution was brought to p H 8.5 with NH4OH; phosphodiesterase was added (about 200 units per IOO mg DNA) and the solution incubated at 37 ° for 24 h. The solution was adjusted to p H 3.5 with 2 N formic acid and the nucleotides separated b y paper electrophoresis. 5'-Ribonucleotides were isolated in a similar manner from RNA, prepared b y the phenol method, b y the action of phosphodiesterase (several additions of the enzyme were required to effect complete degradation) and then application of paper electrophoresis.
Determination o/ speci/ic activities Ribose and deoxyribose were determined respectively b y the orcinol 2e and the diphenylamine 27 methods. Glucose was determined according to the method of SOMOGYI2s and inorganic phosphate b y the method of FISKE AND SUBBAROw (in ref. 29). Estimations of the 5'-nucleotides were carried out by ultraviolet spectrophotometry using the published extinction coefficients z°. 14C-labelled samples were prepared for counting in a windowless gas-flow counter by plating on aluminium planchets and drying, in general, under a lamp. However, in the case of deoxyribose, samples of eluates from paper chromatograms were dried at room temperature in a vacuum desiccator over silica gel; this procedure was followed because drying under a lamp caused a loss of deoxyribose of about 50 % as determined by the diphenylamine method, whereas with drying in a desiccator a complete recovery of the deoxyribose could be obtained. 32P-labelled samples were counted in solution in a liquid-dipping counter. For comparison of various experiments, the results have been expressed in terms of relative specific activity which is referred to I0 000 counts/min of precursor in the medium. In the experiments with 14C-sugars, relative specific activity =
average specific activity of pentose-carbon • i0 a. average specific activity of glucose-carbon
" This t r e a t m e n t w i t h Dowex-5o (H +) liberated the deoxyribose from the p u r i n e nucleosides (see also footnote on page 7; an alternative m e t h o d which could be used is hydrolysis in 0.05 N HC1 at IOO° for 15 min).
Biochim. Biophys. Acta, 87 (1964) 554-563
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S. ITZHAKI, F. D. WHITTLE
(The average specific activities of the pentose-carbon and of glucose-carbon were obtained by dividing the specific activity values (counts/rain per/,mole) of ribose and deoxyribose by 5 and those of glucose added to the medium by 6.) In the case of ~2p experiments, specific activity of nucleotide relative specific activity = specific activity of PI in the medium " IO4. EXPERIMENTAL AND RESULTS
Experiments with 14C Preliminary experiments: In these experiments (Nos. I and 2, Table I) the ribonucleotides were precipitated as their barium salts from the alkaline hydrolysate of the RNA of the defatted tissue. The barium nucleotide fraction was hydrolysed with I N HC1 to yield the ribose originating from the purine nucleotides, leaving the pyrimidine nucleotides intact; the latter were taken up on Dowex-2 (I-ICOs-) TABLE
I
DIFFERENTIAL LABELLING OF THE TWO PYRIMIDINE RIBOSE MOIETIES OF R N A T h e s p e c i f i c a c t i v i t y o f [l~Ce]glucose a d d e d t o t h e i n c u b a t i o n /zrnole.
m e d i u m w a s 22 560 c o u n t s / m i n
Specific activity (counts/rain per/*mole) Expt. No.
I 2
Mixed purine-ribose
259 233
Cytidylic acid-ribose *
6 3
Uridylic acid-ribose
68 58
per
Relative specific activity Mixed purine-ribosc
138 124
Cytidylic acid-ribose *
Uridylic acid-ribose
3 2
36 31
* The t o t a l c o u n t s of t h e c y t i d y l i c a c i d - r i b o s e were too low to g i v e a c c u r a t e values.
from which they were then eluted 1°. Cytidylic and uridylic acids were separated by chromatography on paper (Whatman No. 3MM) with Solvent 3 and further purified by paper electrophoresis before the isolation of ribose from each nucleotide. As is shown in Table I, the specific activity of the ribose obtained from cytidylic acid was much lower than that of uridylic acid-ribose. Distribution o[ 14C among the ribose moieties o/ the individual nucleotides: In Expt. 3 the RNA was obtained as nucleotides by treating the defatted tissue directly with I N KOI-I for 24 h. After acidification with IO N HCI04, the supernatant was collected, neutralized and chromatographed on Whatman paper No. 3~lV[ in Solvent 4 for 48 h. The two bands (one containing guanylic acid and the other adenylic, cytidylic and uridylic acids) were eluted and the individual nucleotides were further purified by paper electrophoresis. In Expt. 4, the nucleotides derived from RNA extracted by the salt method were directly separated by paper electrophoresis. The results (Table II) confirm and extend the observations from the previous experiments. The specific activity of the ribose of adenylic acid is the highest and that of guanylic acid-ribose somewhat lower, while the specific activities of the ribose bound to cytosine and uracil in the RNA are, respectively, of the order of 5 % and 25 % of the value of adenylic acid-ribose. Biochim. Biophys. Acta, 87 (1964) 554-563
559
METABOLIC STUDIES ON NUCLEIC ACID SUGARS. II. TABLE II INCORPORATION OF 14C FROM [14Cs]GLUCOSE INTO THE RIBOSE OF R N A NUCLEOTIDES
The specific a c t i v i t y of the labelled glucose added to the i n c u b a t i o n m e d i u m was 4.62. lO4 c o u n t s / m i n p e r / * m o l e in E x p t . 3 a n d 4.84- lO 4 c o u n t s / m i n p e r / z m o l e in E x p t . 4. Expt. No.
Source o! ribose
3
Adenylic acid Guanylic acid Cytidylic acid Uridylic acid
590 53 ° 36 156
153 138 9 4°
ioo 90 6 26
4
Adenylic Guanylic Cytidylic Uridylic
585 446 16 135
145 iio 4 34
IOO 76 3 23
acid acid acid acid
Speci/ic activity o/ribose (counts/rain per#mole)
Relative specilic activity
Relative 14C incorporation
Incorporation o/14C into ribose and deoxyribose (Table II1): For the isolation of ribose, in both Expts. 5 and 6, the salt method was used followed by paper electrophoresis separation of the individual nucleotides. For the isolation of deoxyribose, in Expt. 5 the mixed purine-deoxyribose and the deoxyribose from deoxycytidine and thymidine were each isolated separately, the degradation of the purine deoxyriboTABLE III DISTRIBUTION OF RADIOACTIVITY IN THE SUGAR MOIETIES BOUND TO THE DIFFERENT BASES IN NUCLEIC ACIDS I n b o t h e x p e r i m e n t s the specific a c t i v i t y of [14C6~glucose added to the m e d i u m was 9.89" lO4 c o u n t s / m i n per /*mole. Expt.
Source o/
No.
sugar
Specific activity o/sugar (counts~rain per ttmole)
RNA: Adenylic acid Guanylic acid Cytidylic acid Uridylic acid
I595 I255 65 495
Relative 14C incorporation
Ribose:
DNA: Mixed p u r i n e nucleosides Deoxycytidine Thymidine
375 7 24
RNA: Adenylic acid Guanylic acid Cytidylic acid Uridylic acid
1665 1295 34 421
DNA: Deoxyadenosine Deoxyguanosine Deoxycytidine Thymidine
Relative specilic activity
194 I52 8 60
IOO 79 4 3I
Deoxyribose:
46 < I 3
ioo 2 6
Ribose:
2o2 157 4 51
IOO 78 2 25
Deoxyribose :
325 420 io 21
39 51 i 2
IOO 129 3 6
B i o c h i m . B i o p h y s . A c t a , 87 (1964) 554-563
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S. ITZHAKI, E. D. WHITTLE
nucleosides was carried out by an ion-exchange resin of an acidic type which is known to cleave the glycosidic linkage of these compounds leaving the pyrimidine nucleosides intact a2. The supernatant from the DNAase-venom digest of DNA was shaken with Dowex-5o (H+) for 4 ° nfin and filtered and the resin washed on the filter. Thus thymidine and deoxyribose (originating from the purine nucleosides) passed through while deoxycytidine was retained by the resin*. The filtrate and washings were deionized, concentrated and chromatographed on W h a t m a n paper No. I in Solvent I for 9 h, and deoxyribose and thymidine were then eluted from their respective bands. Deoxycytidine was eluted from the Dowex-5o with o.5 N N a O H and the eluate neutralized with I-ICI and chromatographed in Solvent 2. In Expt. 6 the four nucleosides were separated from the DNAase-venom digest of DNA on paper chromatography and the deoxyribose isolated from each nucleoside as described under methods. I t can be seen that the pattern of labelling among the ribose moieties here is similar to that of Expts. 3 and 4 (Table II). On the other hand, the difference observed in the extent of labelling of the purine- and pyrimidine-ribose of RNA is even more marked in the respective deoxyribose moieties of DNA. The specific activity of the purine-deoxyribose is considerably higher than that of the pyrimidine-deoxyribose, the incorporation into the deoxyfibose moiety of deoxycytidine being especially low. I t should be noted also that although different procedures were applied for obtaining the nucleotides derived from RNA in Expt. 3 and in Expts. 4-6, the values of the relative specific activity of the corresponding ribose moieties are similar in all experiments (c/. Tables I I and I I I ) .
Uptake o/ a2p In order to test the possibility that, in the preceeding experiments on the uptake of 1'C, there might be a limited formation of cytosine nucleotides in particular (thus causing the observed low specific activity of cytidylic acid-ribose), an experiment was carried out to test the effect of cytidine on the synthesis of nucleic acids as determined by the uptake of 3~p. In Expt. 7 the nucleic acids were extracted b y the salt method and the 2',3'-ribonucleotides thus obtained were assayed for radioactivity. The presence of cytidine in the medium had no effect on the specific activities of the RNA nucleotides and of the deoxyribonucleotides, neither were the patterns of distribution of the label affected (Table IV). This indicates that the formation of cytosine nueleotides is not a limiting factor. Since it was of interest to find also the distribution of 82p into the 5'-nucleotides of RNA, the phenol method of extraction was used. However, it was found that the RNA isolated by this method was contaminated with non-nucleotide compounds strongly labelled with ~lP. Some of these impurities had electrophoretic mobilities similar to those of adenylic and cytidylic acids. Further purification of RNA (which was checked b y comparison of ultraviolet and autoradiographic prints of the electrophoresis papers) was therefore carried out. After treatment of the RNA with * W h e n a s a m p l e of d e o x y a d e n o s i n e w a s s h a k e n s i m i l a r l y w i t h Dowex-5o (H+), t h e yield of free d e o x y r i b o s e in t h e resin effluent w a s 97 ~o a n d t h e a b s o r b a n c y of t h e effluent a t 260 m/~ w a s negligible. However, w h e n t h e e x p e r i m e n t w a s r e p e a t e d w i t h t h y m i d i n e , no d e g r a d a t i o n took place. T h i s w a s c o n f i r m e d b y s u b s e q u e n t p a p e r c h r o m a t o g r a p h y of t h e resin effluent in S o l v e n t I w h i c h s h o w e d no s p o t c o r r e s p o n d i n g to deoxyribose; t h e only s p o t w h i c h g a v e a positive r e a c t i o n w i t h t h e cysteine-H2SO4 s p r a y 33 w a s one i d e n t i c a l to t h y m i d i n e .
Biochim. Biophys. Acta, 87 (1964) 554-563
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METABOLIC STUDIES ON NUCLEIC ACID SUGARS. I1. TABLE IV UPTAKE OF 3~p INTO THE NUCLEOTIDES DERIVED FROM NUCLEIC ACIDS T h e specific a c t i v i t y of E32P!Pt in t h e m e d i u m w a s 1.49. lO5 c o u n t s / m i n p e r # a t o m P. Control Expt. 7
Nucleotide isolated
RNA
Adenylic acid Guanylic acid Cytidylic acid Uridylic acid
DNA
deAMP deGMP deCMP TMP
Specilic activity (countslmin per #mole)
With cytidine (I.I raM)
Relative specific activity
Relative 3~p incorporation
Speci]ic activity (counts]rain per #mole)
Relative speei/ie activity
Relative asp incorporation
174o lO6O 1123 1812
117 71 75 122
ioo 61 65 lO 4
1718 1228 1183 1887
115 82 79 126
ioo 71 69 IiO
465 576 5 °2 647
31 39 34 43
IOO 124 lO8 139
503 542 543 63 °
34 36 36 42
IOO lO8 lO8 125
2-methoxyethanoF a, it was dialysed against distilled water for 3 days with occasional changes of the dialysis water. The RNA was precipitated with ethanol, washed with ethanol and ether and dried. I t was dissolved in a small amount of water and subjected to paper electrophoresis. The RNA remained as a band at the origin and was then eluted. The results (Table V) of the degradation of RNA with phosphoTABLE V SPECIFIC ACTIVITIES OF 5P-NUCLEOTIDES ISOLATED FROM R N A AND D N A AFTER a2p LABELLING T h e specific a c t i v i t y of ~3*P]PI in t h e m e d i u m w a s 1.42. i o s c o u n t s / m i n p e r # a t o m P i n E x p t . 8 a n d 2.86. lO 5 c o u n t s / m i n p e r # a t o m P i n E x p t . 9. Expt. No.
Nudeotide isolated
8
RNA
AMP GMP CMP UMP
9
DNA
deAMP deGMP deCMP TMP
Speci/ic activity (counts]rain per ~mole)
Relative speci/ic activity
Relative 3~p incorporation
407 431 484 443
29 3° 34 31
IOO 106 119 lO 9
75 ° 920 856 1182
26 32 3° 41
IOO 123 114 158
diesterase (Expt. 8) show that the specific activities of the 5'-nucleotides fall within a narrow range; a m a x i m u m difference of 20 % is observed between AMP and CMP. From another batch of tissue (Expt. 9) where the DNA was extracted from the phenol layer (see Methods), no great differences were found between the specificactivity values of the four nucleotides. Thymidylic acid had the highest specific a c t i v i t y - - a b o u t 1. 5 times that of deAMP, the nucleotide with the lowest incorporation. This finding is similar to the results of Expt. 7 where the DNA was obtained from the sodium nucleates extracted directly b y the salt method (c[. Tables IV and V). Degradation o/ ribonucleosides: In view of these results, it was of interest to determine the extent of degradation of purine and pyrimidine nucleosides by rat thymus. The results of these experiments (Table VI) show that after incubation Biochim. Biophys. Acta,
87 (1964) 554-563
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1;,. D .
1TZHAKI,
S.
\VHITTLI,-,
with t h y m u s h o m o g e n a t e , adenosine was e x t e n s i v e l y d e a m i n a t e d to inosine while o n l y traces of h y p o x a n t h i n e were d e t e c t a b l e ; uridine a n d c y t i d i n e on the o t h e r h a n d were recovered unchanged. TABLE VI D E G R A D A T I O N OF N U C L E O S I D E S B Y T H Y M U S HOMOGENATE
Each flask contained 1.2 ml thymus homogenate (equivalent to z2 mg wet wt. thymus), o. 5 ml o.i M sodium phosphate buffer (pH 7.4) and 2o/~moles of the appropriate nucleoside; total volume was made up to 4 ml with water. After incubation for 3° rain at 37.5~' the flasks were cooled in ice and i ml cold 3 N HCIO4 was added. The mixture was centrifuged and 4 ml of the supernatant were neutralized with i IN"KOH, cooled, and the IKCIO4 spun down. An aliquot of the supernatant was chromatographed, together with appropriate markers on Whatman 3 MM paper in Solvent 5 for 45 h. The materials from the ultraviolet-absorbing bands were eluted and determined spectrophotometrlcally " 30. Nucleoside added
Compound recovered
Adenosine
Adenosine Inosine Hypoxanthine
Cytidine Uridine
Cytidine Uridine
o, Recovery ,o
35 59 traces 97 95
DISCUSSION
The results show t h a t t h e r e is a g r e a t e r u t i l i z a t i o n of glucose for the f o r m a t i o n of p u r i n e - b o u n d ribose of R N A t h a n for t h a t of pyrimidine-ribose. I t is k n o w n t h a t in t h e e n z y m i c synthesis of t h e ribose nucleotides c o n s t i t u t i n g t h e precursors of R N A , t h e p e n t o s e moieties of t h e p u r i n e a n d p y r i m i d i n e nncleotides originate in all cases from t h e r i b o s e - 5 - p h o s p h a t e residues of 5-phosphoribosyl p y r o p h o s p h a t e . The differences in t h e degree of labelling of t h e four ribose moieties o b s e r v e d here could be e x p l a i n e d on the basis of the e x t e n t of b r e a k d o w n a n d re-utilization of t h e c o m p o n e n t nucleosides of R N A . I n this respect, it is i n t e r e s t i n g to note t h a t t h e ribose m o i e t y a t t a c h e d to c y t o s i n e has t h e lowest specific a c t i v i t y . This could be due to the r e l a t i v e l y higher degree of m e t a b o l i c s t a b i l i t y of t h e ribosidic b o n d of cytidine. Thus, d u r i n g t h e p e r i o d of labelling, a net u t i l i z a t i o n of [12Clcytidine (formed from the cellular R N A as a result of its renewal processes) for t h e re-synthesis of R N A t a k e s place, which is a p p r e c i a b l y higher t h a n t h e u t i l i z a t i o n of t h e o t h e r nucleosides. This is reflected in the low labelling of t h e isolated ribose m o i e t y of c y t i d y l i c acid. S u p p o r t for this i n t e r p r e t a t i o n comes from t h e results of 3ip u p t a k e ; here, in c o n t r a s t to t h e ribose findings, a s o m e w h a t similar degree of labelling occurs in all four 5'-nucleotides of R N A . I t is k n o w n t h a t c y t i d i n e is an efficient precursor of R N A 34. Also, from w o r k of ROLL et el. 35-37 on t h e u t i l i z a t i o n of r i b o t i d e s for t h e synthesis of nucleic acids in several r a t tissues, it is clear t h a t after extensive d e p h o s p h o r y l a t i o n of t h e nucleotides, t h e resulting nucleosides are i n c o r p o r a t e d into R N A . A m o n g t h e four nucleoside residues, c y t i d i n e is t h e m o s t e x t e n s i v e l y i n c o r p o r a t e d . T h a t c y t i d i n e is m o r e efficiently u t i l i z e d for R N A synthesis t h a n u r i d i n e has been shown also b y o t h e r w o r k e r s 3s,ag. The results of t h e differences in the d e g r a d a t i o n of nucleosides b y t h y m u s h o m o g e n a t e also s u p p o r t this e x p l a n a t i o n since c y t i d i n e a n d uridine r e m a i n i n t a c t d u r i n g t h e i n c u b a t i o n whereas adenosine is e x t e n s i v e l y d e a m i n a t e d . Biochim. Biophys. Acre, 87 (I964) 554-Y~3
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563
The results with DNA show, similarly to those with RNA, that the incorporation of 14C into the purine-bound deoxyribose is higher than that into the pyrimidinedeoxyribose. The differences are even more marked here, the specific activities of thymidine-deoxyribose and deoxycytidine-deoxyribose being about 5 ~o of that of either of the purine-deoxyribose moieties. That the specific activity of thymidinedeoxyribose is also low would in fact be expected if it is assumed that thymidylic acid, at least in part, is formed in the thymus from deoxycytidylic acid by the functioning of deoxycytidylate deaminase 4°. ACKNOWLEDGEMENTS
We are grateful to the British Empire Cancer Campaign and the Medical Research Council for grants supporting this work. REFERENCES 1 ]7I. HOLZER, A n n . /{ev. Biochem., 28 (1959) I 7 I . 2 B. AXBLROD, in D. M. GREENBERG, Metabolic Pathways, Vol. I, A c a d e m i c Press, N e w Y ork, 196o, p. 205. 8 0 . TOUSTER, Ann. Rev. Biochem., 31 (1962) 4o7 . 4 p. REICHARD, Advan. Enzymol., 21 (1959) 263. P. REICHARD, dr. Biol. Chem., 236 (1961) 2511. 6 E. C. MOORE AND R. n . HURLBERT, Biochim. Biophys. Acta, 55 (1962) 651. 7 p . REICHARD, J . Biol. Chem., 237 (1962) 3513 . s L. E. BERTANI, A. H~_GGMARK AND P. REICHARD, J. Biol. Chem., 238 (1963) 34o7 . 9 A. LARSSON, J. Biol. Chem., 238 (1963) 3414 . 10 S. ITZHAKI, Biochim. Biophys. Acta, 87 (1964) 541. 11 S. ITZHAKI, Biochim. Biophys. Acta, 37 (196o) 16o. is S. ITZHAKI, Nature, 185 (196o) 614 . 13 j . F. KOERNER AND R. L. SINSHEIMER, J . Biol, Chem., 228 (1957) lO49. 14 M. A. JERMYN AND F. A. ISHERWOO3, Biochem. J., 44 (1949) 4 °2. 15 C. TAMM, H. S. SHAPIRO, R. LIPSHITZ AND E. CHARGAFF, J. Biol. Chem., 203 (1953) 673. is G. R. V~,TYATT, Biochem. J., 48 (1951) 584 . 17 R. MARKHAM AND J. D. SMITH, Biochem. J., 52 (1952) 552. is R. MARKHAM AND J. D. SMITH, Biochem. J., 45 (1949) 29419 R. MARKHAM, in K. PAECH AND M. V. TRACEY, Modern Methods o[ Plant Analysis, Vol. 4, S p r i n g e r , B e r l i n , 1955, p. 246. 20 S. M. PARTRIDGE, Nature, 164 (1949) 443. 2t j . N. DAVIDSON AND R. M. S. SMELLIE, Biochem. J., 52 (1952) 599. 22 R. B. HURLBERT AND V. R. POTTER, J. Biol. Chem., 195 (1952) 257. 23 j . N. DAVlDSON AND R. M. S. SMELLIE, Biochem. J., 52 (1952) 594. 24 K. S. KIRBY, Biochem. J., 64 (1956) 405 . 25 D. B. DUNN AND J. D. SMITH, Biochem. J., 67 (1957) 494. 2e 1-I. G. ALBAUM AND W. W. UMBREIT, J. Biol. Chem., 167 (1947) 369. 27 Z. DISCHE, in E. CHARGAFF AND J. N. DAVlDSON, The Nucleic Acids, Vol. I, A c a d e m i c Press, N e w York , 1955, p. 287. 22 M. SOMOGYI, J. Biol. Chem., 117 (1937) 771. 29 L. F. LELOIR ANn C. E. CARDINI, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, A c a d e m i c Press, N e w Y o r k , 1957, p. 843. so Vv'. E. COHN, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, A c a d e m i c Press, N e w Y o r k , 1957, p. 724 • 31 G. SCHMIDT AND S. J. THANNHAUSER, J. Biol. Chem., 161 (1945) 83. 32 S. G. LALAND AND W. G. OVERBND, Acta Chem. Scan&, 8 (1954) 192. 33 j . G. BUCHANAN, Nature, 168 (1951) lO91. 34 I. A. ROSE AND B. S. SCHWEIGERT, J. Biol. Chem., 202 (1953) 635. 36 p. M. ROLL, H. VC-EINFELD, E. CARROLL AND G. B. BROWN, J. Biol. Chem., 220 (1956) 439. 36 p. M. ROLL, H. WEINFELD AND E. CARROLL, J. Biol. Chem., 22o (1956) 455. ~ P. M. ROLL, J. Biol. Chem., 231 (1958) 183. 38 p. REICHARD, Acta Chem. Scan&, i i (1957) I i . 2~ O. F. BATZER AND B. S. SCHWEIGERT, Biochim. Biophys. Acta, 47 (1961) 197. 40 G. F. MALEY AND F. MALEY, J . Biol. Chem., 234 (1959) 2975.
Biochim. Biophys. Acta, 87 (1964) 554-563
BIOCHIMICA EF BIOPHYICAS ACTA
BBA 95076
M E T A B O L I C S T U D I E S ON T H E S U G A R S O F N U C L E I C A C I D S II. laC-LABELLING OF R I B O S E AND D E O X Y R I B O S E AND ITS COMPARISON W I T H 32p I N C O R P O R A T I O N I N T O R I B O N U C L E I C ACID AND D E O X Y R I B O N U C L E I C ACID OF RAT-THYMUS CELLS S. ITZHAKI AND ELIZABETH D. WHITTLE Department o/ Radiotherapeutics, University o[ Cambridge, Cambridge (Great Britain) (Received March 24th, 1964)
SUMMARY I. R a t - t h y m u s cells were incubated with [14Ce]glucose. Ribose and deoxyribose moieties bound to the different bases of nucleic acids were isolated and the incorporation of laC into these sugars was determined. 2. Among the ribose moieties, those bound to adenine and guanine had the highest specific activities; that of uracil-bound ribose was lower, while the specific activity of the cytosine-bound ribose was the lowest, amounting to only a few per cent of that of the purine-ribose. 3. In deoxyribonucleic acid, similarly, the specific activity of the purinedeoxyribose was much higher (about 20 times) than that of the pyrimidine-deoxyribose. 4. On the other hand, when 32p was used as a precursor, no such differences were observed in its uptake into the 5'-nucleotides derived from ribonucleic acid, nor in its uptake into the deoxyribonucleic acid nucleotides. 5- These results are discussed in relation to the comparative utilization of the purine and pyrimidine nucleosides as precursors in the formation of nucleic acids.
INTRODUCTION The biosynthesis of the ribose moiety of RNA has been studied in several animal tissues and in microorganisms with the aid of specifically labelled glucose 1-3. These studies have shown that there are two pathways for the formation of ribose, namely the hexosemonophosphate oxidative route and the transaldolase (EC 2.2.1.2)transketolase (EC 2.2.1.1) reactions. The two routes have been shown to function in most cases simultaneously, although in general and depending on type of tissue, one route or the other is predominant. As regards the origin of deoxyribose of DNA, there is now abundant evidence that deoxyribonucleotides are formed from their ribonncleotide analogues 4-0. In a previous paper 1°, the isolation of the pyrimidine-bound ribose and deoxyBiochim. Biophys..4cta, 87 (1964) 554-563
METABOLIC STUDIES ON NUCLEIC ACID SUGARS. II.
555
ribose of nucleic acids has been described. This paper describes the use of this technique for investigating the labelling of the sugar moieties attached to the purine and pyrimidine bases of nucleic acids using [14C61glucose. From these experiments, together with those from parallel experiments on 32p uptake, the renewal of these components in the different types of nucleotides derived from nucleic acids may be compared. The observations reported here, of which brief accounts have appeareda~, 12, show that, while the extent of incorporation of 14C into the sugar moiety of nucleic acids of rat-thymus cells depends on the type of base it is attached to, no such differences are observed in the corresponding incorporation of 32p. MATERIALS AND METHODS
Materials E14Cn~Glucose and [32pJorthophosphate (in isotonic solution, pH 7) were purchased from the Radiochemical Centre, Amersham. The lyophilized snake venom used was that of the eastern diamond rattlesnake (Crotalus adamanteus) obtained from Ross Allen's Reptile Institute, Silver Springs, Florida; phosphodiesterase (EC 3.1.4.1 ) was prepared from the venom according to the method of KOERNER AND SINSHEIMERis. DNAase (EC 3.1.4-5) (once crystallized) was obtained from Worthington Biochemical Corporation, Freehold, New Jersey. I)owex-5o (H+), Dowex-2 (HCOs-) and mixed Dowex (COs-treated) were washed and prepared for use as described previously 1°.
Chromatography and electrophoresis Paper chromatography was carried out by the descending technique using the following solvents. Solvent I: the upper (organic) layer of an equilibrated mixture of ethyl acetate-water-acetic acid (3:3:I,V/V) freshly prepared14; Solvent 2: n-butanol-o.6 N NH4OH (6:1, v/v)15; Solvent 3: isopropanol-conc. HC1 (specific gravity I.I9)-water (17o:41:39, v/v)16; Solvent 4: isopropanol-water (7:3, v/v) with N H 3 in the gas phaselT; Solvent 5: n-butanol-water (86:14, v/v) aS. Paper electrophoresis was done on Whatman paper No. 3MM, 17 cm wide, soaked in 0.o5 M formate buffer (pH 3.5) with an apparatus similar to that of MARKHAMAND SMITH19. Location of purine and pyrimidine compounds on paper was carried out by ultraviolet photography is. Ribose and deoxyribose were detected on paper by spraying with aniline hydrogen phthalate ~°.
Tissue and incubation Thymus glands were obtained from male albino rats weighing about 13o-15o g. Cell suspensions from the thymus were prepared and incubated for 3 h as described earlier 1°. In the experiments with laC, each ml of the incubation medium contained 5 mg of E14Cnlglucose of known specific activity. In the s*l~ experiments Es2Plorthophosphate was added to the medium which contained also non-labelled glucose (5 mg/ml). At the end of incubation the contents of 2 or 3 flasks were pooled and the cells collected by centrifugation for the extraction of nucleic acids; thus each experiment represents tissue from 12-16 thymus glands. Biochim. Biophys. Acta, 87 (1964) 554-563
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S. ITZHAKI, E. D. WHITTLE
Extraction o/ nucleic acids Of the two methods described below', one or the other (as specified) was used in each experiment except Nos. 1-3; the procedures used in the latter are described with the relevant results. For the isolation of the sugars, the salt method was found eventually to be the most satisfactory of all since it yields in a convenient form both the ribonucleotides for subsequent separation by paper electrophoresis, and the DNA for degradation and isolation of the individual nucleosides by paper chromatography. Salf method: The acid-soluble material and tipids were extracted from the cells as described previously 1°. The sodium nucleates were extracted from the dried defatted tissue with hot io o/' Jo NaC1 by essentially the method of DAVlI)SOX axe) SMELLIE 21, and I-~URLBERT AND POTTER 22. Prior to heating at IOO°, the suspension of the tissue residue in IO O/~oNaC1 was brought to pH 6.5-7 by addition of NaOH (British Drug Houses Universal Indicator, used internally). It was left at room temperature with occasional shaking and further addition of NaOH till the pH remained constant; the pH was then maintained at this value throughout the salt extraction procedure. The sodium nucleates were fractionated into RNA (as nucleotides) and DNA by hydrolysis with 0.3 N KOH for 18 h at 37 ° and subsequent acidification with HC10 4 (ref. 23). The ribonucleotide supernatant was neutralized with KOH and concentrated under reduced pressure to a volume of about I ml for subsequent separation of the individual nucleotides by paper electrophoresis. The DNA was recovered from the residue by dissolving in dilute KOH and re-precipitating it at pH 4 with ethanol. Phenol method: KIRBY'S24 method for extraction of RNA from animal tissues with phenol was used. In Expt. 9 where the DNA was also required, it was separated from the phenol layer (which contained the residue of proteins and DNA) as follows. The liquid phase was withdrawn, the residue was homogenized with ethanol, spun down, washed several times with ethanol and finally with ether. After extraction of lipids, the DNA was extracted with hot IO % NaC1, treated with 0.3 N KOH to remove any residual RNA, and recovered by acidification as in the salt method above.
Degradation o[ D N A to nucleosides This was carried out by the combined action of deoxyribonuclease and snake venom zS. The DNA was dissolved in 0.02 M MgSO4 (IO mg DNA in I ml solution) and by adding I N NH4OH (final p H 7.6). A few crystals of DNAase and a few drops of chloroform were added and the mixture incubated at 37 ° for 6 h. NH4OI-I to bring to p]-I 9 and 0.25 volume of o.I M glycine buffer (pIZ 9) were then added, followed by snake venom in the ratio of 1.25 mg to IO mg DNA. After further incubation for 5 h, the mixture was centrifuged and the supernatant containing the deoxyribonucleosides was collected. For the isolation of the individual nucleosides the supernatant was applied to Whatman No. 3MM and chromatographed in Solvent 2 for 48 h. The bands were eluted with water and the eluates from several chromatograms were combined for the isolation of deoxyribose.
Isolation o/ ribose The purine-bound ribose was obtained by hydrolysis of the purine nucleotides with I N I-IC1 at IOO° for i h; for the pyrimidine-ribose, cytidylic and uridylic acids
Biochim. Biophys. Acta, 87 (1964) 554-563
METABOLIC STUDIES ON NUCLEIC ACID SUGARS.
II.
557
were first treated with Br~ and NaOH, then hydrolysed with HC1. In each case the mixture was neutralized with Dowex-2 (HC03-) and then deionized with mixed Dowex (CO2-treated) and the ribose isolated by paper chromatography in Solvent I. Details have been given previously TM.
Isolation o/ deoxyribose To the purine deoxyribonucleoside solution, about 3 ml of a thick suspension of Dowex-5o (H +) were added. The mixture was then shaken for 40 rain ~ and filtered and the resin washed. The combined filtrate and washings were neutralized with Dowex-2 (t-ICO3-) and deionized with mixed Dowex (C02-treated). Deoxycytidine and thymidine were first treated with Br2 and NaOH, then hydrolysed in 0.05 N HC1 at IOO° for 15 min (ref. to) and the mixture was neutralized and deionized with resin as above. In each case deoxyribose was isolated by chromatography on W h a t m a n paper No. I in Solvent I for 15 h in a manner similar to that for ribose.
Isolation o/5'-nucleotides in 32p experiments After digestion of the DNA with DNAase (as a b o v e ) t h e solution was brought to p H 8.5 with NH4OH; phosphodiesterase was added (about 200 units per IOO mg DNA) and the solution incubated at 37 ° for 24 h. The solution was adjusted to p H 3.5 with 2 N formic acid and the nucleotides separated b y paper electrophoresis. 5'-Ribonucleotides were isolated in a similar manner from RNA, prepared b y the phenol method, b y the action of phosphodiesterase (several additions of the enzyme were required to effect complete degradation) and then application of paper electrophoresis.
Determination o/ speci/ic activities Ribose and deoxyribose were determined respectively b y the orcinol 2e and the diphenylamine 27 methods. Glucose was determined according to the method of SOMOGYI2s and inorganic phosphate b y the method of FISKE AND SUBBAROw (in ref. 29). Estimations of the 5'-nucleotides were carried out by ultraviolet spectrophotometry using the published extinction coefficients z°. 14C-labelled samples were prepared for counting in a windowless gas-flow counter by plating on aluminium planchets and drying, in general, under a lamp. However, in the case of deoxyribose, samples of eluates from paper chromatograms were dried at room temperature in a vacuum desiccator over silica gel; this procedure was followed because drying under a lamp caused a loss of deoxyribose of about 50 % as determined by the diphenylamine method, whereas with drying in a desiccator a complete recovery of the deoxyribose could be obtained. 32P-labelled samples were counted in solution in a liquid-dipping counter. For comparison of various experiments, the results have been expressed in terms of relative specific activity which is referred to I0 000 counts/min of precursor in the medium. In the experiments with 14C-sugars, relative specific activity =
average specific activity of pentose-carbon • i0 a. average specific activity of glucose-carbon
" This t r e a t m e n t w i t h Dowex-5o (H +) liberated the deoxyribose from the p u r i n e nucleosides (see also footnote on page 7; an alternative m e t h o d which could be used is hydrolysis in 0.05 N HC1 at IOO° for 15 min).
Biochim. Biophys. Acta, 87 (1964) 554-563
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S. ITZHAKI, F. D. WHITTLE
(The average specific activities of the pentose-carbon and of glucose-carbon were obtained by dividing the specific activity values (counts/rain per/,mole) of ribose and deoxyribose by 5 and those of glucose added to the medium by 6.) In the case of ~2p experiments, specific activity of nucleotide relative specific activity = specific activity of PI in the medium " IO4. EXPERIMENTAL AND RESULTS
Experiments with 14C Preliminary experiments: In these experiments (Nos. I and 2, Table I) the ribonucleotides were precipitated as their barium salts from the alkaline hydrolysate of the RNA of the defatted tissue. The barium nucleotide fraction was hydrolysed with I N HC1 to yield the ribose originating from the purine nucleotides, leaving the pyrimidine nucleotides intact; the latter were taken up on Dowex-2 (I-ICOs-) TABLE
I
DIFFERENTIAL LABELLING OF THE TWO PYRIMIDINE RIBOSE MOIETIES OF R N A T h e s p e c i f i c a c t i v i t y o f [l~Ce]glucose a d d e d t o t h e i n c u b a t i o n /zrnole.
m e d i u m w a s 22 560 c o u n t s / m i n
Specific activity (counts/rain per/*mole) Expt. No.
I 2
Mixed purine-ribose
259 233
Cytidylic acid-ribose *
6 3
Uridylic acid-ribose
68 58
per
Relative specific activity Mixed purine-ribosc
138 124
Cytidylic acid-ribose *
Uridylic acid-ribose
3 2
36 31
* The t o t a l c o u n t s of t h e c y t i d y l i c a c i d - r i b o s e were too low to g i v e a c c u r a t e values.
from which they were then eluted 1°. Cytidylic and uridylic acids were separated by chromatography on paper (Whatman No. 3MM) with Solvent 3 and further purified by paper electrophoresis before the isolation of ribose from each nucleotide. As is shown in Table I, the specific activity of the ribose obtained from cytidylic acid was much lower than that of uridylic acid-ribose. Distribution o[ 14C among the ribose moieties o/ the individual nucleotides: In Expt. 3 the RNA was obtained as nucleotides by treating the defatted tissue directly with I N KOI-I for 24 h. After acidification with IO N HCI04, the supernatant was collected, neutralized and chromatographed on Whatman paper No. 3~lV[ in Solvent 4 for 48 h. The two bands (one containing guanylic acid and the other adenylic, cytidylic and uridylic acids) were eluted and the individual nucleotides were further purified by paper electrophoresis. In Expt. 4, the nucleotides derived from RNA extracted by the salt method were directly separated by paper electrophoresis. The results (Table II) confirm and extend the observations from the previous experiments. The specific activity of the ribose of adenylic acid is the highest and that of guanylic acid-ribose somewhat lower, while the specific activities of the ribose bound to cytosine and uracil in the RNA are, respectively, of the order of 5 % and 25 % of the value of adenylic acid-ribose. Biochim. Biophys. Acta, 87 (1964) 554-563
559
METABOLIC STUDIES ON NUCLEIC ACID SUGARS. II. TABLE II INCORPORATION OF 14C FROM [14Cs]GLUCOSE INTO THE RIBOSE OF R N A NUCLEOTIDES
The specific a c t i v i t y of the labelled glucose added to the i n c u b a t i o n m e d i u m was 4.62. lO4 c o u n t s / m i n p e r / * m o l e in E x p t . 3 a n d 4.84- lO 4 c o u n t s / m i n p e r / z m o l e in E x p t . 4. Expt. No.
Source o! ribose
3
Adenylic acid Guanylic acid Cytidylic acid Uridylic acid
590 53 ° 36 156
153 138 9 4°
ioo 90 6 26
4
Adenylic Guanylic Cytidylic Uridylic
585 446 16 135
145 iio 4 34
IOO 76 3 23
acid acid acid acid
Speci/ic activity o/ribose (counts/rain per#mole)
Relative specilic activity
Relative 14C incorporation
Incorporation o/14C into ribose and deoxyribose (Table II1): For the isolation of ribose, in both Expts. 5 and 6, the salt method was used followed by paper electrophoresis separation of the individual nucleotides. For the isolation of deoxyribose, in Expt. 5 the mixed purine-deoxyribose and the deoxyribose from deoxycytidine and thymidine were each isolated separately, the degradation of the purine deoxyriboTABLE III DISTRIBUTION OF RADIOACTIVITY IN THE SUGAR MOIETIES BOUND TO THE DIFFERENT BASES IN NUCLEIC ACIDS I n b o t h e x p e r i m e n t s the specific a c t i v i t y of [14C6~glucose added to the m e d i u m was 9.89" lO4 c o u n t s / m i n per /*mole. Expt.
Source o/
No.
sugar
Specific activity o/sugar (counts~rain per ttmole)
RNA: Adenylic acid Guanylic acid Cytidylic acid Uridylic acid
I595 I255 65 495
Relative 14C incorporation
Ribose:
DNA: Mixed p u r i n e nucleosides Deoxycytidine Thymidine
375 7 24
RNA: Adenylic acid Guanylic acid Cytidylic acid Uridylic acid
1665 1295 34 421
DNA: Deoxyadenosine Deoxyguanosine Deoxycytidine Thymidine
Relative specilic activity
194 I52 8 60
IOO 79 4 3I
Deoxyribose:
46 < I 3
ioo 2 6
Ribose:
2o2 157 4 51
IOO 78 2 25
Deoxyribose :
325 420 io 21
39 51 i 2
IOO 129 3 6
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S. ITZHAKI, E. D. WHITTLE
nucleosides was carried out by an ion-exchange resin of an acidic type which is known to cleave the glycosidic linkage of these compounds leaving the pyrimidine nucleosides intact a2. The supernatant from the DNAase-venom digest of DNA was shaken with Dowex-5o (H+) for 4 ° nfin and filtered and the resin washed on the filter. Thus thymidine and deoxyribose (originating from the purine nucleosides) passed through while deoxycytidine was retained by the resin*. The filtrate and washings were deionized, concentrated and chromatographed on W h a t m a n paper No. I in Solvent I for 9 h, and deoxyribose and thymidine were then eluted from their respective bands. Deoxycytidine was eluted from the Dowex-5o with o.5 N N a O H and the eluate neutralized with I-ICI and chromatographed in Solvent 2. In Expt. 6 the four nucleosides were separated from the DNAase-venom digest of DNA on paper chromatography and the deoxyribose isolated from each nucleoside as described under methods. I t can be seen that the pattern of labelling among the ribose moieties here is similar to that of Expts. 3 and 4 (Table II). On the other hand, the difference observed in the extent of labelling of the purine- and pyrimidine-ribose of RNA is even more marked in the respective deoxyribose moieties of DNA. The specific activity of the purine-deoxyribose is considerably higher than that of the pyrimidine-deoxyribose, the incorporation into the deoxyfibose moiety of deoxycytidine being especially low. I t should be noted also that although different procedures were applied for obtaining the nucleotides derived from RNA in Expt. 3 and in Expts. 4-6, the values of the relative specific activity of the corresponding ribose moieties are similar in all experiments (c/. Tables I I and I I I ) .
Uptake o/ a2p In order to test the possibility that, in the preceeding experiments on the uptake of 1'C, there might be a limited formation of cytosine nucleotides in particular (thus causing the observed low specific activity of cytidylic acid-ribose), an experiment was carried out to test the effect of cytidine on the synthesis of nucleic acids as determined by the uptake of 3~p. In Expt. 7 the nucleic acids were extracted b y the salt method and the 2',3'-ribonucleotides thus obtained were assayed for radioactivity. The presence of cytidine in the medium had no effect on the specific activities of the RNA nucleotides and of the deoxyribonucleotides, neither were the patterns of distribution of the label affected (Table IV). This indicates that the formation of cytosine nueleotides is not a limiting factor. Since it was of interest to find also the distribution of 82p into the 5'-nucleotides of RNA, the phenol method of extraction was used. However, it was found that the RNA isolated by this method was contaminated with non-nucleotide compounds strongly labelled with ~lP. Some of these impurities had electrophoretic mobilities similar to those of adenylic and cytidylic acids. Further purification of RNA (which was checked b y comparison of ultraviolet and autoradiographic prints of the electrophoresis papers) was therefore carried out. After treatment of the RNA with * W h e n a s a m p l e of d e o x y a d e n o s i n e w a s s h a k e n s i m i l a r l y w i t h Dowex-5o (H+), t h e yield of free d e o x y r i b o s e in t h e resin effluent w a s 97 ~o a n d t h e a b s o r b a n c y of t h e effluent a t 260 m/~ w a s negligible. However, w h e n t h e e x p e r i m e n t w a s r e p e a t e d w i t h t h y m i d i n e , no d e g r a d a t i o n took place. T h i s w a s c o n f i r m e d b y s u b s e q u e n t p a p e r c h r o m a t o g r a p h y of t h e resin effluent in S o l v e n t I w h i c h s h o w e d no s p o t c o r r e s p o n d i n g to deoxyribose; t h e only s p o t w h i c h g a v e a positive r e a c t i o n w i t h t h e cysteine-H2SO4 s p r a y 33 w a s one i d e n t i c a l to t h y m i d i n e .
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METABOLIC STUDIES ON NUCLEIC ACID SUGARS. I1. TABLE IV UPTAKE OF 3~p INTO THE NUCLEOTIDES DERIVED FROM NUCLEIC ACIDS T h e specific a c t i v i t y of E32P!Pt in t h e m e d i u m w a s 1.49. lO5 c o u n t s / m i n p e r # a t o m P. Control Expt. 7
Nucleotide isolated
RNA
Adenylic acid Guanylic acid Cytidylic acid Uridylic acid
DNA
deAMP deGMP deCMP TMP
Specilic activity (countslmin per #mole)
With cytidine (I.I raM)
Relative specific activity
Relative 3~p incorporation
Speci]ic activity (counts]rain per #mole)
Relative speei/ie activity
Relative asp incorporation
174o lO6O 1123 1812
117 71 75 122
ioo 61 65 lO 4
1718 1228 1183 1887
115 82 79 126
ioo 71 69 IiO
465 576 5 °2 647
31 39 34 43
IOO 124 lO8 139
503 542 543 63 °
34 36 36 42
IOO lO8 lO8 125
2-methoxyethanoF a, it was dialysed against distilled water for 3 days with occasional changes of the dialysis water. The RNA was precipitated with ethanol, washed with ethanol and ether and dried. I t was dissolved in a small amount of water and subjected to paper electrophoresis. The RNA remained as a band at the origin and was then eluted. The results (Table V) of the degradation of RNA with phosphoTABLE V SPECIFIC ACTIVITIES OF 5P-NUCLEOTIDES ISOLATED FROM R N A AND D N A AFTER a2p LABELLING T h e specific a c t i v i t y of ~3*P]PI in t h e m e d i u m w a s 1.42. i o s c o u n t s / m i n p e r # a t o m P i n E x p t . 8 a n d 2.86. lO 5 c o u n t s / m i n p e r # a t o m P i n E x p t . 9. Expt. No.
Nudeotide isolated
8
RNA
AMP GMP CMP UMP
9
DNA
deAMP deGMP deCMP TMP
Speci/ic activity (counts]rain per ~mole)
Relative speci/ic activity
Relative 3~p incorporation
407 431 484 443
29 3° 34 31
IOO 106 119 lO 9
75 ° 920 856 1182
26 32 3° 41
IOO 123 114 158
diesterase (Expt. 8) show that the specific activities of the 5'-nucleotides fall within a narrow range; a m a x i m u m difference of 20 % is observed between AMP and CMP. From another batch of tissue (Expt. 9) where the DNA was extracted from the phenol layer (see Methods), no great differences were found between the specificactivity values of the four nucleotides. Thymidylic acid had the highest specific a c t i v i t y - - a b o u t 1. 5 times that of deAMP, the nucleotide with the lowest incorporation. This finding is similar to the results of Expt. 7 where the DNA was obtained from the sodium nucleates extracted directly b y the salt method (c[. Tables IV and V). Degradation o/ ribonucleosides: In view of these results, it was of interest to determine the extent of degradation of purine and pyrimidine nucleosides by rat thymus. The results of these experiments (Table VI) show that after incubation Biochim. Biophys. Acta,
87 (1964) 554-563
562
1;,. D .
1TZHAKI,
S.
\VHITTLI,-,
with t h y m u s h o m o g e n a t e , adenosine was e x t e n s i v e l y d e a m i n a t e d to inosine while o n l y traces of h y p o x a n t h i n e were d e t e c t a b l e ; uridine a n d c y t i d i n e on the o t h e r h a n d were recovered unchanged. TABLE VI D E G R A D A T I O N OF N U C L E O S I D E S B Y T H Y M U S HOMOGENATE
Each flask contained 1.2 ml thymus homogenate (equivalent to z2 mg wet wt. thymus), o. 5 ml o.i M sodium phosphate buffer (pH 7.4) and 2o/~moles of the appropriate nucleoside; total volume was made up to 4 ml with water. After incubation for 3° rain at 37.5~' the flasks were cooled in ice and i ml cold 3 N HCIO4 was added. The mixture was centrifuged and 4 ml of the supernatant were neutralized with i IN"KOH, cooled, and the IKCIO4 spun down. An aliquot of the supernatant was chromatographed, together with appropriate markers on Whatman 3 MM paper in Solvent 5 for 45 h. The materials from the ultraviolet-absorbing bands were eluted and determined spectrophotometrlcally " 30. Nucleoside added
Compound recovered
Adenosine
Adenosine Inosine Hypoxanthine
Cytidine Uridine
Cytidine Uridine
o, Recovery ,o
35 59 traces 97 95
DISCUSSION
The results show t h a t t h e r e is a g r e a t e r u t i l i z a t i o n of glucose for the f o r m a t i o n of p u r i n e - b o u n d ribose of R N A t h a n for t h a t of pyrimidine-ribose. I t is k n o w n t h a t in t h e e n z y m i c synthesis of t h e ribose nucleotides c o n s t i t u t i n g t h e precursors of R N A , t h e p e n t o s e moieties of t h e p u r i n e a n d p y r i m i d i n e nncleotides originate in all cases from t h e r i b o s e - 5 - p h o s p h a t e residues of 5-phosphoribosyl p y r o p h o s p h a t e . The differences in t h e degree of labelling of t h e four ribose moieties o b s e r v e d here could be e x p l a i n e d on the basis of the e x t e n t of b r e a k d o w n a n d re-utilization of t h e c o m p o n e n t nucleosides of R N A . I n this respect, it is i n t e r e s t i n g to note t h a t t h e ribose m o i e t y a t t a c h e d to c y t o s i n e has t h e lowest specific a c t i v i t y . This could be due to the r e l a t i v e l y higher degree of m e t a b o l i c s t a b i l i t y of t h e ribosidic b o n d of cytidine. Thus, d u r i n g t h e p e r i o d of labelling, a net u t i l i z a t i o n of [12Clcytidine (formed from the cellular R N A as a result of its renewal processes) for t h e re-synthesis of R N A t a k e s place, which is a p p r e c i a b l y higher t h a n t h e u t i l i z a t i o n of t h e o t h e r nucleosides. This is reflected in the low labelling of t h e isolated ribose m o i e t y of c y t i d y l i c acid. S u p p o r t for this i n t e r p r e t a t i o n comes from t h e results of 3ip u p t a k e ; here, in c o n t r a s t to t h e ribose findings, a s o m e w h a t similar degree of labelling occurs in all four 5'-nucleotides of R N A . I t is k n o w n t h a t c y t i d i n e is an efficient precursor of R N A 34. Also, from w o r k of ROLL et el. 35-37 on t h e u t i l i z a t i o n of r i b o t i d e s for t h e synthesis of nucleic acids in several r a t tissues, it is clear t h a t after extensive d e p h o s p h o r y l a t i o n of t h e nucleotides, t h e resulting nucleosides are i n c o r p o r a t e d into R N A . A m o n g t h e four nucleoside residues, c y t i d i n e is t h e m o s t e x t e n s i v e l y i n c o r p o r a t e d . T h a t c y t i d i n e is m o r e efficiently u t i l i z e d for R N A synthesis t h a n u r i d i n e has been shown also b y o t h e r w o r k e r s 3s,ag. The results of t h e differences in the d e g r a d a t i o n of nucleosides b y t h y m u s h o m o g e n a t e also s u p p o r t this e x p l a n a t i o n since c y t i d i n e a n d uridine r e m a i n i n t a c t d u r i n g t h e i n c u b a t i o n whereas adenosine is e x t e n s i v e l y d e a m i n a t e d . Biochim. Biophys. Acre, 87 (I964) 554-Y~3
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563
The results with DNA show, similarly to those with RNA, that the incorporation of 14C into the purine-bound deoxyribose is higher than that into the pyrimidinedeoxyribose. The differences are even more marked here, the specific activities of thymidine-deoxyribose and deoxycytidine-deoxyribose being about 5 ~o of that of either of the purine-deoxyribose moieties. That the specific activity of thymidinedeoxyribose is also low would in fact be expected if it is assumed that thymidylic acid, at least in part, is formed in the thymus from deoxycytidylic acid by the functioning of deoxycytidylate deaminase 4°. ACKNOWLEDGEMENTS
We are grateful to the British Empire Cancer Campaign and the Medical Research Council for grants supporting this work. REFERENCES 1 ]7I. HOLZER, A n n . /{ev. Biochem., 28 (1959) I 7 I . 2 B. AXBLROD, in D. M. GREENBERG, Metabolic Pathways, Vol. I, A c a d e m i c Press, N e w Y ork, 196o, p. 205. 8 0 . TOUSTER, Ann. Rev. Biochem., 31 (1962) 4o7 . 4 p. REICHARD, Advan. Enzymol., 21 (1959) 263. P. REICHARD, dr. Biol. Chem., 236 (1961) 2511. 6 E. C. MOORE AND R. n . HURLBERT, Biochim. Biophys. Acta, 55 (1962) 651. 7 p . REICHARD, J . Biol. Chem., 237 (1962) 3513 . s L. E. BERTANI, A. H~_GGMARK AND P. REICHARD, J. Biol. Chem., 238 (1963) 34o7 . 9 A. LARSSON, J. Biol. Chem., 238 (1963) 3414 . 10 S. ITZHAKI, Biochim. Biophys. Acta, 87 (1964) 541. 11 S. ITZHAKI, Biochim. Biophys. Acta, 37 (196o) 16o. is S. ITZHAKI, Nature, 185 (196o) 614 . 13 j . F. KOERNER AND R. L. SINSHEIMER, J . Biol, Chem., 228 (1957) lO49. 14 M. A. JERMYN AND F. A. ISHERWOO3, Biochem. J., 44 (1949) 4 °2. 15 C. TAMM, H. S. SHAPIRO, R. LIPSHITZ AND E. CHARGAFF, J. Biol. Chem., 203 (1953) 673. is G. R. V~,TYATT, Biochem. J., 48 (1951) 584 . 17 R. MARKHAM AND J. D. SMITH, Biochem. J., 52 (1952) 552. is R. MARKHAM AND J. D. SMITH, Biochem. J., 45 (1949) 29419 R. MARKHAM, in K. PAECH AND M. V. TRACEY, Modern Methods o[ Plant Analysis, Vol. 4, S p r i n g e r , B e r l i n , 1955, p. 246. 20 S. M. PARTRIDGE, Nature, 164 (1949) 443. 2t j . N. DAVIDSON AND R. M. S. SMELLIE, Biochem. J., 52 (1952) 599. 22 R. B. HURLBERT AND V. R. POTTER, J. Biol. Chem., 195 (1952) 257. 23 j . N. DAVlDSON AND R. M. S. SMELLIE, Biochem. J., 52 (1952) 594. 24 K. S. KIRBY, Biochem. J., 64 (1956) 405 . 25 D. B. DUNN AND J. D. SMITH, Biochem. J., 67 (1957) 494. 2e 1-I. G. ALBAUM AND W. W. UMBREIT, J. Biol. Chem., 167 (1947) 369. 27 Z. DISCHE, in E. CHARGAFF AND J. N. DAVlDSON, The Nucleic Acids, Vol. I, A c a d e m i c Press, N e w York , 1955, p. 287. 22 M. SOMOGYI, J. Biol. Chem., 117 (1937) 771. 29 L. F. LELOIR ANn C. E. CARDINI, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, A c a d e m i c Press, N e w Y o r k , 1957, p. 843. so Vv'. E. COHN, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, A c a d e m i c Press, N e w Y o r k , 1957, p. 724 • 31 G. SCHMIDT AND S. J. THANNHAUSER, J. Biol. Chem., 161 (1945) 83. 32 S. G. LALAND AND W. G. OVERBND, Acta Chem. Scan&, 8 (1954) 192. 33 j . G. BUCHANAN, Nature, 168 (1951) lO91. 34 I. A. ROSE AND B. S. SCHWEIGERT, J. Biol. Chem., 202 (1953) 635. 36 p. M. ROLL, H. VC-EINFELD, E. CARROLL AND G. B. BROWN, J. Biol. Chem., 220 (1956) 439. 36 p. M. ROLL, H. WEINFELD AND E. CARROLL, J. Biol. Chem., 22o (1956) 455. ~ P. M. ROLL, J. Biol. Chem., 231 (1958) 183. 38 p. REICHARD, Acta Chem. Scan&, i i (1957) I i . 2~ O. F. BATZER AND B. S. SCHWEIGERT, Biochim. Biophys. Acta, 47 (1961) 197. 40 G. F. MALEY AND F. MALEY, J . Biol. Chem., 234 (1959) 2975.
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