THE BIOSYNTHESIS OF NUCLEIC ACID COMPONENTS STUDIED WITH Cl4

I. PURINES AND PYRIMIDINES IN THE RAT*

BY MILTON R. HEINRICHt AND D. WRIGHT WILSON

(From the Department of Physiological Chemistry, School of Medicine, University of Pennsylvania, Philadelphia)

(Received for publication, March 6, 1959)

Studies of the metabolism of purines and pyrimidines have been made in a variety of ways. Man, rats, pigeons, yeast, Neurospora, and bacteria have been used as experimental organisms. Edson, Krebs, and Model (2) have shown that ammonia and lactate stimulate the production of hypoxanthine by pigeon liver slices. Ammonia labeled with N15 was shown by Barnes and Schoenheimer (3) to be utilized in viva in the forma- tion of tissue purines and pyrimidines.

In studies on uric acid excreted by pigeons, it was found by Buchanan et al. (4-7) that carbon 4 of uric acid is derived from the carboxyl carbon of glycine, carbon 6 from carbon dioxide, carbons 2 and 8 from formic acid, and carbons 4 and 5 from the carboxyl and a-carbons of lactate. In these experiments the possible sources of each carbon atom of the uric acid molecule were demonstrated. These results were confirmed by Karlsson and Barker (8).

The amino group of glycine was found to contribute nitrogen 7 in uric acid of man (9), as well as in the nucleic acid purines of yeast (10) and of the rat (11). Greenberg (12) has demonstrated the incorporation of formate and carbon dioxide into hypoxanthine in pigeon liver homogenates.

The metabolism of birds is different from that of mammals in that birds form uric acid as the chief end-product of nitrogenous metabolism, while mammals form urea. In mammals the excretory product of the purines, allantoin in most animals and uric acid in man, constitutes a very small part of the total nitrogen excretion. The differences between the relative extent of purine metabolism in birds and mammals suggest the possibil-

* Aided by a grant from the American Cancer Society administered by the Com- mittee on Growth of the National Research Council and a grant from the Com- mittee on the Advancement of Research of the University of Pennsylvania. The carbon 14 used in this investigation was supplied by the Isotopes Division, United States Atomic Energy Commission. Preliminary reports of portions of this work have appeared (1).

t Postdoctorate Research Fellow, National Institutes of Health, 194749. Pres- ent address, Amherst College, Amherst, Massachusetts.

447

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448 BIOSYNTHESIS OF NUCLEIC ACID% I

ity that the paths of metabolism and the precursors for purines may be dif- ferent .in these different animals.

Since the work to be discussed below was first described (l), Marsh (13) has reported that formate is a precursor of nucleic acid in both rats and pigeons, and MacLeod and Lardy (14) have shown that the incorpora- tion of CO2 into purines of rats is stimulated by biotin.

The biological synthesis of pyrimidines has been studied very little, owing in part to the difhculty of their isolation. Ammonia (3) and the nitrogen of glycine (15) and erotic acid (16) are reported to be incorporated into uracil; preformed uracil nitrogen is not (17). Other small molecules have been suggested by Mitchell and Houlahan (18) to be concerned with pyrimidine synthesis on the basis of experiments with Neurospora.

The present investigations concern the carbon precursors of tissue purines and pyrimidines in mammals, studied by administering to rats compounds labeled with radioactive carbon. The compounds tested as precursors were carbon dioxide, carboxyl-labeled acetate, carboxyl-labeled glycine, doubly labeled glycine, and formate. Guanine and adenine were isolated from tissue nucleic acids, and another fraction of adenine was obtained from purine derivatives soluble in cold trichloroacetic acid. Pyrimidines were isolated from the nucleic acids. Guanine and uracil were degraded to determine the location of some of the Cl4 in the molecules.

Methods

Animals-Growing rats of the Wistar strain were kept in wire metab- olism cages and fed a ground stock diet during the experimental periods. Labeled compounds were mixed with the food or injected. Urine from all rats in each experiment was collected under toluene and pooled daily. The radioactivity of excreted urea was determined by removing free CO2 from an aliquot of urine by aeration, then digesting with urease, and col- lecting the COZ in barium hydroxide. Respiratory COZ was collected by placing a rat in a glass container fitted with inlet and outlet tubes. After sweeping with COS-free air for 10 to 15 minutes, a sample was collected by bubbling the air through dilute COz-free NaOH, and precipitated with barium chloride.

Synthesis of Labeled Compounds-Sodium bicarbonate was made from barium carbonate containing C14. Carboxyl-labeled acetate was made by the Grignard reaction, and carboxyl-labeled glycine from ethyl bromo- acetate and potassium phthalimide, according to the modified procedure of Sakami, Evans, and Gurin (19). Doubly labeled glycine was synthesized from CYJ-barium carbide by way of acetylene and acetaldehyde, which was oxidized to acetic acid with silver oxide and converted to glycine as described above (19). Formate was obtained by hydrolysis of hydrogen cyanide by the method of Krieble and McNally (20).

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M. R. HEINRICH AND D. W. WILSON 449

Isolation of Compounds-Two fractions were isolated from the rat tis- sues : (a) “nucleotides,” extracted with cold trichloroacetic acid, which yielded adenine after hydrolysis, and (b) mixed nucleic acids, extracted with sodium chloride, precipitated, and hydrolyzed to free the purines and pyrimidines.

Rats were killed by a blow on the head. The skin, feet, head, spine, and tail were rapidly removed and discarded; the gastrointestinal tract was removed, slit lengthwise, washed carefully, and treated with the rest of the carcass. Each carcass was passed through a meat grinder and then treated for 5 minutes in a Waring blendor with 250 ml. of ice-cold 5 per cent trichloroacetic acid. The tissue residue was filtered, washed twice with cold trichloroacetic acid, and treated with lipide solvents as described below. The above operations were carried out in a cold room at 5’.

After adding l/90 volume of concentrated H80a, the trichloroacetic acid extract was boiled for 2 hours to hydrolyze the nucleotides and destroy the trichloroacetic acid. The solution was neutralized with 1: 1 NaOH after partial cooling, and the calcium phosphate filtered and washed. Free purine was precipitated by the method of Kruger and Schmid with copper sulfate and sodium bisulfite (see Hitching5 (21)). After removal of the copper with hydrogen sulfide in acid solution, the purine was reprecipitated with copper as before. The filtrate from a second treatment with hydro- gen sulfide was concentrated to a few ml. by distillation in vacua. When the solution was decolorized with charcoal and chilled, adenine hydro- chloride crystallized. A second crop was obtained by concentrating the supernatant and adding about 2 volumes of alcohol. Both portions were combined and recrystallized until pure from 3 per cent hydrochloric acid. As pointed out by Kerr (22), this adenine is derived almost entirely from free and phosphorylated adenylic acid, other adenine nucleotides being present in very small amounts.

Lipides were removed from the trichloroacetic acid-extracted tissue resi- due by several extractions with boiling 3 : 1 alcohol-ether and ether.

Nucleic acids were extracted from the dry, lipide-free tissue in a 24 hour extraction with hot 10 per cent sodium chloride solution, as described by Barnes and Schoenheimer (3). The nucleates were precipitated with ethanol and washed with ethanol and ether. It was found that stickiness of the precipitate could be avoided by keeping the solution cold during centrifugation and omitting washing with dilute alcohol. The sodium nucleates were white and powdery, and gave a weak biuret test. Yields ranged from 3.9 to 5.1 per cent of the dry tissue weight. These nucleates were a mixture of pentose and desoxypentose types and were hydrolyzed without further purification.

The procedure for isolation of purines and pyrimidines was essentially that of Plentl and Schoenheimer (17). After hydrolysis of the nucleic acids

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450 BIOSYNTHESIS OF NUCLEIC ACIDS. I

with hydrogen chloride in methanol, guanine was isolated as the sulfate, and adenine as the picrate. Pyrimidines were isolated after hydrolysis of the nucleotides in 20 per cent HCl at 180”. No appreciable amount of cytosine was isolated because of its conversion to uracil. A partial separa- tion of the uracil-thymine mixture was accomplished by repeated fractional crystallization. The presence of thymine was confirmed by a strongly positive Woodhouse (23) diazo test on the pyrimidine solution of each ex- periment.

Degradation of Guanine-Two procedures were used: (CL) hydrolysis with hydrochloric acid to glycine, in which the carboxyl and methylene carbons are derived from carbons 4 and 5, respectively, of guanine (24, 25), and (b) permanganate oxidation to carbon dioxide, urea, and guanidine.

Procedure A-Hydrolysis of about 100 mg. of guanine sulfate was carried out with concentrated HCI at 200”, according to the procedure of Wulff (26) as modified by Abrams, Hammarsten, and Shemin (10). In order to avoid dilution by non-radioactive carbon, the glycine was isolated as the copper salt rather than as an organic derivative. After hydrolysis the chloride and sulfate were removed, an excess of cupric hydroxide added, and the mixture heated in a boiling water bath for 10 to 15 minutes. The supernatant and washings were concentrated to a small volume, chilled, and absolute ethanol added to precipitate the copper glycine. The salt was recrystallized several times from small volumes of water. The crystals were blue needles containing 1 mole of water of crystallization.

The copper glycine was dissolved in approximately 10 ml. of water, acidified, and treated with hydrogen sulfide. One portion of the filtrate from copper sulfide was evaporated to dryness and oxidized to CO2 with the Van Slyke-Folch solution. The radioactivity of this fraction represents the average concentration of isotope from carbons 4 and 5 of the original guanine. Another portion of the glycine filtrate from copper sulfide was aerated to remove HZS, and decarboxylated with ninhydrin. The radio- activity of this BaC03 is due to carbon 4 of the original guanine.

Procedure B-Permanganate oxidation of guanine under the conditions described below gives rise to guanidine containing carbon 2, urea (mainly carbon S), and carbon dioxide formed principally from carbons 4, 5, and 6. In a somewhat similar oxidation, Strecker (27) obtained guanidine, urea, oxaluric acid, and parabanic acid after treating guanine in HCl with KClOs.

Guanine sulfate (30 to 60 mg.) was dissolved in a few ml. of COZ-free water in a small flask fitted with a dropping funnel, an inlet tube extending to the bottom, and an outlet tube. 0.4 N (3 e) potassium permanganate was added to the flask in small portions as long as it was decolorized. The solution in the flask was aerated and the pH kept between 1 and 2. The

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M. R. HEINRICH AND D. W. WILSON 451

flask was first kept in cracked ice until completion of the slow reaction, then briefly at 100” with the addition of more permanganate and acid. The liberated CO% was collected but will not be reported (except in Table II), since it does not represent specific carbon atoms. After aeration was stopped, the solution was decolorized with a drop or two of dilute hydrogen peroxide, neutralized to pH 5, and the manganese dioxide removed by filtration and washed with hot water. Urea present in the filtrate was digested with urease in the presence of acetate buffer at pH 5. The COZ of this fraction was mainly but not entirely derived from carbon 8 of guanine.

Solid trichloroacetic acid was added to the solution containing urease and the protein filtered. An excess of silver nitrate was added, and silver guanidine precipitated by making alkaline with saturated barium hydrox- ide. The precipitate was centrifuged, washed, and decomposed with hy- drogen sulfide. The filtrate was concentrated to a small volume in vacua, and guanidine precipitated as the picrate. It was recrystallized until pure, as shown by the melting point. For low activity samples the picric acid was extracted with toluene and ether, and the colorless solution evap- orated to dryness before oxidizing to COZ.

The yields of urea by this procedure were 30 to 40 per cent of a mole per mole of guanine. Probably similar amounts of guanidine were formed because, in one experiment, 14 per cent of a molar equivalent was obtained as pure recrystallized guanidine picrate. Unless some rearrangement has occurred, this compound will contain only carbon 2 of guanine. Only small amounts of free urea and guanidine are oxidized by heating for 1

hour in a boiling water bath at pH 1 with an excess of permanganate (8 per cent for urea and 5 per cent for guanidine, with no guanidine converted to urea).

Degradation of Urucil-Uracil has often been oxidized to oxaluric acid (28-30). We have oxidized uracil with permanganate to carbon dioxide and oxaluric acid. The latter compound was hydrolyzed with alkali to urea and oxalic acid. The three products were obtained in yields of 90 per cent or higher. Oxaluric acid was isolated from a larger scale oxidation and identified as the anhydrous potassium salt (m.p. 224-225”, 16.4 per cent N), free acid (m.p. 205” with decomposition), and ethyl ester (m.p. 174” with decomposition).

The mechanism of this oxidation of uracil is not definitely known; there- fore at present it is impossible to decide which carbon of uracil gives rise to COZ in the degradation. For this reason we shall report the average radioactivity of the three carbons, 4, 5, and 6.

20 to 30 mg. of uracil were dissolved in a few ml. of COz-free water in the aerating flask fitted with a dropping funnel. After sweeping with COz-free

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452 BIOSYNTHEBIS OF NUCLEIC ACIDS. I

air, a tube of saturated barium hydroxide was connected to collect the COZ liberated in the first step. Portions of 0.5 or 1 ml. of 0.4 N (3 e) potas- sium permanganate and a few drops of 0.5 N sulfuric acid were added at room temperature, the pH being kept at 6, as long as the permanganate was decolorized. 25 mg. of uracil required about 5 ml. of permanganate and 0.5 ml. of acid. After removing the barium hydroxide tube, any excess of permanganate was decolorized with a drop or two of dilute hydrogen peroxide, and the manganese dioxide filtered and washed with hot water. At this stage there was no free urea or oxalate in the solution. The solu- tion was made strongly alkaline with 5 drops of 10 per cent sodium hy- droxide and heated for 5 minutes in a boiling water bath. The tube was removed from the bath and neutralized to pH 6 to 6.5 with a few drops of 5 per cent acetic acid. 10 per cent calcium chloride was added dropwise until precipitation of the oxalate was complete, and the solution was neu- tralized to pH 7.5 with dilute ammonia. The calcium oxalate was cen- trifuged and washed thoroughly, then dissolved in 2 N sulfuric acid, and oxidized to CO* with permanganate. The supernatant and washings from the calcium oxalate contained urea. This was digested with urease in the usual wzagr, and the COZ derived from carbon 2 of uracil was collected. The slight excess of calcium in this solution did not inactivate urease.

Thymine was a contaminant of the uracil isolated from animal tissues in these experiments. It was found that thymine, when oxidized under these conditions (kept at pH 6), gave about 40 per cent of the theoretical COZ for 1 equivalent of carbon. About the same fraction of urea was liberated after alkaline hydrolysis, but no oxalate was found.

The purity of each compound was determined by micro-Kjeldahl analysis. This is believed to be adequate, especially when combined with recrystal- lization to constant radioactivity.

Calculated nitrogen contents areas follows: guaninesulfate, (CaHr,NtO)a*- HzS04, 35.0 per cent; adenine hydrochloride, CsHsNa.HCl, 40.8 per cent; adenine picrate, C6HbN6,C8H3N303, 30.8 per cent; uracil, C~H~NZO~, 25.0 per cent; thymine, CaHeNzOz, 22.2 per cent.

Radioactivity measurements on samples were made after combustion by the wet oxidation mixture of Van Slyke and Folch (31),-and collection of the CO2 in saturated barium hydroxide solution. The barium carbonate was centrifuged, washed until free from alkali, and dried. An acetone slurry of the barium carbonate was evaporated slowly to form a uniform layer on metal disks 1 inch in diameter. Counting was done with a thin mica end window Geiger tube.

Observed counts were corrected by factors between 1.0 and 2.0 to a stand- ard plate of “infinite thickness” (maximum self-absorption). With our equipment this is a plate containing 90 mg. of barium carbonate, or 5.47

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M. R. HEINRICH AND D. W. WlLBON 453

mg. of carbon. All counts were then converted to counts per minute per mM of carbon. Samples were counted to a standard error of 5 per cent or less, except when the calculated values were low (viz. 10,20). These latter values are given in Tables I through IV because it is believed that they rep- resent traces of radioactivity, although the values may have a standard error of from 10 to 25 per cent. In each case in which sufficient compound was available, it was recrystallized to constant radioactivity. The major- ity of purine samples was checked in this way.

TABLE I

Administration of Labeled Bicarbonate

Sodium bicarbonate (350,000 c.p.m. per mM of carbon) in solution, 13 mg. per ml. or 54,000 counts per ml. Rat A received six injections, 1 ml. per hour; Rat B, six injections, 2 ml. per hour; Rat C, eleven injections, 1 ml. per hour; Rat D, four injections, 3 ml. per 3 hours. Total isotope 2.2 X lOa c.p.m. Tissues combined after nucleotide extraction.

Nucleotide adenine, Rat A I‘ “ ‘( B U “ “ c “ “ “ D

Nucleic acid Adenine Guanine

C-2 (guanidine) C-3 (urea) C-4, C-5 (glycine) C-0 (calculated)

Uracil C-2 (urea) c-4, c-5, C-6

C.p.m. C.p.m.

!iib:: ttib?

60 150 125 135

Urinary urea Respiratory CO%

Rat A. 5-15 min. after injection I‘ “

5,400

130 170 10 0

35 770

15-30 “ “ I‘ ‘I u 3045 C‘ I‘ “ “ u 45-60 “ #I ‘I “ B* 5-15 u H ‘, 1‘ I‘ 1530 “ “ ‘4 I, “ 30-45 ‘4 “ 4‘ “ I‘ 45-60 “ ‘C I‘

19,000 14,300 9,800 7,900

27,000 21,000 19,000 13,600

650 45

Results

Labeled Carbon Dioxde-Four male rats, average starting weight 130 gm., were given intraperitoneal injections of isotonic sodium bicarbonate solution, as shown in Table I. Rats A, B, and C were sacrificed an hour after the last injections. Rat D was sacrificed 2 hours after the fourth in- jection.

Respiratory carbon dioxide was collected separately from Rats A and B in four portions during the 55 minutes preceding sacrifice. There was high radioactivity in the expired air. In the rat receiving injections of 1 ml. of bicarbonate, 27 per cent of the CP in one injection was exhaled in

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454 BIOSYNTHESIS OF NUCLEIC ACIDS. I

the 55 minute period. The rat receiving 2 ml. per injection exhaled 23 per cent in the same time.

The adenines of the nucleotide fractions were isolated as described and oxidized to CO2 for isotope assay without analyses. The specific activities indicated that incorporation of labeled COz varied with the total amount given, rather than with the size or the times of individual injections.

The tissues of all the rats were combined after nucleotide extraction. Bases isolated from the nucleic acids were recrystallized until the following analyses were obtained: guanine sulfate 34.8 per cent N, adenine picrate 30.7 per cent N, uracil 24.7 per cent N.

The two degradations of guanine showed most of the isotope to be in carbon 6. Of particular interest is the finding of a high concentration of

TABLE II

Administration of Carboxyl-Labeled Acetate

Sodium acetate (26,000 c.p.m. per mM of carbon) given to five rats by subcuta- neous injection twice daily; 40 mg. per rat per day for 5 days. Total isotope 6.3 X lo6 c.p.m.

Nucleotide adenine Nucleic acid

Adenine Guanine

co2 C-2 (guanidine) C-8 (urea)

Uracil C-2 (urea) c-4, c-5, C-6

10

15 0 0

65 15

Urinary urea 2nd day 3rd “ 4th “ 5th I‘

Respiratory CO2 1.5 hrs. after first injection 1.5 “ “ last “ Before sacrificing

C.p.m. per UIM carbon

130 210 200 160

320 75

0

Cl4 in carbon 2 of uracil. The specific activity of this carbon of uracil was similar to that calculated for carbon 6 of guanine. They both were much lower than the specific activity of the carbon of the mixed urinary urea. The bicarbonate carbon as indicated by the expired CO2 had a still higher specific activity.

CarboxyE-Labeled Acetate (Table II)-Five rats, average starting weight 186 gm., were fed a powdered stock diet for 5 days while receiving sub- cutaneous injections of 20 mg. of labeled sodium acetate in 1 ml. of solu- tion twice daily. The animals were sacrificed on the morning of the 6th day, about 16 hours after the last injection; average weight, 191 gm.

Respiratory CO2 was collected from one rat at various times as shown in Table II.

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M. R. HEINRICH AND D. W. WILSON 455

Analyses of the compounds isolated are as follows: nucleotide adenine picrate 30.5 per cent N, nucleic acid adenine picrate 30.0 per cent N, guanine sulfate 34.5 per cent N, uracil 24.6 per cent N. Traces of radio- activity appeared in the whole purines but guanidine and urea from guanine showed no radioactivity.

Radioactivity was found in carbon 2 of uracil, probably owing to the in- corporation of CO*. The radioactivity of the urea from the daily pooled urine was considerably higher than that of the carbon of the ureide group of uracil.

TABLE III

Administration of Carboxyl-Labeled Glycine

Glycine (10,700 c.p.m. per mM of carbon) fed to nine rats, 50 mg. per rat per day for 10 days. Total isotope 1.3 X lo6 c.p.m.

Nucleotide adenine . , . . . . . . . . . . . . . . . . . . Nucleic acid

Adenine.................................................. Guanine . . . . _ . . . . . . . . . . . . . . . . .

C-2 (guanidine) . . . . . . . . . . . . . . C-8 (urea) . . . . . . . . . . . . . . C-4, C-5 (glycine) . . . . . . . . . C-4 (glycine COOH) . . . . . . . . . . .

Uracil C-2 (urea). . . . . . . . C-4, C-5, C-6.. . . . .

Urinary urea.. . . . . . . . . . Feces................... Respiratory CO*. . . .

. . . . . . .

. . . .

. . . .

. . . .

. . . .

. . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . .

. . .

. ,

C.p.m. per InM carbon

300

270 300

10

10

530 1220

10

0

x-20 10 10

Carboxyl-Labeled Glycine (Tabb III)-Nine rats, average starting weight 99 gm., were fed labeled glycine. The animals were sacrificed 12 hours after the last feeding, when the average weight was 128 gm.

The two samples of respiratory COS had only a trace of radioactivity, as did the urea. The same was true for the ureide carbon of uracil. Not more than 1 per cent of the isotope was lost by way of the feces. There appears to be a high efliciency in metabolic utilization of the glycine when fed.

The tissues of all animals were pooled, and the purines and pyrimidines isolated by the usual methods. The analyses are as follows: nucleotide adenine picrate 30.6 per cent N, nucleic acid guanine sulfate 34.6 per cent N, adenine picrate 30.8 per cent N, uracil24.8 per cent N.

Good incorporation of Cl4 into all the purines is evident. Glycine de- rived from guanine by treatment with HCl was found to have all of its

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456 BIOSYNTHESIS OF NTJCLEIC ACIDS. I

Cl4 in the carboxyl carbon. The data show that most of the isotope is in carbon 4 of guanine.

Doubly Labeled Glycine (Table IV)-Five male rats, average starting weight 110 gm., were fed the stock diet containing glycine labeled in both carbons. The animals were sacrificed on the morning of the 6th day, the average weight at this time being 123 gm.

Respiratory COZ was collected from one rat for 15 minute periods during the lst, 2nd, and 5th days, and at 2 hour intervals for 24 hours during the 4th day. Little or no activity was found in these samples or in urinary urea.

Analyses of the compounds are as follows: nucleotide adenine hydro- chloride 40.8 per cent N, nucleic acid guanine sulfate 34.9 per cent N,

TABLE IV

Administration of Doubly Labeled Glycine

Glycine (7900 c.p.m. per mM of carbon) fed to five rats, 18 mg. per rat per day for 5 days. Total isotope 9.5 X lo4 c.p.m.

Nucleotide adenine ............. Nucleic acid

Adenine ...................... Guanine .....................

C-2 (guanidine). ........... C-S (urea). ................ C-4, C-5 (glycine). ......... C-4 (glycine COOH). ......

Uracil ....................... Thymine. ....................

Urinary urea .................. Respiratory COe. ..............

. . . . . . . .

. . . . . . . .

........

........

........

........

........

........

........ ........ ........

..,............

............... ............... ............... ...............

............... ............... ............... ...............

C.p.m. per maa carbon

70

65 75

0 20

115 135

10 0 O-10 O-10

adenine picrate 30.8 per cent N, uracil24.7 per cent N. Thymine was not isolated in large enough quantity to analyze chemically but was found to have no radioactivity.

The carbons of glycine were incorporated into the purines of nucleic acids and of the nucleotides in the rat. These purines had approximately the same Cl4 content, with nucleic acid guanine slightly higher than the others. The analyses of the degradation products of guanine indicated that the carbons of glycine were incorporated into positions 4 and 5 of the purine. It is probable that the specific activity of the glycine administered was too low to demonstrate the incorporation of the a-carbon into positions 2 and 8, as shown by Karlsson and Barker (8) in the pigeon.

Uracil contained practically no radioactivity. This is not in agreement

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hf. FL HEINRICH AND D. W. WILSON 457

with Bergstrand et al. (15) who concluded that glycine was incorporated into the uracil ring when glycine labeled with NX6 was fed.’

Labeled Formate (Table V)-Five male rats, average starting weight 118 gm., were fed the stock diet including labeled sodium formate. The diet with formate was put in the cages each afternoon, and the animals were sacrificed on the morning of the 5th day.

A sample of respiratory COz on the 4th afternoon had an activity of 50 c.p.m. per mM of carbon, and a sample before sacrificing on the 5th morning showed an activity of 230 counts. Urea in the urine of the last day con- tained 360 c.p.m. per mrvr of carbon. Formate appears not to enter the urea molecule in rats, except as it is oxidized to form CO*,

TABLE V

Administration of Labeled Formate

Sodium formate (173,000 c.p.m. per mM of carbon) fed to five rats, 50 mg. per rat per day for 4 days. Total isotope 2.5 X LOB c.p.m.

Nucleotide adenine Nucleic acid

Adenine Guanine

C-2 (guanidine) C-3 (urea) C-4, C-5 (glycine)

Uracil C-2 (urea) c-4, c-5, c-6

Thymine

4,490

4,550 5,760

16,400 12,500

0

190 60 60

-

-- Urinary urea, last day Feces Respiratory CO*

Before last feeding After “ ‘(

360 140

50 230

Analyses of the compounds isolat,ed are as follows: nucleotide adenine hydrochloride 40.5 per cent N, nucleic acid guanine sulfate 35.0 per cent N, adenine hydrochloride 40.8 per cent N. These three compounds were recrystallized several times after the above analyses were obtained, to check for constant radioactivity; uracil 24.8 per cent N, thymine 23.2 per cent N,

Formate is ari excellent precursor of purines in the rat. It contributes largely to carbons 2 and 8, as shown by the permanganate degradation. Carbons 4 and 5, obtained as glycine in the HCl degradation, had no radioactivity.

Formate is apparently not a direct precursor of pyrimidines, since uracil

* A private communication from Professor Hammarsten states that he has already found that the a-carbon of glycine is not incorporated into the pyrimidines.

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458 BIOSYNTHESIS OF NUCLEIC ACIDS. I

and thymine were very slightly active as compared with the purines. The highest concentration of isotope in uracil was in the ureide carbon, probably owing to incorporation of COS. The specific activity of thymine was less than that of uracil. This is in agreement with the suggestion that the desoxyribonucleic acids turn over less readily than the ribonu- cleic acids (32).

DISCUSSION

Our experiments demonstrate the incorporation of the carbon of COz, glycine, and formic acid into the purines of the nucleic acids and the adenine of the tissue nucleotides of rats. COz contributes carbon mainly to position 6 and not to positions 2 and 8. The carboxyl and the a-carbons of glycine may enter, respectively, positions 4 and 5. Formic acid contributes carbon to positions 2 and 8. These results are in agree- ment with the observations of Buchanan et al. (6, 7), made on the uric acid excreted by pigeons. We have failed to find the incorporation of the carboxyl group of acetate in carbons 2 and 8 of the purine structure.2 The total amount of Cl4 administered as acetate by us was small for such an active metabolite, even though it was given in amounts 6 times those used in the experiment with doubly labeled glycine.

Although metabolic “dilution” figures do not have great significance in experiments carried out under such widely varying conditions, they are given here for comparison. Ratios of the specific activity of the compound as administered to that of isolated guanine are as follows: bicarbonate 2070, carboxyl-labeled glycine 37, doubly labeled glycine 109, formate 29.

The ratios of the specific radioactivities of guanine and adenine are of considerable interest. The finding of Brown et al. (33) that labeled die- tary adenine is incorporated into nucleic acids as adenine, and converted to guanine with about 40 per cent less isotope, has been interpreted to in- dicate that adenine is a precursor of guanine. In the present studies,

‘and in the work of others (3, 10, 11, 14, 15), the use of labeled precursors having small molecules usually leads to a similar but somewhat greater in- corporation of the isotope in guanine than in adenine. In our experiments, the ratios of specific radioactivities of guanine and adenine of the nucleic acids ranged from 1.09 to 1.33. This suggests that adenine is not the pre- cursor of guanine in these experiments.

Little is known concerning the metabolism of the pyrimidines. In their catabolism in mammals, the nitrogen is converted into urea (34, 17), while the purines, with somewhat similar structure, are excreted mainly as al-

* In a nersonal communication, Buchanan has stated that he has obtained results with pigeons similar to ours with rats. The source of error of the earlier experiments has been shown to have been due to the presence of labeled formate in the synthetic labeled acetate.

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M. R. HEINRICR AND D. W. WILSON 459

lantoin or uric acid. The experiment with labeled sodium bicarbonate proves that the carbon of COZ becomes the ureide carbon of uracil. The extent of incorporation of radioactive carbon appears to be about the same in position 2 of uracil as in position 6 of guanine. In both instances the incorporation of Cl* depends upon the concentration of C1*02 in the body. This utilization of CO2 in the biological synthesis of uracil constitutes another example of the fixation of COZ in compounds important in metab- olism in the body. The carbon of glycine, formate, and acetate is not used directly in the synthesis of uracil.

After feeding labeled glycine, very small amounts of radioactivity ap- peared in the expired COZ. Apparently the glycine, when fed in small amounts, is incorporated into the body to a very large extent and is broken down very slowly. As little radioactive CO, was formed, there was little incorporation of Cl4 into carbon 6 of guanine, carbon 2 of uracil, or the carbon of urinary urea.

The finding of radioactivity in the expired CO2 of the formate experiment indicates that formic acid is oxidized in the rat. This is in agreement with the work of Sakami (35). It was oxidized very slowly in the experiment of Buchanan on the pigeon. Nevertheless it is obvious that formate en- tered readily into metabolic reactions in the experiments of both investi- gators as well as in ours.

The authors are greatly indebted to Dr. Samuel Gurin and Dr. Adelaide M. Delluva for the synthesis of the labeled compounds and to Mrs. Oveida E. Mayo for her invaluable technical assistance throughout this work.

SUMMARY

The biological synthesis of purines and pyrimidines in rats was studied by administering precursors labeled with radioactive carbon. Adenine, guanine, uracil, and thymine were isolated from tissue nucleic acids, and adenine from nucleotides. Guanine and uracil were degraded to deter- mine the position of the isotope within the molecule.

Carbon dioxide was found to contribute to carbon 6 of guanine, glycine contributed its carboxyl carbon to carbon 4 and its a-carbon to carbon 5, and formate was incorporated into positions 2 and 8. While CO2 was in- corporated into position 6 of the purines, it entered at a similar rate into position 2 of uracil, viz. the ureide carbon. This constitutes a new reaction for the fixation of CO2 in a compound necessary for the body.

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Milton R. Heinrich and D. Wright WilsonTHE RAT

: I. PURINES AND PYRIMIDINES IN14ACID COMPONENTS STUDIED WITH C

THE BIOSYNTHESIS OF NUCLEIC

1950, 186:447-460.J. Biol. Chem. 

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