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Incorporation of pyrimidine deoxyribonucleosides into liver lipids and other components
6 Io
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96722
I N C O R P O R A T I O N OF P Y R I M I D I N E D E O X Y R I B O N U C L E O S I D E S I N T O L I V E R L I P I D S AND O T H E R COMPONENTS W A L T E R C. S C H N E I D E R ANn A N T O I N E T T E E. G R E C O
Laboratory o/ Biochemistry, National Cancer Institute, National Institutes o] Health, Department o/ Health, Education, and Wel/are, Bethesda, Md. 2OOl4 (U.S.A.) (Received A u g u s t i 7 t h , 197 o)
SUMMARY
I. Labeled thymidine or deoxycytidine were injected into rats or mice and the distribution of the radioactivity in the nuclei, mitochondria, microsomes or soluble fraction isolated from the livers was determined. I t was found that the microsomes contained the largest amount of particulate-bound radioactivity and the nuclei the least and that the lipid and protein fractions of the mitochondria and microsomes contained the bulk of their radioactivity while the DNA fraction contained very little label. 2. Incorporation of labeled thymidine or deoxycytidine into DNA, lipids and proteins was diminished, but not to the same degree in each, b y the prior administration of unlabeled forms of these compounds. The incorporation of thymidine into lipids and proteins but not into DNA was also diluted by the prior injection of the thymidine catabolities, fl-aminoisobutyric acid or dihydrothymine. 3. Chromatography of the particulate lipid fraction obtained after labeling with [*H]thymidine or EaHldeoxycytidine revealed that lecithin, phosphatidyl ethanolamine and neutral glycerides contained most of the radioactivity. In the case of the lipids obtained from the liver soluble fraction, the radioactivity was associated only with the neutral glyceride fraction. 4- When the particulate lipid fractions were hydrolyzed and chromatographed the main radioactive products corresponded to glycerol, glycerylphosphoryl choline and glycerylphosphoryl ethanolamine when L~H]thymidine was the precursor. With [3H~deoxycytidine, however, two main radioactive products, apparently derived from the f a t t y acid protion of the glycerides, were obtained. 5- Hydrolysis of the microsomal protein fraction obtained after labeling with E~Hlthymidine and fractionation of the amino acids released revealed that serine, aspartic acid and glutamic acid contained the largest amount of label, alanine and methionine were much less radioactive, and the other amino acids were unlabeled.
INTRODUCTION
Thymidine is currently widely employed as a precursor of DNA. This usage is based on the initial experiments of REICHARD AND ESTBORN1 and REICHARD2 which demonstrated that this compound as well as deoxycytidine were incorporated as a unit into the DNA of regenerating rat liver but not to a significant extent into RNA. Biochim. Biophys. Acta, 228 (1971) 6 i o - 6 2 6
DEOXYRIBONUCLEOSIDE INCORPORATION
611
On the other hand, it has been known for some time that thymidine is degraded in biological systems and reports have appeared in the literature on the labeling of amino acids 3, tricarboxylic acids 3, glycogen4, RNA 5, and proteins e after administration of radioactive thymidine or thymine. I t is not entirely clear, however, to what extent the labeling of other cellular components interferes with the use of thymidine as a precursor of DNA. In a recent study on the association of DNA with liver mitochondria and microsomes 7, we noted that the amount of radioactivity incorporated from thymidine into the DNA of these organelles was only a minor fraction of the radioactivity they contained. The present experiments were undertaken to define the nature of the components other than DNA labeled b y thymidine and to establish whether this labeling was real or artifactual. The results of these studies indicate t h a t both the lipids and the proteins obtained from liver cytoplasmic fractions were heavily labeled when thymidine or deoxycytidine were given to animals.
EXPERIMENTAL
The animals used were male Holtzman or Sprague--Dawley rats 200-300 g in weight or C3H mice, 2-3 months old, and were either fed ad libitum a commercial pellet diet or were fasted for 18-24 h before use. The labeled precursor was administered intraperitoneally and 5 min-24 h later the animals were killed and the livers removed. In some cases other compounds were also given by intraperitoneal injection prior to the precursor. The livers were homogenized in 0.25 M sucrose and nuclei, mitochondria and microsomes were isolated b y differential centrifugation as described previously 7. In some cases the homogenate was simply centrifuged for I h at 4 ° ooo rev./min in the type A-ITO rotor of the International B-6o centrifuge to obtain a pellet of the particulate material (nuclei, mitochondria and microsomes) and a soluble or supernatant fraction. The liver fractions were then separated essentially as described previously s into acid-soluble compounds, lipids, RNA, DNA, and protein. Sufficient HC104 was added to each fraction at o ° to bring the concentration to 0.6 M. The precipitates were centrifuged down and washed twice b y resuspension and resedimentation in cold 0.3 M HCI04. The three extracts constituted the acid-soluble fraction of the tissue. The lipids were then extracted from the washed pellet b y resuspension in absolute ethanol, neutralization of the mixture to litmus with NH4OH, and resedimentation*. Extraction with ethanol was repeated twice more and after the final extraction the pellet was mixed with o.3-1.o M N a O H and heated for I h at 37 °. Sufficient HC10, was then added to neutralize the N a O H and to provide an excess concentration of 0.6 M and after cooling, the precipitate was sedimented and washed once * U n p u b l i s h e d studies in this l a b o r a t o r y showed t h a t neutralization of the e t h a n o l - t i s s u e m i x t u r e reduced the loss of p r o t e i n and R N A to a m i n i m u m and this procedure was adopted as standard. I n one experiment, the ethanol e x t r a c t i o n s were followed b y t w o extractions with e t h a n o l - c h l o r o f o r m (2 : I, b y voi.); t h e latter e x t r a c t i o n s were omitted in s u b s e q u e n t experiments because the e x t r a c t s contained less t h a n IO % of the r a d i o a c t i v i t y recovered in the ethanol extracts.
Biochim. Biophys. Acta, 228 (1971) 61o-626
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W. C. SCHNEIDER, A. E. GRECO
with cold o.3 M HC104. The two extracts obtained from this alkaline hydrolysis contained the RNA in the form of nucleotides. DNA was extracted from the pellet b y resuspension in 0.3 M HC104 and heating for 15 rain at 9 o°. This process was repeated once and the precipitate was washed once with cold 0. 3 M HC104. The final pellet, which should contain only protein, was dissolved in o.5 M NaOH. The extraction procedures were monitored b y measuring the radioactivity of each extract and it was noted that a burst of radioactivity appeared in the first extract of each series and was followed b y a decline to insignificant levels in the final extraction. For further examination of the lipids, the ethanol extracts were combined and mixed with 0.25 vol. of carbon tetrachloride and then equilibrated with an equal volume of 2 M KC19,1°. The carbon tetrachloride phase which separated out was washed twice with additional 2 M KC1 and once with distilled water. In some cases the ethanol extracts were evaporated on a rotating evaporator instead and the residue was redissolved in chloroform. The lipids were separated by thin-layer chromatogr a p h y on plates of Silica gel G using a preliminary development with chloroform follow ed b y drying and development with chloroform-methanol-water (65:25:4, b y vol.)11. The lipid spots were located by exposing the plates to iodine vapor and b y spraying with ninhydrin. Hydrolysis of the lipid solutions was carried out b y mild alkaline and acid hydrolysis as described b y DAWSONlz, An aliquot of the lipid concentrate was mixed with 5.0 ml of ethanol, 0.65 ml of water and 0.25 ml of I M NaOH. After 20 rain incubation at 37 °, o.14 ml of IOO °/o (w/v) trichloroacetic acid was added and the heating continued for another 30 min. The ethanol was removed under a stream of N 2 and the aqueous phase was extracted 3 times with petroleum ether. Aliquots of the aqueous phase were chromatographed on a thin layer of DEAE-cellulose (Bakerflex) with isopropanol-acetic acid-water (55:25:20, b y vol.) as the developing solvent. The petroleum ether extracts of the hydrolysate were chromatographed on Bakerflex silica gel thin-layer sheets with benzene as the developing solvent. Phosphorus was determined colorimetrically b y the method of AMESla on samples hydrolyzed with H2SO 4 (ref. 14). Tritium and 14C were measured witlithe liquid scintillation spectrometer. Samples adjusted to contain 2.0 ml of water were mixed with 15 ml of a scintillator solution (40 ml of Liquifluor and 333 ml of Triton X-IOO diluted to I 1 with toluene). In the case of the alkaline solutions of the protein samples, 0.05 ml of 9 ° °/o formic acid was added to each vial in addition. The counts obtained were corrected to the same efficiency by the internal standardization method. Counting efficiency in unquenched samples was about 20 % for tritium. 1~5I activity was measured in a g a m m a spectrometer. The following labeled compounds were used in these experiments: EMe-~H] thymidine, E2-14C] thymidine, and CSHldeoxycytidine, Schwarz Bioresearch, Inc., [6-SH]thymidine and E5-3Hldeoxycytidine, Amersham-Searle Corp.; phosphatidyl EI,2-14C2]choline, phospliatidyl Ei,2-14C21ethanolamine, phosphatidyl [3-14Clserine, and [4-14C~cholesterol, Tracerlab; [I,3-14C~ glycerol and [ c a r b o x y - 1 4 C l t r i p a l m i t i n , New England Nuclear; E125IJiododeoxyuridine, International Chemical and Nuclear Co. Unlabeled phospholipids were obtained from General Biochemicals and used as chromatographic standards. Glycerylphosphoryl choline, glycerylphosphoryl ethanolamine and glycerylphosphoryl serine labeled with 14C were obtained from the labeled lipids b y mild alkaline and acid hydrolysis as described above. Biochim. Biophys. Acta, 228 (1971) 61o-626
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DEXYRIBONUCLEOSIDE INCORPORATION
The amino acid analyses were performed chromatographically w~th a Beckman I2oC amino acid analyzer programmed to complete the separation of the amino acids in 19o min using a starting buffer of sodium citrate (pH 3.25) a pH 4.30 limit buffer of sodium citrate (both buffers were 0.2 M in Na+), and monitoring the effluent by ninhydrin color development. Amino acid content was calculated from the magnitude of the ninhydrin color and individual amino acids were identified by the time at which they emerged from the column as well as by ninhydrin color spectra. The column was calibrated with a standard mixture of amino acids. In order to determine radioactivity of individual amino acids, the column was used with a stream splitter which allowed part of the effluent to pass through the analyzer part of the machine and the remainder to be collected with a fraction collector. Peak tubes of radioactivity were rechromatographed with standard amino acid markers to confirm identification and establish purity. RESULTS
In the first experiment, Table I, mitochondria and microsomes were isolated from the livers of rats 24 h after they received either E3H]thymidine or EaH]deoxycytidine. The data show that both particulate fractions contained substantial amounts of radioactivity with either precursor but the nucleic acid fraction accounted TABLE I INCORPORATION OF E3H]THYMIDINE AND [SH]DEOXYCYTIDINE INTO LIVER MITOCHONDRIA AND MICRO$OMES Two rats were given either 25o/,C of EMe-3H]thymidine (io C/mmole) or 250/~C [SH]deoxycytidine (2. 5 C]mmole, Schwarz Bioresearch) intraperitoneally and 24 h later t h e livers were r e m o v ed and fractionated as described in the text.
Precursor
Liver fraction
Total incorporation (cov.nls]min)
Distribution of rad$oactivity (counts]min) Acid soluble
Lipid
RNA
DNA
Protein
Thymidine
Mitochondria Microsomes
63 900 131 60o
14oo 132o
22 17o 66 98o
224o 671o
23 39o 18 95 °
14 7 ° o 37 640
Deoxycytidine
Mitochondria Microsomes
72 600 157 40o
661o 567 o
23 23o 71 14o
3 I9O" 39 57 ° 14 OlO * 66 58o
R N A a n d D N A were e x t r a c t e d t o g e t h e r in this e x p e r i m e n t b y using h o t HC104.
for less than IO °/o of the total label when deoxycytidine was the precursor. When thymidine was the precursor, the DNA fraction accounted for only about 15 % of the radioactivity of the microsomes and 36 °/o of the mitochondrial label. The lipid and the protein fractions of these organelles contained radioactivity that accounted for more than 50 % of the incorporation. The labeling of the lipids and the proteins of the mitochondria and microsomes was unexpected both because of the high proportion of radioactivity they represented and also because the labeling of lipids by these precursors had not previously been reported ". " While this m a n u s c r i p t was in preparation, a note appeared in which the labeling of the lipid fraction of tissues after the a d m i n i s t r a t i o n of radioactive t h y m i d i n e was described 15.
Biochim. Biophys. Acta, 228 (1971) 61o-626
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w.c.
SCHNEIDER,
A. E. G R E C O
In order to determine if the labeling of lipids and proteins was limited to the cytoplasm and was related to DNA synthesis, shorter incorporation times were used and purified nuclei were examined as well. The results in Table I I represent incorporation times of 5 and 30 min and show that the nuclei contained the lowest amount of total radioactivity of the three particulate fractions isolated, while the microsomes
TABLE
It
INCORPORATION
OF THYMIDINE
INTO RAT LIVER PARTICULATE FRACTIONS
E a c h of t w o f a s t e d r a t s w a s g i v e n 2 5 o , u C of E M e - S H ] t h y m i d i n e ( i o # C / m m o ] e ) later the livers were removed and fractionated as described in the text.
Time (rain)
Liver fraction
Total incorporation (counts/mi n)
5
Nuclei Mitochondria Microsomes
30 700 64 8oo 85 6 o o
4 020 58 o6o 2o 54 °
7 860 3 24o 24 4°0
3°
Nuclei Mitochondria Microsomes
47 200 76 5oo 248 ooo
i 320 51 5 6 0 17 8 6 0
8 73 ° IO 9 4 ° lO6 6o0
a n d 5 o r 3° m i n
Distribution o/radioactivity (counts/rain) Acid soluble
Lipid
RNA
DNA
Protein
I 81o 97 ° 9 16o
IO 6 2 0 39o 4 280
6 39o 2 14o 27 2 2 o
33 ° I 99o 23 3 1 o
25 9 6 0 5 280 12 9 0 0
io 860 6 739 87 3 0 0
contained the most. When the distribution of the label between the various classes of compounds was examined, it was noted t h a t the acid-soluble fraction was already at peak levels at 5 rain in all three particulate fractions, declined somewhat at 30 min and reached near zero levels at 24 h (Table I). The mitochondrial fraction contained the highest levels of the particulate-bound acid-soluble precursors. The labeling of the lipids and the proteins of all fractions was already evident at 5 min and increased in amount at 30 min. The increase in labeling between 5 and 30 min was especially marked in the microsomal fraction where the radioactivity reached peak levels in the proteins and lipids and then appeared to decline to a lower level at 24 h (Table I). The labeling of the DNA fraction exceeded that of the proteins and lipids only in the case of the nuclear fraction; the DNA fraction of the mitochondria and the microsomes contained less radioactivity than did any of the other fractions obtained from these particulates except at 24 h when the radioactivity of the RNA fraction was the lowest. It is noteworthy, however, that the radioactivity of the RNA fraction of the microsomes exceeded that of the DNA fraction after 5 and 3 ° rain of incorporation.
Labeling o/subcellular components with [14Clthymidine In order to deternfine whether the position of the label in the thymidine molecule might be critical in determining the specificity of labeling, experiments were also conducted with [2-14C]thymidine. Preliminary studies with rats indicated that the lipids of microsomes were also labeled by this precursor, but the radioactivity of all fractions were very low due undoubtedly to the lower specific activity of the 14C-labeled precursor and to the fact that less radioactivity was given than was the case with the [3H]thymidine. By using mice instead of rats, a higher ratio of 14C Biochim. Biophys. Acta, 2 2 8 ( 1 9 7 1 ) 6 1 o - 6 2 6
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DEOXYRIBONUCLEOSIDE INCORPORATION
administered to body weight could be achieved more easily and the radioactivity of t h e f r a c t i o n s w a s c o r r e s p o n d i n g l y g r e a t e r . T h e r e s u l t s of t h e s e e x p e r i m e n t s a r e given in Table III. The data were similar in many respects to those obtained with the [ a H j t h y m i d i n e , a l t h o u g h t h e p r o p o r t i o n of r a d i o a c t i v i t y f o u n d i n t h e D N A f r a c t i o n s w a s c o n s i d e r a b l y g r e a t e r w i t h t h e 14C-labeled p r e c u r s o r . B o t h p r o t e i n s a n d l i p i d s of all t h r e e f r a c t i o n s w e r e l a b e l e d a t b o t h t i m e p o i n t s b u t t h e p r o t e i n s c o n t a i n e d m o r e r a d i o a c t i v i t y t h a n t h e lipids. T h e r a d i o a c t i v i t y of t h e m i c r o s o m a l p r o t e i n s a n d l i p i d s also declined between 30 min and 24 h after administering the precursor, just as had been observed with {aH]thymidine. TABLE III I N C OR P O R AT IO N OF [ l a C I T H Y M I D I N E I N T O MOUSE L I V E R P A R T I C U L A T E FRACTIONS
Each of four C3H mice was given 5.o/*C of E2-14C]thymidine (47.9 mC/mmole). Two mice were sacrificed 3 ° rain or 24 h later and the livers were removed and fractionated as described in the text.
Labeling time
Tissue /raction
Total incorporation (counts/min)
Distribution o/radioactivity (counts/rain) Acid soluble
Lipid
RNA
3 ° min
Nuclei Mitochondria Microsomes
8 300 i i 3oo 18 8o0
25 642o 81o
83 565 436o
224 226 124o
6 99o 2 o7 ° 2 54 °
98o 2o2o 985o
24 h
Nuclei Mitochondria Microsomes
16 34 ° 7 800 12 20o
115 175 45 °
165 650 16oo
280 135 47 °
13 920 3 760 4 47 °
186o 3 °80 521°
DNA
Protein
E//ects o] added compounds on the incorporation o / t h y m i d i n e and deoxycytidine The above experiments indicated that both thymidine and deoxycytidine were s e r v i n g a s p r e c u r s o r s of l i p i d a n d p r o t e i n s . A l t h o u g h t h e r a d i o c h e m i c a l s u s e d i n t h e s e T A B L E IV E F F E C T OF O T H E R COMPOUNDS U P O N T H E INCORPORATION OF [ S H ] T H Y M I D I N E I N T O MOUSE L I V E R
Each C3H mouse received 22.5/*C of [Me-SH]thymidine (2 nmoles) or 37.5/*C [aH]deoxycytidine (2.75 nmoles), b y injection. Unlabeled compounds, in the amounts indicated below, were injected 5 rain prior to radioactive material. The animals were sacrificed I h after the final injection and the livers removed and separated into particulate and soluble fractions as described in the text
Compounds administered
Soluble radioactivity (counts~rain)
[3H]Thymidine 321 500 Thymidine (4°/*moles) + [aH]thymidine 4Ol 85o DL-fl-Aminoisobutyric acid (4 ° pmo]es) + [SH]thymidine i 559 ooo Dihydrothymine (4°/*moles) + [SH]thymidine 468 5o0 [aH]Deoxycytidine 9o 4 ooo Deoxycytidine (8/*moles) + [aH]deoxycytidine I 073 ooo
Particulate radioactivity (counts/rain) A cid soluble
Lipid
RNA
DNA
Protein
15 390
92 57 ° 19oo
25 880
12 66o
19 4oo
13 46o 164o
92o
2 39o
69 93 °
26 27o 115o
20 23o
6 93 °
27 97 ° 58 300
14 94 ° 124o 114 80o 98o0
28 61o 7° 7o0
7 38o 51 55 °
63 IOO
88 IOO 772o
8 73 °
38 500
Biochim. Biophys. Acta, 228 (1971) 6 I O ~ 2 6
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S C H N E I D E R , A. E. GRECO
experiments were assayed by the manufacturer and reported to be more than 99 % pure and independent tests in our laboratory also indicated high purity, it was necessary to consider that the incorporation of label into lipid and protein might not be a property of the parent molecule, but of some contaminant or breakdown product. This possibility was tested by injecting either the unlabeled precursor itself or a precursor metabolite just prior to administration of the radioactive compound to see whether dilution of incorporation occurred. In these experiments, for convenience a single particulate fraction containing all the nuclei, mitochondria and microsomes of the liver was used instead of the individual fractions. The results in Table IV show that unlabeled thymidine reduced the incorporation of label into DNA, lipid and protein. However, the dilution of DNA labeling that occurred (28-fold) was much greater than the dilution of either lipid or protein labeling (5-7-fold). Two natural products of thymidine catabolism, fl-aminoisobutyric acid and dihydrothymine also reduced the incorporation of label into lipid and protein but the dilution of the latter incorporation was not as great as that observed with thymidine itself. Neither of these metabolites affected the labeling of the DNA fraction to a marked extent. The fl-aminoisobutyric acid, however, did result in a marked increase in the pool of labeled precursors (soluble radioactivity+particulate acid-soluble radioactivity). When unlabeled deoxycytidine was given together with the labeled compound dilution of incorporation into all fractions was observed. Since the amount of unlabeled deoxycytidine given was only one-fifth that of the unlabeled thymidine, the dilutions that occurred were correspondingly lower; 8-fold for DNA and only 25 % for lipid and protein.
Incorporation o/radiolysis products o/ thymidine Since tritiated compounds are known to undergo a slow self-destruction, it was of some interest to test the incorporation of the radiolysis products. When an old sample of [3H]thymidine was submitted to thin-layer chromatography, three TABLE
V
INCORPORATION OF RADIOLYSIS PRODUCTS OF THYMIDINE INTO MOUSE LIVER A s a m p l e of [ M e - 3 H ] t h y m i d i n e , w h i c h h a d b e e n s t o r e d i n a r e f r i g e r a t o r for a b o u t a y e a r , w a s a p p l i e d t o a s h e e t of B a k e r f l e x c e l l u l o s e F a n d c h r o m a t o g r a p h e d t w i c e w i t h b u t a n o l - w a t e r (86:14, b y v o l . ) . A b a n d of m a t e r i a l a t 1 2 . 5 - 1 3 . 5 c m f r o m t h e o r i g i n w a s o b s e r v e d u n d e r u l t r a v i o l e t l i g h t a n d c o r r e s p o n d e d t o t h a t of u n l a b e l e d t h y m i d i n e o n a c o m p a n i o n c h r o m a t o g r a m . W h e n t h e c h r o m a t o g r a m w a s s c a n n e d for r a d i o a c t i v i t y , t w o s l o w l y m i g r a t i n g b a n d s of r a d i o a c t i v i t y ( i a n d 2) w e r e o b s e r v e d i n a d d i t i o n t o o n e c o r r e s p o n d i n g t o t h e o n e s e e n u n d e r u l t r a v i o l e t l i g h t (3). T h e t h r e e b a n d s w e r e e l u t e d a n d a l i q u o t s w e r e i n j e c t e d i n t o f a s t e d C 3 H m i c e . i h l a t e r the livers were removed and particulate and soluble fractions were isolated as described in the text.
Band No.
I 2 3 3"
Amount recovered (/*C)
Amount in/ected (l~C)
Soluble radioactivity (counts/min)
177 257 490 286
27.2 27. 4 28.1 21.2
25I 366 473 638
900 500 3o0 670
Particulate radioactivity (counts/rain) Acid soluble
Lipid
RNA
DNA
Protein
25 25 18 35
I I 25 3°
199o 955 267 ° 3309
500 43 ° 6540 9425
2 I 8 12
320 240 7o0 670
19o 680 3o0 720
33 ° ooo o30 14o
* B a n d 3 w a s r e c h r o m a t o g r a p h e d o n a B a k e r f l e x s i l i c a gel s h e e t w i t h e t h y l a c e t a t e - f o r m i c a c i d - w a t e r (60 : 5 : 35, u p p e r p h a s e ) a s t h e s o l v e n t .
Biochim. Biophys. Acta, 228 (1971) 6 1 o - 6 2 6
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radioactive components were obtained, one of which was visible under ultraviolet light and corresponded to thymidine. The latter was rechromatographed on a thin layer of silica gel and found to be 97 ~o pure. When equivalent amounts of these radioactive materials were given to mice, it was found that only the one corresponding to thymidine, (Band 3, Table V), was incorporated into DNA. This material was also incorporated strongly into lipid and protein. Further chromatography on silica gel failed to change the pattern or extent of incorporation. Since the other bands of radioactive material that were obtained gave only low levels of radioactivity in all fractions, it could be concluded that they were not responsible for the labeling of proteins and lipids. The data in Tables IV and V also show that the liver supernatant or soluble fraction contained much more radioactivity than the particulate fraction. This was not unexpected since the former might be expected to contain the bulk of the administered precursor at such a short time after administration to the animal. It was somewhat surprising therefore to find that large part of the supernatant radioactivity appeared to be lipid also. In the experiment in which thymidine Fraction 3" was given for example (Table V) it was found that the bulk of the supernatant radioactivity was present in the acid-soluble fraction but that 25.5 % (163 ooo counts/rain) was recovered in the ethanol or lipid extract of the supernatant fraction. The source of this lipid m a y have been the f a t t y layer that accumulated at the top of the tube after centrifugation of the homogenate, since care was taken to include it with the supernatant fluid. The large amount of lipid-associated radioactivity observed in the supernatant fraction in this experiment was unusual since only 44 000-54 ooo counts/ rain (11.7-13. 7 % of the supernatant fluid radioactivity) were recovered in this fraction in three other experiments. Nevertheless, the results show that the supernatant fluid did contain labeled f a t t y material t h a t rivaled in amount that found in the lipid extracts of the particulate fraction of the liver.
Nature o/ the labeling o~ the lipid/raction When carbon tetrachloride was added to the ethanol extracts of the mitochondrial and microsomal fractions or of the combined particulate fraction and the mixture was equilibrated and washed with 2 M KC1 and water as described above, it was found that the bulk of the radioactivity was retained in the carbon tetrachloride. In several experiments with [SH]thymidine or [3H]deoxycytidine, the recovery of radioactivity in the carbon tetrachloride ranged between 87 and 99 % of that present in the ethanol extracts. When the lipid extracts of the particulate fraction of the liver homogenate were chromatographed on thin layers of Silica gel G, spots detected b y exposure to iodine vapors and corresponding in position to authentic lysolecithin, sphingomyelin, lecithin, phosphatidyl ethanolamine, and cardiolipin were obtained. In addition, a band of neutral fats was also found at the solvent front. When these spots were examined for phosphorus content and radioactivity, the results presented in Table VI were obtained. The data show that the lecithin and phosphatidyl ethanolamine bands accounted for most of the radioactivity associated with phospholipids. The lecithin spots contained the largest amount of phospholipid radioactivity and appeared to contain more radioactivity per unit of phosphorus than the phosphatidyl ethanolamine. The data also show that large amounts of radioactivity rivalling that assoBiochim. Biophys. Acta, 228 (1971) 61o-626
O
o0
CHROMATOGRAPHY
OF THE
MOUSE LIVER
PARTICULATE
LIPIDS
R a d i o a c t i v i t y (counts/rain) P h o s p h o r u s (/*g)
Radioactivity (counts/min) P h o s p h o r u s (/~g)
Radioactivity !counts/min) P h o s p h o r u s {,ug)
[6_SH]Thymidine
EMe-aH~Thymidine
[5_3HlDeoxycytidine
454 3.1
144 o
315 1.7
462 9.4
181 o
384 9.3
Lipid componene" LL Sph
I 555 lO9
9 342 128
I i 920 ioi
L
2806 47.3
592 46.6
28Ol 32.9
PEA
304 9.9
116 8.9
149 7.1
C
14 92o o
2 622 o
3 034 o
Gly
lO2 82
89 71
97 ioo
(%)
Recovery
* LL, lysolecithin; Sph, s p h i n g o m y e l i n ; L, lecithin; P E A , p h o s p h a t i d y l e t h a n o l a m i n e ; C, cardiolipin; Gly, glycerides a n d n e u t r a l fats f o u n d at s o l v e n t front.
Measurement
Precursor in~ected
Samples of t h e lipid e x t r a c t s in chloroform were applied to io c m × 20 c m Silica gel G plates a n d d e v e l o p e d with c h l o r o f o r m followed b y c h l o r o f o r m - m e t h a n o l w a t e r ( 6 5 : 2 5 : 4 , b y vol.). T h e s p o t s were l o c a t e d b y e x p o s u r e to iodine v a p o r s , s c r a p e d off a n d e l u t e d 3-4 t i m e s with m e t h a n o l . R a d i o a c t i v i t y a n d t o t a l p h o s p h o r u s were m e a s u r e d on t h e m e t h a n o l e x t r a c t s . T h e s p o t s were identified b y c o m p a r i s o n with t h e position of reference lipids on c o m p a n i o n c h r o m a t o g r a m s .
THIN-LAYER
T A B L E VI
©
Oo
DEOXYRIBONUCLEOSIDE INCORPORATION
619
c i a t e d w i t h lecithin were f o u n d in t h e m a t e r i a l a c c u m u l a t i n g a t t h e s o l v e n t front a n d k n o w n to c o n t a i n t h e n e u t r a l fats, as well as o t h e r n e u t r a l f a t t y materials. Several o t h e r t h i n - l a y e r c h r o m a t o g r a p h i c p r o c e d u r e s were also used to e x a m i n e t h e c o m p o n e n t s of the lipid e x t r a c t s . These i n c l u d e d t h e c h l o r o f o r m - m e t h a n o l a m m o n i a solvent w i t h silica gel G TM t h e c h l o r o f o r m - m e t h a n o l - w a t e r - a c e t i c acid syst e m s described b y SKIPSKI et al. 1~, a n d t h e NH4NO3-containing silica gel G p l a t e s y s t e m described b y HOFMANN TM. The a m o u n t of r a d i o a c t i v i t y a s s o c i a t e d w i t h t h e lecithin, p h o s p h a t i d y l e t h a n o l a m i n e , a n d n e u t r a l fat spots was in all cases t h e s a m e as h a d been o b s e r v e d w i t h t h e s y s t e m described. No evidence was o b t a i n e d for labeling of p h o s p h a t i d y l serine, which t h e a b o v e s y s t e m s p e r m i t to s e p a r a t e from phosphatidyl ethanolamine. I t has been n o t e d a b o v e t h a t a considerable fraction of the r a d i o a c t i v i t y recovered in t h e soluble or s u p e r n a t a n t fraction of t h e liver h o m o g e n a t e was also ext r a c t a b l e w i t h ethanol. W h e n this e x t r a c t e d m a t e r i a l was c h r o m a t o g r a p h e d on silica gel G as described in T a b l e VI, 95 % of t h e r a d i o a c t i v i t y was recovered in t h e n e u t r a l fat b a n d a c c u m u l a t i n g at the s o l v e n t front. F u r t h e r m o r e when t h e e t h a n o l e x t r a c t of t h e liver s u p e r n a t a n t fluid was c h r o m a t o g r a p h e d on a sheet of B a k e r flex silica gel w i t h benzene as t h e d e v e l o p i n g solvent, 92 % of t h e r a d i o a c t i v i t y was p r e s e n t in a b a n d with an R F of 0.5, c o r r e s p o n d i n g to t h e position of [~4C~tripalmitin in t h e s a m e solvent system.
Hydrolysis o/ lipids W h e n t h e e t h a n o l e x t r a c t s were h y d r o l y z e d b y t h e m i l d a l k a l i n e a n d acid p r o c e d u r e of DAWSONTM as described above, t h e results p r e s e n t e d in T a b l e V I I were o b t a i n e d . The d a t a show t h a t t h e h y d r o l y s i s of t h e p h o s p h o l i p i d s was a l m o s t c o m p l e t e since 95 % or more of t h e p h o s p h o r u s of t h e p a r t i c u l a t e fractions was recovered in t h e a q u e o u s phase after hydrolysis. W h e n either [ 6 - 3 H l t h y m i d i n e or EMe-ZHlthymidine was t h e precursor, t h e largest fraction of t h e t o t a l r a d i o a c t i v i t y was recovered in t h e a q u e o u s phase a f t e r h y d r o l y s i s whereas w i t h [5-3HJdeoxycytidine 88 °/o of t h e r a d i o a c t i v i t y was recovered TABLE VII HYDROLYSIS
OF LIVER
LIPIDS
Aliquots of the ethanol extracts of the tissue fractions indicated were hydrolyzed with NaOH and trich]oroacetic acid and then were evaporated and extracted with petroleum ether as described in the text. Portions of the aqueous and the organic phases were then assayed for radioactivity and total phosphorus
Precursor in#cted
Liver fraction
[Me-*H]Thymidine lVticrosomes Particulate Soluble [6-3H~Thymidine Particulate [5-SH]Deoxycytidine Particulate
A queous phase
Organic phase
Radioactivity (counts~rain)
Phosphorus (~g)
Radioactivity (counts/rain)
Phosphorus (l~g)
15 400 3 775 4 9o5 i i 35° 3 075
381 * * 159 193
I 43 ° i 513 1°3 3 900 22 800
o "
* 9.0 8.5
* Not measured.
Biochim. Biophys. Acta, 228 (i97 z) 61o-626
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SCHNEIDER, A. E. GRECO
in the organic solvent phase. These results indicated that the fatty acid components of the glycerides were being labeled by the latter precursor while the remainder of the glyceride molecule was the major recipient of the label when [aH]thymidine was used. The fact that 98 °/o of the radioactivity of the lipids of the soluble fraction, Table VII, also appeared in the aqueous phase after hydrolysis suggests that the glyceryl portion of the lipids was being labeled by thymidine since, as was noted above, thin-layer chromatography indicated that the soluble fraction lipids were neutral fats. The aqueous phase obtained after hydrolysis of the lipids was also examined chromatographically to determined whether the products corresponded to the lipid composition. As shown in Fig. i, 3H-labeled bands corresponding in migration rate to glycerylphosphoryl ethanolamine, glycerylphosphoryl choline and glycerol were obtained. These products agreed with the lipid composition reported in Table VI. 0®Q
QD
20C
® I
G2) C ~ 5O
,oo E
~
.~ 3oc
I! / I, II
I0(3
Z
o
4
8
12
~
2'o
cm
~2
-
1~
"
2'o
¢m
Fig. i. C h r o m a t o g r a p h y of t h e a q u e o u s p h a s e of t h e lipid h y d r o l y s a t e . A s a m p l e of t h e a q u e o u s p h a s e o b t a i n e d a f t e r h y d r o l y s i s of t h e lipids ( [ 6 - 3 H ] t h y m i d i n e precursor, Table vii) w a s c h r o m a t o g r a p h e d on a s h e e t of B a k e r f l e x D E A E - c e l l u l o s e w i t h i s o p r o p a n o l - a c e t i c a c i d - w a t e r as described in t h e t e x t . A f t e r d r y i n g , t h e s h e e t w a s c u t into I - c m - w i d e s t r i p s a n d a s s a y e d for radioa c t i v i t y . T h e position of t h e 14C-labeled reference c o m p o u n d s is i n d i c a t e d b y t h e r o u n d e d s p o t s at t h e t o p of t h e figure; S, g l y c e r o p h o s p h o r y l serine; E, g l y c e r o p h o s p h o r y l e t h a n o l a m i n e ; C, glyc e r o p h o s p h o r y l choline; G, glycerol. T h e s o l v e n t f r o n t w a s a t 16. 7 cm. Fig. 2. T h i n - l a y e r c h r o m a t o g r a p h y of lipids labeled w i t h [SH]deoxycytidine. A n a l i q u o t of t h e organic p h a s e a f t e r h y d r o l y s i s , T a b l e VII, w a s applied to a s h e e t of l:lakerflex silica gel a n d chrom a t o g r a p h e d w i t h benzene. A f t e r d r y i n g , t h e s h e e t w a s e x p o s e d to iodine v a p o r s to locate t h e lipid s p o t s a n d t h e n t h e s h e e t w a s c u t into i - c m s t r i p s a n d a s s a y e d for r a d i o a c t i v i t y . T h e s o l v e n t f r o n t w a s a t 16. 5 cm. T h e p o s i t i o n of t h e s p o t s revealed b y t h e iodine v a p o r s are i n d i c a t e d in solid b l a c k a t t h e t o p of t h e figure; t h e positions of [14C]palmitate (P) a n d t r i p a l m i t i n (T), as d e t e r m i n e d on a s e p a r a t e c h r o m a t o g r a m , are also s h o w n here.
Since most of the lipid radioactivity obtained after labeling with [aH]deoxycytidine appeared in the organic solvent phase after hydrolysis, this fraction was also examined by chromatography. As shown in Fig. 2, two bands of radioactivity were observed. One migrated at a rate just slightly greater than that of [14C]palmitate, suggesting that this component may be a fatty acid. When the unhydrolyzed lipids were chromatographed under the same conditions, two bands of radioactivity were also observed (not illustrated). One of these was at the origin of the chromatogram and contained 90 % of the label while the other contained i o % of the radioactivity Biochim. Biophys. Acta, 228 (i97 I) 6 1 o - 6 2 6
DEOXYRIBONUCLEOSIDE INCORPORATION
621
and migrated at the same rate as the [l*C]tripalmitin. It is apparent that the rapidly migrating component in the hydrolyzed sample was not present originally and did not correspond to either tripalmitin or palmitic acid; the nature of this component must remain unresolved.
Labeling o/proteins The results presented in Tables I - I I I indicated that the protein fraction of the nuclei, mitochondria and microsomes also appeared to be labeled after administration of the pyrimidine nucleosides. Since the microsomes contained the largest amount of radioactivity, they were selected for further study to determine whether the labeling of the proteins was real or an artifact. In one experiment, a portion of the protein residue of the microsomal fraction obtained after labeling with [Me-3H]thymi dine, was mixed with 25 °/zg of unlabeled thymidine and deoxycytidine and then hydrolyzed with formic acid as described b y WYATT19. When the hydrolyzate was chromatographed on paper to separate the bases, three bands of radioactivity were obtained, none of which corresponded to the added cytosine or thymine. This finding would appear to rule out nucleic acids or their pyrimidine bases as being directly associated with the protein fraction radioactivity. Another sample of the microsomal protein was then hydrolyzed with HC1 to obtain the constituent amino acids. The hydrolysate was fractionated chromatographically with an amino acid analyzer equipped with a stream splitting attachment. The results of this experiment are presented in Table V I I I and show that 5 amino acids were radioactive. Serine, aspartic acid and glutamic acid contained about the TABLE VIII INCORPORATION
OF R A D I O A C T I V I T Y
FROM
[Me-3H]THYMIDINE INTO THE AMINO ACIDS OF
MICRO-
SOMAL PROTEINS
The dried protein residue r e m a i n i n g after r e m o v a l of acid-soluble compounds, lipids and nucleic acids from mierosomes was hydrolyzed for 22 h at IiO ° with c o n s t a n t boiling HCI. After evaporation and drying, the sample was extracted w i t h s t a r t i n g buffer. A sample of the e x t r a c t was used for r o u t i n e analysis of the a m i n o acid c o n t e n t w i t h the B e c k m a n a m i n o acid analyzer. The rem a i n d e r of the sample was t h e n fractionated in t h e s a m e m a c h i n e using t h e s t r e a m splitting feature to send a p o r t i o n of the effluent to t h e analyzer and the r e m a i n d e r to a fraction collector. The collected fractions were assayed for radioactivity. The results r e p o r t e d represent the values obtained w i t h 21.6 m g of microsomal protein.
Amino acid
Content (t~moles)
Radioactivity (counts /min )
Aspartic acid Threonine Serine G l u t a m i c acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine leucine Tyrosine Phenylalanine
12.51 5.72 6.86 14.2o 3.78 8.34 9.oo o.54 6.12 1.84 4.27 13.65 2.94 4.75
198o o 211 o 22o5 o o 33 ° o o 237 o o o o
Biochim. Biophys. Acta, 228 (1971) 61o-626
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w.c.
SCHNEIDER, A. E. GRECO
same amount of radioactivity but the specific activity of the former was the highest. Alanine and methionine were also labeled but to a much smaller extent than the other three amino acids. The basic amino acids were retained on the column in the experiment reported in Table V I I I and were eluted unresolved with dilute NaOH; they did not contain significant radioactivity.
Iododeoxyuridine Since this nucleoside analogue has been used instead of thymidine for labeling DNA ~°, it was of interest to determine if it would permit a more precise labeling of liver DNA. The data in Table I X show that this was not the case and that the pattern TABLE IX INCORPORATION OF IODODEOXYURIDINE INTO LIVER FRACTIONS E a c h of t w o C3 H mice w a s g i v e n IO/~C of [ ' 2 H ] i o d o d e o x y u r i d i n e (97 C / m m o l e ) a n d 24 h l a t e r t h e livers w e r e r e m o v e d a n d f r a c t i o n a t e d .
Liver ]faction
Nuclei Mitochondria Microsomes Supernatant
Total radioactivity (counts~rain) 2 3 15 198
311 564 483 700
Distribution o] radioactivity (counts~rain) fl cid soluble lO 7 678 688 193 ooo
Lipid
5° 822 5812 329 °
RNA
DNA
Protein
131 25o 1289
19o2 1135 1154
121 679 654 ° 237o*
* N u c l e i acids were n o t r e m o v e d in t h i s a n a l y s i s .
of labeling was very similar to that reported in Tables I - I I I for the other precursors. With E12Hliododeoxyuridine, the microsomes again contained the largest amount of particulate radioactivity and the lipids and proteins accounted for most of the microsomal label. These two fractions were selected for further study. It was found that 85-93 % of the label in the lipid extract was lost into the aqueous phase when it was treated with carbon tetrachloride and 2 M KC1 as described in EXPERIMENTAL. I t was concluded that the labeling of the lipid fraction was an artifact. In the case of the microsomal protein, hydrolysis and fractionation with the amino acid analyzer showed that the entire radioactivity appeared in a ninhydrin positive peak similar in position to that of cysteic acid. Since this amino acid is poorly adsorbed and is the first to emerge from the chromatographic column, it is possible that the radioactivity was recovered here only because it was not adsorbed b y the column and not because it was incorporated into cysteic acid. If this were true, it would mean that the proteins, like the lipids, were not actually labeled by the iododeoxyuridine. Nevertheless, it is apparent that iododeoxyuridine was no more specific a precursor of liver cytoplasmic DNA t h a n the other pyrimidine nucleosides tested. Only in the nuclear fraction was the DNA preferentially labeled. DISCUSSION
The findings reported here on the incorporation of thymidine and deoxycytidine are important for several reasons. In the first place, they show that in normal rodent Biochim. •iophys. Acta, 228 (1971) 61o-626
DEOXYRIBONUCLEOSIDE INCORPORATION
623
liver most of the deoxyribonucleoside incorporated does not appear in the expected receptor, DNA, but is recovered instead in lipids and proteins. Furthermore, the present results show that the labeling of the latter by thymidine was not an artifact since the radioactivity of the proteins and the lipids was shown to be restricted to certain amino acids present in the proteins and to definite glycerides present in the lipid mixture. Consequently the possibility that the labeling of these fractions was due to contamination by or incomplete extraction of DNA or nucleoside derivatives need no longer considered. The present results also lend additional insight into the events that are occurring within the liver cell with respect to thymidine incorporation. Acid-soluble precursor molecules rapidly accumulate in both mitochondria and microsomes, especially the former, and reach peak levels in 5 min after administration. Whether these precursors are associated with particulate material so that they may be phosphorylated prior to incorporation into DNA or while they are degraded is not indicated by these results. It should be noted however that the soluble or nonparticulate fraction of the liver contains even larger amounts of acid-soluble radioactive precursors and one might speculate that the compounds present in this pool differ from those found in the particulate acid-soluble fraction. In this connection, CHANG AND LOONEY21 reported that degradation products of thymidine reached peak levels in 2 min in the liver acid-soluble fraction while thymidine nucleotides did not reach maximum levels until IO min. The labeling of the lipids and proteins occurred extremely rapidly especially in the microsomes. Large amounts of radioactivity were present after only 5 min of incorporation and reached the highest level after 30 min. The decline in microsomal radioactivity which was noted at 2 4 h with both [3Hlthymidine and [14C]thymidine may indicate either that these proteins and lipids have a rapid turnover or that they are synthesized in the microsomes prior to transfer to other parts of the cell. The latter explanation would agree with experiments which have demonstrated that the synthesis of proteins and phospholipids occurs in microsomes z2-~ and also with the data indicating that the labeling of the nuclear and mitochondrial lipids and proteins occurs more slowly and progressively (Tables II and III). Thymidine is known to be degraded in the liver by a pathway involving thymine, dihydrothymine and fl-aminoisobutyric acid 25. The latter can be excreted by the animal 28 or further metabolized via the tricarboxylic acid cycle to a wide variety of products. The labeling of the liver lipids and proteins after the administration of thymidine can apparently by explained by the operation of these pathways. This was shown by the dilution of incorporation that occurred when dihydrothymine and fl-aminoisobutyric acid were given prior to the isotope. Also, according to CHANG AND LOONEY 21, fl-aminoisobutyric acid and related degradation products accounted for 39-58 % of the acid-soluble radioactivity in the liver at periods of 2 rain- 5 h after the injection of thymidine. Similarly POTTER3 tentatively identified labeled succinic, malic, glutamic and aspartic acids in the acid-soluble fraction of rat liver after the administration of thymidine. The amino acids that were labeled in the microsomal proteins could be obtained directly from such labeled pools or readily derived by related pathways. The labeling of the lipids probably also occurs by the operation of such pathways since either the glycerol or the fatty acid portions of the lipid molecules were the main recipients of radioactivity. Biochim. Biophys. Acta, 228 (1971) 61o-626
624
w.c.
SCHNEIDER, A. E. GRECO
One disquieting feature in the incorporation of thymidine into lipids, proteins and DNA was the enormous concentrations of metabolites required relative to the labeled thymidine given (20 000:I) to achieve dilution and the failure of unlabeled thymidine to dilute incorporation into lipid and protein to the same extent as into DNA. The former can be explained b y the fact that the tracer dose of thymidine (about 2.5 nmoles) was far below that required to saturate the DNA synthetic pathway (about 1.0 #mole for a I80-g rat~7). Therefore a large fraction of the unlabeled thymidine that was given was apparently required to achieve saturation of the incorporation system and could not lead to dilution of labeling. The fact that the labeling of the lipids and proteins was diluted to a lesser extent than was incorporation into DNA indicates that the catabolic pathways require an even higher level of thymidine for saturation than does the p a t h w a y leading to DNA. The tremendous capacity of the liver for the degradation of thymidine is well known 21. Although the results of the present experiments showed that thymidine was not very specific as a precursor of DNA in the liver, how seriously does this affect the use of thymidine as a precursor? In the case of biochemical experiments, it simply means that DNA can only be studied after rigorous purification, and imposes limitations on the type of information that can be obtained. If the labeling of DNA were completely specific, it would be possible to calculate the total DNA content of mitochondria and microsomes from measurements of their radioactivity. This would be valuable information to have but obviously cannot be obtained under the circumstances. From the standpoint of autoradiography it is obvious that great care must also be exercised in establishing that the labeled material is actually DNA ~. Here too sorely needed experiments appear unfeasible because of the lack of labeling specificity. For example, autoradiography could have provided a simple means of establishing whether the DNA which we isolate from liver microsomes 7 was associated with the microsomes in the intact liver cell. It can be argued that adult normal rodent liver should not be used in studies of DNA synthesis because of the extremely low rate of cell division that occurs. Other tissues such as spleen and thymus and various tumors have much higher rates of mitosis and would seem to be more suitable subjects of study. However, it has been found that thymidine is also incorporated into the proteins and lipids of these tissues s,15, although to a lesser extent relative to DNA than was the case in liver. Nonetheless these results show that in no case was thymidine completely specific as a precursor of DNA. It seemed possible that the specificity of thymidine might be increased if the label were localized in some other part of the molecule. The results with L2-14C]thymidine were favorable in this regard since a greater proportion of the label was recovered in the DNA relative to that present in lipids and proteins with this precursor. In mitochondria and microsomes, however, large amounts of radioactivity were still recovered in the lipids and the proteins. The results with E125Iliododeoxyuridine were no more encouraging since they showed that proteins and lipids were also labeled, especially in the microsomes (Table IX). The labeling of proteins by this precursor had been noted earlier 20 and in a very recent paper 2s the labeling of lipids was also noted. Our results indicate that the labeling of the lipids and proteins by iododeoxyuridine was not a true labeling since the label was readily lost from the lipids and m a y or m a y not be associated with a definite amino acid component of the Biochim. Biophys. Acta, 228 1197 I) 61o-626
DEOXYRIBONUCLEOSIDE INCORPORATION
625
proteins. Nevertheless the labeling of these fractions does occur and poses the same problems as were encountered with thymidine and deoxycytidine. It was also hoped that the administration of catabolites to the animals prior to the labeled thymidine would reduce the incorporation into proteins and lipids to such an extent that the specificity for labeling D N A would be satisfactory. The results with E~Hlthymidine showed that this was not the case. However, since similar experiments with [2-14Clthymidine and with deoxycytidine and iododeoxyuridine were not attempted, it is possible that a solution to this problem could be devised. The fact that the R N A fraction of the liver was in some cases more heavily labeled than the D N A fraction (e.g. the microsomal R N A fraction, Table II) rsises the question of the nature of the labeling of these fractions. Our previous experiments 7, in which nuclear, mitochondrial and microsomal D N A were extensively purified, showed that the D N A was being labeled by [3Hlthymidine. Whether R N A is also being labeled cannot be determined from our experiments since no attempt was made to purify the R N A or to determine the nature of the labeled products in the R N A fraction. In this connection, however, it should be noted that the formation of thymine riboside by liver extracts has been reported 3 and that the labeling of R N A in another system s by [2-14Clthymidine has been established to occur as conversion to uridylate. It would therefore appear to be entirely possible that the labeling of the R N A fraction could involve either the direct incorporation of the thymine into R N A or the incorporation of thymidine breakdown products into RNA. Further experiments would be required to establish this point.
ACKNOWLEDGMENT
The authors are indebted to Mr. James F. Williams for the amino acid analyses.
REFERENCES I P. REICHARD AND B. J. ESTBORN, J. Biol. Chem., 188 (1951) 839. 2 P. REICHARD, Acta Chem. Seand., 9 (1955) 1275. 3 V. R. POTTER, in F. STOHLMAN, JR., The Kinetics o/Cellular Proli[eration, Grune and Stratton, N e w York, 1959, p. IiO. 4 W. B. COUNTS AND W. C. FLAMM, Biochim. Biophys. Acta, 114 {I966) 628. 5 R. ROYCHOUDHOURYAND S. P. SEN, Biochem. Biophys. Res. Commun., 14 {I964) 76 B. J. BRYANT, J. Cell Biol., 29 (1966) 29. 7 W. C. SCHNEIDER AND E. L. HUFF, J . Biol. Chem., 244 (1969) 4843 • 8 VV. C. SCHNEIDER, J. Biol. Chem., 164 (1946) 747. 9 S. B. WEISS, S. W. SMITH AND E. P. KENNEDY, J . Biol. Chem., 231 (1958) 53IO W. C. SCHNEIDER AND R. •. BEHKI, J. Biol. Chem., 238 (1963) 3565 • I I H. WAGNER, L. HbRHAMMER AND H. WOLFF, t~iochem. Z., 334 (1961) 175. 12 R. M. C. DAWSON, Biochem. J., 75 (196o) 45. 13 B. N. AMES, in E. F. NEUFELD AND V. GINSBURG, Methods in Enzymology, Vol. 8, Academic Press, New York, 1966, p. 115. 14 C. H. ]~ISKF. AND Y. SUBBAROW, J. Biol. Chem., 66 (1925) 37515 D. K. MYERS AND S. RAM, Can. J. Physiol. Pharm., 47 (1969) 732. 16 ~Ar. D. SKIDMORE AND C. J. ENTENMAN, J. Lipid Res., 3 (1962) 47117 V. P. SKIPSKI, R. F. PETERSON AND M. BARCLAY, Biochem. J., 9o (196o) 374. 18 A. F. HOFMANN, in A. C. FRAZER, Biochemical Problems o/Lipids, Elsevier, Amsterdam, 1963, p.I. 19 G. R. WYATT, Biochem. J., 48 (1951) 584. 20 B. W. F o x AND W. H. PRUSOFF, Cancer Res., 25 (1965) 234.
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626 21 22 23 24 25 26 27 28 29
W. C. SCHNEIDER, A. E. GRECO L. O. CHANG AND V~. B. LOONEY, Cancer Res., 25 (1965) 1817. P. SIEKEVITZ, J. Biol. Chem., 195 (1952) 549. G. F. VqlLGRAM AND E. P. KENNEDY, J. Biol. Chem., 238 (1963) 2615. W. C. SCHNEIDER, .[. Biol. Chem., 238 (1963) 3572. K. FINK, R. E. CLINE, R. B. HENDEnSON AND R. 1V~.FINK, J. Biol. Chem., 221 (1956) 425 . K. FINK, R. 13. HENDERSON AND R. M. FINK, r . Biol. Chem., 197 (1952) 441. O. F. NYGAARD AND P~. L. POTTER, Radiation Res., IO (1959) 462. C. P. FUSSELL, J. Cell Biol., 39 (1968) 264. L. A. DETHLEFSEN, Cancer Res., 29 (1969) 1717.
Bioehim. Biophys. Acta, 228 (1971) 61o-026
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96722
I N C O R P O R A T I O N OF P Y R I M I D I N E D E O X Y R I B O N U C L E O S I D E S I N T O L I V E R L I P I D S AND O T H E R COMPONENTS W A L T E R C. S C H N E I D E R ANn A N T O I N E T T E E. G R E C O
Laboratory o/ Biochemistry, National Cancer Institute, National Institutes o] Health, Department o/ Health, Education, and Wel/are, Bethesda, Md. 2OOl4 (U.S.A.) (Received A u g u s t i 7 t h , 197 o)
SUMMARY
I. Labeled thymidine or deoxycytidine were injected into rats or mice and the distribution of the radioactivity in the nuclei, mitochondria, microsomes or soluble fraction isolated from the livers was determined. I t was found that the microsomes contained the largest amount of particulate-bound radioactivity and the nuclei the least and that the lipid and protein fractions of the mitochondria and microsomes contained the bulk of their radioactivity while the DNA fraction contained very little label. 2. Incorporation of labeled thymidine or deoxycytidine into DNA, lipids and proteins was diminished, but not to the same degree in each, b y the prior administration of unlabeled forms of these compounds. The incorporation of thymidine into lipids and proteins but not into DNA was also diluted by the prior injection of the thymidine catabolities, fl-aminoisobutyric acid or dihydrothymine. 3. Chromatography of the particulate lipid fraction obtained after labeling with [*H]thymidine or EaHldeoxycytidine revealed that lecithin, phosphatidyl ethanolamine and neutral glycerides contained most of the radioactivity. In the case of the lipids obtained from the liver soluble fraction, the radioactivity was associated only with the neutral glyceride fraction. 4- When the particulate lipid fractions were hydrolyzed and chromatographed the main radioactive products corresponded to glycerol, glycerylphosphoryl choline and glycerylphosphoryl ethanolamine when L~H]thymidine was the precursor. With [3H~deoxycytidine, however, two main radioactive products, apparently derived from the f a t t y acid protion of the glycerides, were obtained. 5- Hydrolysis of the microsomal protein fraction obtained after labeling with E~Hlthymidine and fractionation of the amino acids released revealed that serine, aspartic acid and glutamic acid contained the largest amount of label, alanine and methionine were much less radioactive, and the other amino acids were unlabeled.
INTRODUCTION
Thymidine is currently widely employed as a precursor of DNA. This usage is based on the initial experiments of REICHARD AND ESTBORN1 and REICHARD2 which demonstrated that this compound as well as deoxycytidine were incorporated as a unit into the DNA of regenerating rat liver but not to a significant extent into RNA. Biochim. Biophys. Acta, 228 (1971) 6 i o - 6 2 6
DEOXYRIBONUCLEOSIDE INCORPORATION
611
On the other hand, it has been known for some time that thymidine is degraded in biological systems and reports have appeared in the literature on the labeling of amino acids 3, tricarboxylic acids 3, glycogen4, RNA 5, and proteins e after administration of radioactive thymidine or thymine. I t is not entirely clear, however, to what extent the labeling of other cellular components interferes with the use of thymidine as a precursor of DNA. In a recent study on the association of DNA with liver mitochondria and microsomes 7, we noted that the amount of radioactivity incorporated from thymidine into the DNA of these organelles was only a minor fraction of the radioactivity they contained. The present experiments were undertaken to define the nature of the components other than DNA labeled b y thymidine and to establish whether this labeling was real or artifactual. The results of these studies indicate t h a t both the lipids and the proteins obtained from liver cytoplasmic fractions were heavily labeled when thymidine or deoxycytidine were given to animals.
EXPERIMENTAL
The animals used were male Holtzman or Sprague--Dawley rats 200-300 g in weight or C3H mice, 2-3 months old, and were either fed ad libitum a commercial pellet diet or were fasted for 18-24 h before use. The labeled precursor was administered intraperitoneally and 5 min-24 h later the animals were killed and the livers removed. In some cases other compounds were also given by intraperitoneal injection prior to the precursor. The livers were homogenized in 0.25 M sucrose and nuclei, mitochondria and microsomes were isolated b y differential centrifugation as described previously 7. In some cases the homogenate was simply centrifuged for I h at 4 ° ooo rev./min in the type A-ITO rotor of the International B-6o centrifuge to obtain a pellet of the particulate material (nuclei, mitochondria and microsomes) and a soluble or supernatant fraction. The liver fractions were then separated essentially as described previously s into acid-soluble compounds, lipids, RNA, DNA, and protein. Sufficient HC104 was added to each fraction at o ° to bring the concentration to 0.6 M. The precipitates were centrifuged down and washed twice b y resuspension and resedimentation in cold 0.3 M HCI04. The three extracts constituted the acid-soluble fraction of the tissue. The lipids were then extracted from the washed pellet b y resuspension in absolute ethanol, neutralization of the mixture to litmus with NH4OH, and resedimentation*. Extraction with ethanol was repeated twice more and after the final extraction the pellet was mixed with o.3-1.o M N a O H and heated for I h at 37 °. Sufficient HC10, was then added to neutralize the N a O H and to provide an excess concentration of 0.6 M and after cooling, the precipitate was sedimented and washed once * U n p u b l i s h e d studies in this l a b o r a t o r y showed t h a t neutralization of the e t h a n o l - t i s s u e m i x t u r e reduced the loss of p r o t e i n and R N A to a m i n i m u m and this procedure was adopted as standard. I n one experiment, the ethanol e x t r a c t i o n s were followed b y t w o extractions with e t h a n o l - c h l o r o f o r m (2 : I, b y voi.); t h e latter e x t r a c t i o n s were omitted in s u b s e q u e n t experiments because the e x t r a c t s contained less t h a n IO % of the r a d i o a c t i v i t y recovered in the ethanol extracts.
Biochim. Biophys. Acta, 228 (1971) 61o-626
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W. C. SCHNEIDER, A. E. GRECO
with cold o.3 M HC104. The two extracts obtained from this alkaline hydrolysis contained the RNA in the form of nucleotides. DNA was extracted from the pellet b y resuspension in 0.3 M HC104 and heating for 15 rain at 9 o°. This process was repeated once and the precipitate was washed once with cold 0. 3 M HC104. The final pellet, which should contain only protein, was dissolved in o.5 M NaOH. The extraction procedures were monitored b y measuring the radioactivity of each extract and it was noted that a burst of radioactivity appeared in the first extract of each series and was followed b y a decline to insignificant levels in the final extraction. For further examination of the lipids, the ethanol extracts were combined and mixed with 0.25 vol. of carbon tetrachloride and then equilibrated with an equal volume of 2 M KC19,1°. The carbon tetrachloride phase which separated out was washed twice with additional 2 M KC1 and once with distilled water. In some cases the ethanol extracts were evaporated on a rotating evaporator instead and the residue was redissolved in chloroform. The lipids were separated by thin-layer chromatogr a p h y on plates of Silica gel G using a preliminary development with chloroform follow ed b y drying and development with chloroform-methanol-water (65:25:4, b y vol.)11. The lipid spots were located by exposing the plates to iodine vapor and b y spraying with ninhydrin. Hydrolysis of the lipid solutions was carried out b y mild alkaline and acid hydrolysis as described b y DAWSONlz, An aliquot of the lipid concentrate was mixed with 5.0 ml of ethanol, 0.65 ml of water and 0.25 ml of I M NaOH. After 20 rain incubation at 37 °, o.14 ml of IOO °/o (w/v) trichloroacetic acid was added and the heating continued for another 30 min. The ethanol was removed under a stream of N 2 and the aqueous phase was extracted 3 times with petroleum ether. Aliquots of the aqueous phase were chromatographed on a thin layer of DEAE-cellulose (Bakerflex) with isopropanol-acetic acid-water (55:25:20, b y vol.) as the developing solvent. The petroleum ether extracts of the hydrolysate were chromatographed on Bakerflex silica gel thin-layer sheets with benzene as the developing solvent. Phosphorus was determined colorimetrically b y the method of AMESla on samples hydrolyzed with H2SO 4 (ref. 14). Tritium and 14C were measured witlithe liquid scintillation spectrometer. Samples adjusted to contain 2.0 ml of water were mixed with 15 ml of a scintillator solution (40 ml of Liquifluor and 333 ml of Triton X-IOO diluted to I 1 with toluene). In the case of the alkaline solutions of the protein samples, 0.05 ml of 9 ° °/o formic acid was added to each vial in addition. The counts obtained were corrected to the same efficiency by the internal standardization method. Counting efficiency in unquenched samples was about 20 % for tritium. 1~5I activity was measured in a g a m m a spectrometer. The following labeled compounds were used in these experiments: EMe-~H] thymidine, E2-14C] thymidine, and CSHldeoxycytidine, Schwarz Bioresearch, Inc., [6-SH]thymidine and E5-3Hldeoxycytidine, Amersham-Searle Corp.; phosphatidyl EI,2-14C2]choline, phospliatidyl Ei,2-14C21ethanolamine, phosphatidyl [3-14Clserine, and [4-14C~cholesterol, Tracerlab; [I,3-14C~ glycerol and [ c a r b o x y - 1 4 C l t r i p a l m i t i n , New England Nuclear; E125IJiododeoxyuridine, International Chemical and Nuclear Co. Unlabeled phospholipids were obtained from General Biochemicals and used as chromatographic standards. Glycerylphosphoryl choline, glycerylphosphoryl ethanolamine and glycerylphosphoryl serine labeled with 14C were obtained from the labeled lipids b y mild alkaline and acid hydrolysis as described above. Biochim. Biophys. Acta, 228 (1971) 61o-626
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DEXYRIBONUCLEOSIDE INCORPORATION
The amino acid analyses were performed chromatographically w~th a Beckman I2oC amino acid analyzer programmed to complete the separation of the amino acids in 19o min using a starting buffer of sodium citrate (pH 3.25) a pH 4.30 limit buffer of sodium citrate (both buffers were 0.2 M in Na+), and monitoring the effluent by ninhydrin color development. Amino acid content was calculated from the magnitude of the ninhydrin color and individual amino acids were identified by the time at which they emerged from the column as well as by ninhydrin color spectra. The column was calibrated with a standard mixture of amino acids. In order to determine radioactivity of individual amino acids, the column was used with a stream splitter which allowed part of the effluent to pass through the analyzer part of the machine and the remainder to be collected with a fraction collector. Peak tubes of radioactivity were rechromatographed with standard amino acid markers to confirm identification and establish purity. RESULTS
In the first experiment, Table I, mitochondria and microsomes were isolated from the livers of rats 24 h after they received either E3H]thymidine or EaH]deoxycytidine. The data show that both particulate fractions contained substantial amounts of radioactivity with either precursor but the nucleic acid fraction accounted TABLE I INCORPORATION OF E3H]THYMIDINE AND [SH]DEOXYCYTIDINE INTO LIVER MITOCHONDRIA AND MICRO$OMES Two rats were given either 25o/,C of EMe-3H]thymidine (io C/mmole) or 250/~C [SH]deoxycytidine (2. 5 C]mmole, Schwarz Bioresearch) intraperitoneally and 24 h later t h e livers were r e m o v ed and fractionated as described in the text.
Precursor
Liver fraction
Total incorporation (cov.nls]min)
Distribution of rad$oactivity (counts]min) Acid soluble
Lipid
RNA
DNA
Protein
Thymidine
Mitochondria Microsomes
63 900 131 60o
14oo 132o
22 17o 66 98o
224o 671o
23 39o 18 95 °
14 7 ° o 37 640
Deoxycytidine
Mitochondria Microsomes
72 600 157 40o
661o 567 o
23 23o 71 14o
3 I9O" 39 57 ° 14 OlO * 66 58o
R N A a n d D N A were e x t r a c t e d t o g e t h e r in this e x p e r i m e n t b y using h o t HC104.
for less than IO °/o of the total label when deoxycytidine was the precursor. When thymidine was the precursor, the DNA fraction accounted for only about 15 % of the radioactivity of the microsomes and 36 °/o of the mitochondrial label. The lipid and the protein fractions of these organelles contained radioactivity that accounted for more than 50 % of the incorporation. The labeling of the lipids and the proteins of the mitochondria and microsomes was unexpected both because of the high proportion of radioactivity they represented and also because the labeling of lipids by these precursors had not previously been reported ". " While this m a n u s c r i p t was in preparation, a note appeared in which the labeling of the lipid fraction of tissues after the a d m i n i s t r a t i o n of radioactive t h y m i d i n e was described 15.
Biochim. Biophys. Acta, 228 (1971) 61o-626
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w.c.
SCHNEIDER,
A. E. G R E C O
In order to determine if the labeling of lipids and proteins was limited to the cytoplasm and was related to DNA synthesis, shorter incorporation times were used and purified nuclei were examined as well. The results in Table I I represent incorporation times of 5 and 30 min and show that the nuclei contained the lowest amount of total radioactivity of the three particulate fractions isolated, while the microsomes
TABLE
It
INCORPORATION
OF THYMIDINE
INTO RAT LIVER PARTICULATE FRACTIONS
E a c h of t w o f a s t e d r a t s w a s g i v e n 2 5 o , u C of E M e - S H ] t h y m i d i n e ( i o # C / m m o ] e ) later the livers were removed and fractionated as described in the text.
Time (rain)
Liver fraction
Total incorporation (counts/mi n)
5
Nuclei Mitochondria Microsomes
30 700 64 8oo 85 6 o o
4 020 58 o6o 2o 54 °
7 860 3 24o 24 4°0
3°
Nuclei Mitochondria Microsomes
47 200 76 5oo 248 ooo
i 320 51 5 6 0 17 8 6 0
8 73 ° IO 9 4 ° lO6 6o0
a n d 5 o r 3° m i n
Distribution o/radioactivity (counts/rain) Acid soluble
Lipid
RNA
DNA
Protein
I 81o 97 ° 9 16o
IO 6 2 0 39o 4 280
6 39o 2 14o 27 2 2 o
33 ° I 99o 23 3 1 o
25 9 6 0 5 280 12 9 0 0
io 860 6 739 87 3 0 0
contained the most. When the distribution of the label between the various classes of compounds was examined, it was noted t h a t the acid-soluble fraction was already at peak levels at 5 rain in all three particulate fractions, declined somewhat at 30 min and reached near zero levels at 24 h (Table I). The mitochondrial fraction contained the highest levels of the particulate-bound acid-soluble precursors. The labeling of the lipids and the proteins of all fractions was already evident at 5 min and increased in amount at 30 min. The increase in labeling between 5 and 30 min was especially marked in the microsomal fraction where the radioactivity reached peak levels in the proteins and lipids and then appeared to decline to a lower level at 24 h (Table I). The labeling of the DNA fraction exceeded that of the proteins and lipids only in the case of the nuclear fraction; the DNA fraction of the mitochondria and the microsomes contained less radioactivity than did any of the other fractions obtained from these particulates except at 24 h when the radioactivity of the RNA fraction was the lowest. It is noteworthy, however, that the radioactivity of the RNA fraction of the microsomes exceeded that of the DNA fraction after 5 and 3 ° rain of incorporation.
Labeling o/subcellular components with [14Clthymidine In order to deternfine whether the position of the label in the thymidine molecule might be critical in determining the specificity of labeling, experiments were also conducted with [2-14C]thymidine. Preliminary studies with rats indicated that the lipids of microsomes were also labeled by this precursor, but the radioactivity of all fractions were very low due undoubtedly to the lower specific activity of the 14C-labeled precursor and to the fact that less radioactivity was given than was the case with the [3H]thymidine. By using mice instead of rats, a higher ratio of 14C Biochim. Biophys. Acta, 2 2 8 ( 1 9 7 1 ) 6 1 o - 6 2 6
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DEOXYRIBONUCLEOSIDE INCORPORATION
administered to body weight could be achieved more easily and the radioactivity of t h e f r a c t i o n s w a s c o r r e s p o n d i n g l y g r e a t e r . T h e r e s u l t s of t h e s e e x p e r i m e n t s a r e given in Table III. The data were similar in many respects to those obtained with the [ a H j t h y m i d i n e , a l t h o u g h t h e p r o p o r t i o n of r a d i o a c t i v i t y f o u n d i n t h e D N A f r a c t i o n s w a s c o n s i d e r a b l y g r e a t e r w i t h t h e 14C-labeled p r e c u r s o r . B o t h p r o t e i n s a n d l i p i d s of all t h r e e f r a c t i o n s w e r e l a b e l e d a t b o t h t i m e p o i n t s b u t t h e p r o t e i n s c o n t a i n e d m o r e r a d i o a c t i v i t y t h a n t h e lipids. T h e r a d i o a c t i v i t y of t h e m i c r o s o m a l p r o t e i n s a n d l i p i d s also declined between 30 min and 24 h after administering the precursor, just as had been observed with {aH]thymidine. TABLE III I N C OR P O R AT IO N OF [ l a C I T H Y M I D I N E I N T O MOUSE L I V E R P A R T I C U L A T E FRACTIONS
Each of four C3H mice was given 5.o/*C of E2-14C]thymidine (47.9 mC/mmole). Two mice were sacrificed 3 ° rain or 24 h later and the livers were removed and fractionated as described in the text.
Labeling time
Tissue /raction
Total incorporation (counts/min)
Distribution o/radioactivity (counts/rain) Acid soluble
Lipid
RNA
3 ° min
Nuclei Mitochondria Microsomes
8 300 i i 3oo 18 8o0
25 642o 81o
83 565 436o
224 226 124o
6 99o 2 o7 ° 2 54 °
98o 2o2o 985o
24 h
Nuclei Mitochondria Microsomes
16 34 ° 7 800 12 20o
115 175 45 °
165 650 16oo
280 135 47 °
13 920 3 760 4 47 °
186o 3 °80 521°
DNA
Protein
E//ects o] added compounds on the incorporation o / t h y m i d i n e and deoxycytidine The above experiments indicated that both thymidine and deoxycytidine were s e r v i n g a s p r e c u r s o r s of l i p i d a n d p r o t e i n s . A l t h o u g h t h e r a d i o c h e m i c a l s u s e d i n t h e s e T A B L E IV E F F E C T OF O T H E R COMPOUNDS U P O N T H E INCORPORATION OF [ S H ] T H Y M I D I N E I N T O MOUSE L I V E R
Each C3H mouse received 22.5/*C of [Me-SH]thymidine (2 nmoles) or 37.5/*C [aH]deoxycytidine (2.75 nmoles), b y injection. Unlabeled compounds, in the amounts indicated below, were injected 5 rain prior to radioactive material. The animals were sacrificed I h after the final injection and the livers removed and separated into particulate and soluble fractions as described in the text
Compounds administered
Soluble radioactivity (counts~rain)
[3H]Thymidine 321 500 Thymidine (4°/*moles) + [aH]thymidine 4Ol 85o DL-fl-Aminoisobutyric acid (4 ° pmo]es) + [SH]thymidine i 559 ooo Dihydrothymine (4°/*moles) + [SH]thymidine 468 5o0 [aH]Deoxycytidine 9o 4 ooo Deoxycytidine (8/*moles) + [aH]deoxycytidine I 073 ooo
Particulate radioactivity (counts/rain) A cid soluble
Lipid
RNA
DNA
Protein
15 390
92 57 ° 19oo
25 880
12 66o
19 4oo
13 46o 164o
92o
2 39o
69 93 °
26 27o 115o
20 23o
6 93 °
27 97 ° 58 300
14 94 ° 124o 114 80o 98o0
28 61o 7° 7o0
7 38o 51 55 °
63 IOO
88 IOO 772o
8 73 °
38 500
Biochim. Biophys. Acta, 228 (1971) 6 I O ~ 2 6
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S C H N E I D E R , A. E. GRECO
experiments were assayed by the manufacturer and reported to be more than 99 % pure and independent tests in our laboratory also indicated high purity, it was necessary to consider that the incorporation of label into lipid and protein might not be a property of the parent molecule, but of some contaminant or breakdown product. This possibility was tested by injecting either the unlabeled precursor itself or a precursor metabolite just prior to administration of the radioactive compound to see whether dilution of incorporation occurred. In these experiments, for convenience a single particulate fraction containing all the nuclei, mitochondria and microsomes of the liver was used instead of the individual fractions. The results in Table IV show that unlabeled thymidine reduced the incorporation of label into DNA, lipid and protein. However, the dilution of DNA labeling that occurred (28-fold) was much greater than the dilution of either lipid or protein labeling (5-7-fold). Two natural products of thymidine catabolism, fl-aminoisobutyric acid and dihydrothymine also reduced the incorporation of label into lipid and protein but the dilution of the latter incorporation was not as great as that observed with thymidine itself. Neither of these metabolites affected the labeling of the DNA fraction to a marked extent. The fl-aminoisobutyric acid, however, did result in a marked increase in the pool of labeled precursors (soluble radioactivity+particulate acid-soluble radioactivity). When unlabeled deoxycytidine was given together with the labeled compound dilution of incorporation into all fractions was observed. Since the amount of unlabeled deoxycytidine given was only one-fifth that of the unlabeled thymidine, the dilutions that occurred were correspondingly lower; 8-fold for DNA and only 25 % for lipid and protein.
Incorporation o/radiolysis products o/ thymidine Since tritiated compounds are known to undergo a slow self-destruction, it was of some interest to test the incorporation of the radiolysis products. When an old sample of [3H]thymidine was submitted to thin-layer chromatography, three TABLE
V
INCORPORATION OF RADIOLYSIS PRODUCTS OF THYMIDINE INTO MOUSE LIVER A s a m p l e of [ M e - 3 H ] t h y m i d i n e , w h i c h h a d b e e n s t o r e d i n a r e f r i g e r a t o r for a b o u t a y e a r , w a s a p p l i e d t o a s h e e t of B a k e r f l e x c e l l u l o s e F a n d c h r o m a t o g r a p h e d t w i c e w i t h b u t a n o l - w a t e r (86:14, b y v o l . ) . A b a n d of m a t e r i a l a t 1 2 . 5 - 1 3 . 5 c m f r o m t h e o r i g i n w a s o b s e r v e d u n d e r u l t r a v i o l e t l i g h t a n d c o r r e s p o n d e d t o t h a t of u n l a b e l e d t h y m i d i n e o n a c o m p a n i o n c h r o m a t o g r a m . W h e n t h e c h r o m a t o g r a m w a s s c a n n e d for r a d i o a c t i v i t y , t w o s l o w l y m i g r a t i n g b a n d s of r a d i o a c t i v i t y ( i a n d 2) w e r e o b s e r v e d i n a d d i t i o n t o o n e c o r r e s p o n d i n g t o t h e o n e s e e n u n d e r u l t r a v i o l e t l i g h t (3). T h e t h r e e b a n d s w e r e e l u t e d a n d a l i q u o t s w e r e i n j e c t e d i n t o f a s t e d C 3 H m i c e . i h l a t e r the livers were removed and particulate and soluble fractions were isolated as described in the text.
Band No.
I 2 3 3"
Amount recovered (/*C)
Amount in/ected (l~C)
Soluble radioactivity (counts/min)
177 257 490 286
27.2 27. 4 28.1 21.2
25I 366 473 638
900 500 3o0 670
Particulate radioactivity (counts/rain) Acid soluble
Lipid
RNA
DNA
Protein
25 25 18 35
I I 25 3°
199o 955 267 ° 3309
500 43 ° 6540 9425
2 I 8 12
320 240 7o0 670
19o 680 3o0 720
33 ° ooo o30 14o
* B a n d 3 w a s r e c h r o m a t o g r a p h e d o n a B a k e r f l e x s i l i c a gel s h e e t w i t h e t h y l a c e t a t e - f o r m i c a c i d - w a t e r (60 : 5 : 35, u p p e r p h a s e ) a s t h e s o l v e n t .
Biochim. Biophys. Acta, 228 (1971) 6 1 o - 6 2 6
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617
radioactive components were obtained, one of which was visible under ultraviolet light and corresponded to thymidine. The latter was rechromatographed on a thin layer of silica gel and found to be 97 ~o pure. When equivalent amounts of these radioactive materials were given to mice, it was found that only the one corresponding to thymidine, (Band 3, Table V), was incorporated into DNA. This material was also incorporated strongly into lipid and protein. Further chromatography on silica gel failed to change the pattern or extent of incorporation. Since the other bands of radioactive material that were obtained gave only low levels of radioactivity in all fractions, it could be concluded that they were not responsible for the labeling of proteins and lipids. The data in Tables IV and V also show that the liver supernatant or soluble fraction contained much more radioactivity than the particulate fraction. This was not unexpected since the former might be expected to contain the bulk of the administered precursor at such a short time after administration to the animal. It was somewhat surprising therefore to find that large part of the supernatant radioactivity appeared to be lipid also. In the experiment in which thymidine Fraction 3" was given for example (Table V) it was found that the bulk of the supernatant radioactivity was present in the acid-soluble fraction but that 25.5 % (163 ooo counts/rain) was recovered in the ethanol or lipid extract of the supernatant fraction. The source of this lipid m a y have been the f a t t y layer that accumulated at the top of the tube after centrifugation of the homogenate, since care was taken to include it with the supernatant fluid. The large amount of lipid-associated radioactivity observed in the supernatant fraction in this experiment was unusual since only 44 000-54 ooo counts/ rain (11.7-13. 7 % of the supernatant fluid radioactivity) were recovered in this fraction in three other experiments. Nevertheless, the results show that the supernatant fluid did contain labeled f a t t y material t h a t rivaled in amount that found in the lipid extracts of the particulate fraction of the liver.
Nature o/ the labeling o~ the lipid/raction When carbon tetrachloride was added to the ethanol extracts of the mitochondrial and microsomal fractions or of the combined particulate fraction and the mixture was equilibrated and washed with 2 M KC1 and water as described above, it was found that the bulk of the radioactivity was retained in the carbon tetrachloride. In several experiments with [SH]thymidine or [3H]deoxycytidine, the recovery of radioactivity in the carbon tetrachloride ranged between 87 and 99 % of that present in the ethanol extracts. When the lipid extracts of the particulate fraction of the liver homogenate were chromatographed on thin layers of Silica gel G, spots detected b y exposure to iodine vapors and corresponding in position to authentic lysolecithin, sphingomyelin, lecithin, phosphatidyl ethanolamine, and cardiolipin were obtained. In addition, a band of neutral fats was also found at the solvent front. When these spots were examined for phosphorus content and radioactivity, the results presented in Table VI were obtained. The data show that the lecithin and phosphatidyl ethanolamine bands accounted for most of the radioactivity associated with phospholipids. The lecithin spots contained the largest amount of phospholipid radioactivity and appeared to contain more radioactivity per unit of phosphorus than the phosphatidyl ethanolamine. The data also show that large amounts of radioactivity rivalling that assoBiochim. Biophys. Acta, 228 (1971) 61o-626
O
o0
CHROMATOGRAPHY
OF THE
MOUSE LIVER
PARTICULATE
LIPIDS
R a d i o a c t i v i t y (counts/rain) P h o s p h o r u s (/*g)
Radioactivity (counts/min) P h o s p h o r u s (/~g)
Radioactivity !counts/min) P h o s p h o r u s {,ug)
[6_SH]Thymidine
EMe-aH~Thymidine
[5_3HlDeoxycytidine
454 3.1
144 o
315 1.7
462 9.4
181 o
384 9.3
Lipid componene" LL Sph
I 555 lO9
9 342 128
I i 920 ioi
L
2806 47.3
592 46.6
28Ol 32.9
PEA
304 9.9
116 8.9
149 7.1
C
14 92o o
2 622 o
3 034 o
Gly
lO2 82
89 71
97 ioo
(%)
Recovery
* LL, lysolecithin; Sph, s p h i n g o m y e l i n ; L, lecithin; P E A , p h o s p h a t i d y l e t h a n o l a m i n e ; C, cardiolipin; Gly, glycerides a n d n e u t r a l fats f o u n d at s o l v e n t front.
Measurement
Precursor in~ected
Samples of t h e lipid e x t r a c t s in chloroform were applied to io c m × 20 c m Silica gel G plates a n d d e v e l o p e d with c h l o r o f o r m followed b y c h l o r o f o r m - m e t h a n o l w a t e r ( 6 5 : 2 5 : 4 , b y vol.). T h e s p o t s were l o c a t e d b y e x p o s u r e to iodine v a p o r s , s c r a p e d off a n d e l u t e d 3-4 t i m e s with m e t h a n o l . R a d i o a c t i v i t y a n d t o t a l p h o s p h o r u s were m e a s u r e d on t h e m e t h a n o l e x t r a c t s . T h e s p o t s were identified b y c o m p a r i s o n with t h e position of reference lipids on c o m p a n i o n c h r o m a t o g r a m s .
THIN-LAYER
T A B L E VI
©
Oo
DEOXYRIBONUCLEOSIDE INCORPORATION
619
c i a t e d w i t h lecithin were f o u n d in t h e m a t e r i a l a c c u m u l a t i n g a t t h e s o l v e n t front a n d k n o w n to c o n t a i n t h e n e u t r a l fats, as well as o t h e r n e u t r a l f a t t y materials. Several o t h e r t h i n - l a y e r c h r o m a t o g r a p h i c p r o c e d u r e s were also used to e x a m i n e t h e c o m p o n e n t s of the lipid e x t r a c t s . These i n c l u d e d t h e c h l o r o f o r m - m e t h a n o l a m m o n i a solvent w i t h silica gel G TM t h e c h l o r o f o r m - m e t h a n o l - w a t e r - a c e t i c acid syst e m s described b y SKIPSKI et al. 1~, a n d t h e NH4NO3-containing silica gel G p l a t e s y s t e m described b y HOFMANN TM. The a m o u n t of r a d i o a c t i v i t y a s s o c i a t e d w i t h t h e lecithin, p h o s p h a t i d y l e t h a n o l a m i n e , a n d n e u t r a l fat spots was in all cases t h e s a m e as h a d been o b s e r v e d w i t h t h e s y s t e m described. No evidence was o b t a i n e d for labeling of p h o s p h a t i d y l serine, which t h e a b o v e s y s t e m s p e r m i t to s e p a r a t e from phosphatidyl ethanolamine. I t has been n o t e d a b o v e t h a t a considerable fraction of the r a d i o a c t i v i t y recovered in t h e soluble or s u p e r n a t a n t fraction of t h e liver h o m o g e n a t e was also ext r a c t a b l e w i t h ethanol. W h e n this e x t r a c t e d m a t e r i a l was c h r o m a t o g r a p h e d on silica gel G as described in T a b l e VI, 95 % of t h e r a d i o a c t i v i t y was recovered in t h e n e u t r a l fat b a n d a c c u m u l a t i n g at the s o l v e n t front. F u r t h e r m o r e when t h e e t h a n o l e x t r a c t of t h e liver s u p e r n a t a n t fluid was c h r o m a t o g r a p h e d on a sheet of B a k e r flex silica gel w i t h benzene as t h e d e v e l o p i n g solvent, 92 % of t h e r a d i o a c t i v i t y was p r e s e n t in a b a n d with an R F of 0.5, c o r r e s p o n d i n g to t h e position of [~4C~tripalmitin in t h e s a m e solvent system.
Hydrolysis o/ lipids W h e n t h e e t h a n o l e x t r a c t s were h y d r o l y z e d b y t h e m i l d a l k a l i n e a n d acid p r o c e d u r e of DAWSONTM as described above, t h e results p r e s e n t e d in T a b l e V I I were o b t a i n e d . The d a t a show t h a t t h e h y d r o l y s i s of t h e p h o s p h o l i p i d s was a l m o s t c o m p l e t e since 95 % or more of t h e p h o s p h o r u s of t h e p a r t i c u l a t e fractions was recovered in t h e a q u e o u s phase after hydrolysis. W h e n either [ 6 - 3 H l t h y m i d i n e or EMe-ZHlthymidine was t h e precursor, t h e largest fraction of t h e t o t a l r a d i o a c t i v i t y was recovered in t h e a q u e o u s phase a f t e r h y d r o l y s i s whereas w i t h [5-3HJdeoxycytidine 88 °/o of t h e r a d i o a c t i v i t y was recovered TABLE VII HYDROLYSIS
OF LIVER
LIPIDS
Aliquots of the ethanol extracts of the tissue fractions indicated were hydrolyzed with NaOH and trich]oroacetic acid and then were evaporated and extracted with petroleum ether as described in the text. Portions of the aqueous and the organic phases were then assayed for radioactivity and total phosphorus
Precursor in#cted
Liver fraction
[Me-*H]Thymidine lVticrosomes Particulate Soluble [6-3H~Thymidine Particulate [5-SH]Deoxycytidine Particulate
A queous phase
Organic phase
Radioactivity (counts~rain)
Phosphorus (~g)
Radioactivity (counts/rain)
Phosphorus (l~g)
15 400 3 775 4 9o5 i i 35° 3 075
381 * * 159 193
I 43 ° i 513 1°3 3 900 22 800
o "
* 9.0 8.5
* Not measured.
Biochim. Biophys. Acta, 228 (i97 z) 61o-626
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SCHNEIDER, A. E. GRECO
in the organic solvent phase. These results indicated that the fatty acid components of the glycerides were being labeled by the latter precursor while the remainder of the glyceride molecule was the major recipient of the label when [aH]thymidine was used. The fact that 98 °/o of the radioactivity of the lipids of the soluble fraction, Table VII, also appeared in the aqueous phase after hydrolysis suggests that the glyceryl portion of the lipids was being labeled by thymidine since, as was noted above, thin-layer chromatography indicated that the soluble fraction lipids were neutral fats. The aqueous phase obtained after hydrolysis of the lipids was also examined chromatographically to determined whether the products corresponded to the lipid composition. As shown in Fig. i, 3H-labeled bands corresponding in migration rate to glycerylphosphoryl ethanolamine, glycerylphosphoryl choline and glycerol were obtained. These products agreed with the lipid composition reported in Table VI. 0®Q
QD
20C
® I
G2) C ~ 5O
,oo E
~
.~ 3oc
I! / I, II
I0(3
Z
o
4
8
12
~
2'o
cm
~2
-
1~
"
2'o
¢m
Fig. i. C h r o m a t o g r a p h y of t h e a q u e o u s p h a s e of t h e lipid h y d r o l y s a t e . A s a m p l e of t h e a q u e o u s p h a s e o b t a i n e d a f t e r h y d r o l y s i s of t h e lipids ( [ 6 - 3 H ] t h y m i d i n e precursor, Table vii) w a s c h r o m a t o g r a p h e d on a s h e e t of B a k e r f l e x D E A E - c e l l u l o s e w i t h i s o p r o p a n o l - a c e t i c a c i d - w a t e r as described in t h e t e x t . A f t e r d r y i n g , t h e s h e e t w a s c u t into I - c m - w i d e s t r i p s a n d a s s a y e d for radioa c t i v i t y . T h e position of t h e 14C-labeled reference c o m p o u n d s is i n d i c a t e d b y t h e r o u n d e d s p o t s at t h e t o p of t h e figure; S, g l y c e r o p h o s p h o r y l serine; E, g l y c e r o p h o s p h o r y l e t h a n o l a m i n e ; C, glyc e r o p h o s p h o r y l choline; G, glycerol. T h e s o l v e n t f r o n t w a s a t 16. 7 cm. Fig. 2. T h i n - l a y e r c h r o m a t o g r a p h y of lipids labeled w i t h [SH]deoxycytidine. A n a l i q u o t of t h e organic p h a s e a f t e r h y d r o l y s i s , T a b l e VII, w a s applied to a s h e e t of l:lakerflex silica gel a n d chrom a t o g r a p h e d w i t h benzene. A f t e r d r y i n g , t h e s h e e t w a s e x p o s e d to iodine v a p o r s to locate t h e lipid s p o t s a n d t h e n t h e s h e e t w a s c u t into i - c m s t r i p s a n d a s s a y e d for r a d i o a c t i v i t y . T h e s o l v e n t f r o n t w a s a t 16. 5 cm. T h e p o s i t i o n of t h e s p o t s revealed b y t h e iodine v a p o r s are i n d i c a t e d in solid b l a c k a t t h e t o p of t h e figure; t h e positions of [14C]palmitate (P) a n d t r i p a l m i t i n (T), as d e t e r m i n e d on a s e p a r a t e c h r o m a t o g r a m , are also s h o w n here.
Since most of the lipid radioactivity obtained after labeling with [aH]deoxycytidine appeared in the organic solvent phase after hydrolysis, this fraction was also examined by chromatography. As shown in Fig. 2, two bands of radioactivity were observed. One migrated at a rate just slightly greater than that of [14C]palmitate, suggesting that this component may be a fatty acid. When the unhydrolyzed lipids were chromatographed under the same conditions, two bands of radioactivity were also observed (not illustrated). One of these was at the origin of the chromatogram and contained 90 % of the label while the other contained i o % of the radioactivity Biochim. Biophys. Acta, 228 (i97 I) 6 1 o - 6 2 6
DEOXYRIBONUCLEOSIDE INCORPORATION
621
and migrated at the same rate as the [l*C]tripalmitin. It is apparent that the rapidly migrating component in the hydrolyzed sample was not present originally and did not correspond to either tripalmitin or palmitic acid; the nature of this component must remain unresolved.
Labeling o/proteins The results presented in Tables I - I I I indicated that the protein fraction of the nuclei, mitochondria and microsomes also appeared to be labeled after administration of the pyrimidine nucleosides. Since the microsomes contained the largest amount of radioactivity, they were selected for further study to determine whether the labeling of the proteins was real or an artifact. In one experiment, a portion of the protein residue of the microsomal fraction obtained after labeling with [Me-3H]thymi dine, was mixed with 25 °/zg of unlabeled thymidine and deoxycytidine and then hydrolyzed with formic acid as described b y WYATT19. When the hydrolyzate was chromatographed on paper to separate the bases, three bands of radioactivity were obtained, none of which corresponded to the added cytosine or thymine. This finding would appear to rule out nucleic acids or their pyrimidine bases as being directly associated with the protein fraction radioactivity. Another sample of the microsomal protein was then hydrolyzed with HC1 to obtain the constituent amino acids. The hydrolysate was fractionated chromatographically with an amino acid analyzer equipped with a stream splitting attachment. The results of this experiment are presented in Table V I I I and show that 5 amino acids were radioactive. Serine, aspartic acid and glutamic acid contained about the TABLE VIII INCORPORATION
OF R A D I O A C T I V I T Y
FROM
[Me-3H]THYMIDINE INTO THE AMINO ACIDS OF
MICRO-
SOMAL PROTEINS
The dried protein residue r e m a i n i n g after r e m o v a l of acid-soluble compounds, lipids and nucleic acids from mierosomes was hydrolyzed for 22 h at IiO ° with c o n s t a n t boiling HCI. After evaporation and drying, the sample was extracted w i t h s t a r t i n g buffer. A sample of the e x t r a c t was used for r o u t i n e analysis of the a m i n o acid c o n t e n t w i t h the B e c k m a n a m i n o acid analyzer. The rem a i n d e r of the sample was t h e n fractionated in t h e s a m e m a c h i n e using t h e s t r e a m splitting feature to send a p o r t i o n of the effluent to t h e analyzer and the r e m a i n d e r to a fraction collector. The collected fractions were assayed for radioactivity. The results r e p o r t e d represent the values obtained w i t h 21.6 m g of microsomal protein.
Amino acid
Content (t~moles)
Radioactivity (counts /min )
Aspartic acid Threonine Serine G l u t a m i c acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine leucine Tyrosine Phenylalanine
12.51 5.72 6.86 14.2o 3.78 8.34 9.oo o.54 6.12 1.84 4.27 13.65 2.94 4.75
198o o 211 o 22o5 o o 33 ° o o 237 o o o o
Biochim. Biophys. Acta, 228 (1971) 61o-626
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SCHNEIDER, A. E. GRECO
same amount of radioactivity but the specific activity of the former was the highest. Alanine and methionine were also labeled but to a much smaller extent than the other three amino acids. The basic amino acids were retained on the column in the experiment reported in Table V I I I and were eluted unresolved with dilute NaOH; they did not contain significant radioactivity.
Iododeoxyuridine Since this nucleoside analogue has been used instead of thymidine for labeling DNA ~°, it was of interest to determine if it would permit a more precise labeling of liver DNA. The data in Table I X show that this was not the case and that the pattern TABLE IX INCORPORATION OF IODODEOXYURIDINE INTO LIVER FRACTIONS E a c h of t w o C3 H mice w a s g i v e n IO/~C of [ ' 2 H ] i o d o d e o x y u r i d i n e (97 C / m m o l e ) a n d 24 h l a t e r t h e livers w e r e r e m o v e d a n d f r a c t i o n a t e d .
Liver ]faction
Nuclei Mitochondria Microsomes Supernatant
Total radioactivity (counts~rain) 2 3 15 198
311 564 483 700
Distribution o] radioactivity (counts~rain) fl cid soluble lO 7 678 688 193 ooo
Lipid
5° 822 5812 329 °
RNA
DNA
Protein
131 25o 1289
19o2 1135 1154
121 679 654 ° 237o*
* N u c l e i acids were n o t r e m o v e d in t h i s a n a l y s i s .
of labeling was very similar to that reported in Tables I - I I I for the other precursors. With E12Hliododeoxyuridine, the microsomes again contained the largest amount of particulate radioactivity and the lipids and proteins accounted for most of the microsomal label. These two fractions were selected for further study. It was found that 85-93 % of the label in the lipid extract was lost into the aqueous phase when it was treated with carbon tetrachloride and 2 M KC1 as described in EXPERIMENTAL. I t was concluded that the labeling of the lipid fraction was an artifact. In the case of the microsomal protein, hydrolysis and fractionation with the amino acid analyzer showed that the entire radioactivity appeared in a ninhydrin positive peak similar in position to that of cysteic acid. Since this amino acid is poorly adsorbed and is the first to emerge from the chromatographic column, it is possible that the radioactivity was recovered here only because it was not adsorbed b y the column and not because it was incorporated into cysteic acid. If this were true, it would mean that the proteins, like the lipids, were not actually labeled by the iododeoxyuridine. Nevertheless, it is apparent that iododeoxyuridine was no more specific a precursor of liver cytoplasmic DNA t h a n the other pyrimidine nucleosides tested. Only in the nuclear fraction was the DNA preferentially labeled. DISCUSSION
The findings reported here on the incorporation of thymidine and deoxycytidine are important for several reasons. In the first place, they show that in normal rodent Biochim. •iophys. Acta, 228 (1971) 61o-626
DEOXYRIBONUCLEOSIDE INCORPORATION
623
liver most of the deoxyribonucleoside incorporated does not appear in the expected receptor, DNA, but is recovered instead in lipids and proteins. Furthermore, the present results show that the labeling of the latter by thymidine was not an artifact since the radioactivity of the proteins and the lipids was shown to be restricted to certain amino acids present in the proteins and to definite glycerides present in the lipid mixture. Consequently the possibility that the labeling of these fractions was due to contamination by or incomplete extraction of DNA or nucleoside derivatives need no longer considered. The present results also lend additional insight into the events that are occurring within the liver cell with respect to thymidine incorporation. Acid-soluble precursor molecules rapidly accumulate in both mitochondria and microsomes, especially the former, and reach peak levels in 5 min after administration. Whether these precursors are associated with particulate material so that they may be phosphorylated prior to incorporation into DNA or while they are degraded is not indicated by these results. It should be noted however that the soluble or nonparticulate fraction of the liver contains even larger amounts of acid-soluble radioactive precursors and one might speculate that the compounds present in this pool differ from those found in the particulate acid-soluble fraction. In this connection, CHANG AND LOONEY21 reported that degradation products of thymidine reached peak levels in 2 min in the liver acid-soluble fraction while thymidine nucleotides did not reach maximum levels until IO min. The labeling of the lipids and proteins occurred extremely rapidly especially in the microsomes. Large amounts of radioactivity were present after only 5 min of incorporation and reached the highest level after 30 min. The decline in microsomal radioactivity which was noted at 2 4 h with both [3Hlthymidine and [14C]thymidine may indicate either that these proteins and lipids have a rapid turnover or that they are synthesized in the microsomes prior to transfer to other parts of the cell. The latter explanation would agree with experiments which have demonstrated that the synthesis of proteins and phospholipids occurs in microsomes z2-~ and also with the data indicating that the labeling of the nuclear and mitochondrial lipids and proteins occurs more slowly and progressively (Tables II and III). Thymidine is known to be degraded in the liver by a pathway involving thymine, dihydrothymine and fl-aminoisobutyric acid 25. The latter can be excreted by the animal 28 or further metabolized via the tricarboxylic acid cycle to a wide variety of products. The labeling of the liver lipids and proteins after the administration of thymidine can apparently by explained by the operation of these pathways. This was shown by the dilution of incorporation that occurred when dihydrothymine and fl-aminoisobutyric acid were given prior to the isotope. Also, according to CHANG AND LOONEY 21, fl-aminoisobutyric acid and related degradation products accounted for 39-58 % of the acid-soluble radioactivity in the liver at periods of 2 rain- 5 h after the injection of thymidine. Similarly POTTER3 tentatively identified labeled succinic, malic, glutamic and aspartic acids in the acid-soluble fraction of rat liver after the administration of thymidine. The amino acids that were labeled in the microsomal proteins could be obtained directly from such labeled pools or readily derived by related pathways. The labeling of the lipids probably also occurs by the operation of such pathways since either the glycerol or the fatty acid portions of the lipid molecules were the main recipients of radioactivity. Biochim. Biophys. Acta, 228 (1971) 61o-626
624
w.c.
SCHNEIDER, A. E. GRECO
One disquieting feature in the incorporation of thymidine into lipids, proteins and DNA was the enormous concentrations of metabolites required relative to the labeled thymidine given (20 000:I) to achieve dilution and the failure of unlabeled thymidine to dilute incorporation into lipid and protein to the same extent as into DNA. The former can be explained b y the fact that the tracer dose of thymidine (about 2.5 nmoles) was far below that required to saturate the DNA synthetic pathway (about 1.0 #mole for a I80-g rat~7). Therefore a large fraction of the unlabeled thymidine that was given was apparently required to achieve saturation of the incorporation system and could not lead to dilution of labeling. The fact that the labeling of the lipids and proteins was diluted to a lesser extent than was incorporation into DNA indicates that the catabolic pathways require an even higher level of thymidine for saturation than does the p a t h w a y leading to DNA. The tremendous capacity of the liver for the degradation of thymidine is well known 21. Although the results of the present experiments showed that thymidine was not very specific as a precursor of DNA in the liver, how seriously does this affect the use of thymidine as a precursor? In the case of biochemical experiments, it simply means that DNA can only be studied after rigorous purification, and imposes limitations on the type of information that can be obtained. If the labeling of DNA were completely specific, it would be possible to calculate the total DNA content of mitochondria and microsomes from measurements of their radioactivity. This would be valuable information to have but obviously cannot be obtained under the circumstances. From the standpoint of autoradiography it is obvious that great care must also be exercised in establishing that the labeled material is actually DNA ~. Here too sorely needed experiments appear unfeasible because of the lack of labeling specificity. For example, autoradiography could have provided a simple means of establishing whether the DNA which we isolate from liver microsomes 7 was associated with the microsomes in the intact liver cell. It can be argued that adult normal rodent liver should not be used in studies of DNA synthesis because of the extremely low rate of cell division that occurs. Other tissues such as spleen and thymus and various tumors have much higher rates of mitosis and would seem to be more suitable subjects of study. However, it has been found that thymidine is also incorporated into the proteins and lipids of these tissues s,15, although to a lesser extent relative to DNA than was the case in liver. Nonetheless these results show that in no case was thymidine completely specific as a precursor of DNA. It seemed possible that the specificity of thymidine might be increased if the label were localized in some other part of the molecule. The results with L2-14C]thymidine were favorable in this regard since a greater proportion of the label was recovered in the DNA relative to that present in lipids and proteins with this precursor. In mitochondria and microsomes, however, large amounts of radioactivity were still recovered in the lipids and the proteins. The results with E125Iliododeoxyuridine were no more encouraging since they showed that proteins and lipids were also labeled, especially in the microsomes (Table IX). The labeling of proteins by this precursor had been noted earlier 20 and in a very recent paper 2s the labeling of lipids was also noted. Our results indicate that the labeling of the lipids and proteins by iododeoxyuridine was not a true labeling since the label was readily lost from the lipids and m a y or m a y not be associated with a definite amino acid component of the Biochim. Biophys. Acta, 228 1197 I) 61o-626
DEOXYRIBONUCLEOSIDE INCORPORATION
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proteins. Nevertheless the labeling of these fractions does occur and poses the same problems as were encountered with thymidine and deoxycytidine. It was also hoped that the administration of catabolites to the animals prior to the labeled thymidine would reduce the incorporation into proteins and lipids to such an extent that the specificity for labeling D N A would be satisfactory. The results with E~Hlthymidine showed that this was not the case. However, since similar experiments with [2-14Clthymidine and with deoxycytidine and iododeoxyuridine were not attempted, it is possible that a solution to this problem could be devised. The fact that the R N A fraction of the liver was in some cases more heavily labeled than the D N A fraction (e.g. the microsomal R N A fraction, Table II) rsises the question of the nature of the labeling of these fractions. Our previous experiments 7, in which nuclear, mitochondrial and microsomal D N A were extensively purified, showed that the D N A was being labeled by [3Hlthymidine. Whether R N A is also being labeled cannot be determined from our experiments since no attempt was made to purify the R N A or to determine the nature of the labeled products in the R N A fraction. In this connection, however, it should be noted that the formation of thymine riboside by liver extracts has been reported 3 and that the labeling of R N A in another system s by [2-14Clthymidine has been established to occur as conversion to uridylate. It would therefore appear to be entirely possible that the labeling of the R N A fraction could involve either the direct incorporation of the thymine into R N A or the incorporation of thymidine breakdown products into RNA. Further experiments would be required to establish this point.
ACKNOWLEDGMENT
The authors are indebted to Mr. James F. Williams for the amino acid analyses.
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626 21 22 23 24 25 26 27 28 29
W. C. SCHNEIDER, A. E. GRECO L. O. CHANG AND V~. B. LOONEY, Cancer Res., 25 (1965) 1817. P. SIEKEVITZ, J. Biol. Chem., 195 (1952) 549. G. F. VqlLGRAM AND E. P. KENNEDY, J. Biol. Chem., 238 (1963) 2615. W. C. SCHNEIDER, .[. Biol. Chem., 238 (1963) 3572. K. FINK, R. E. CLINE, R. B. HENDEnSON AND R. 1V~.FINK, J. Biol. Chem., 221 (1956) 425 . K. FINK, R. 13. HENDERSON AND R. M. FINK, r . Biol. Chem., 197 (1952) 441. O. F. NYGAARD AND P~. L. POTTER, Radiation Res., IO (1959) 462. C. P. FUSSELL, J. Cell Biol., 39 (1968) 264. L. A. DETHLEFSEN, Cancer Res., 29 (1969) 1717.
Bioehim. Biophys. Acta, 228 (1971) 61o-026