The incorporation of formate into pentosenucleic acids in various organs of the rat

The Incorporation of Formate into Pentosenucleic in Various Organs of the Rat’

Acids

Pierre Drochmans,2 Denis H. Marrian3 and George Bosworth Brown From the Laboratories

of the Sloan-Kettering Institute New York, New York Received

March

for Cancer Research,

4; 1952

Certain purines, notably adenine, are readily incorporated into the nucleic acids (1, 2). Studies of the origin of purines synthesized de novo (3, 4) have suggested an over-all mechanism wherein a mononucleotide (inosinic acid) is the first detectable purine derivative. The confluence of the pathways of utilization of exogenous purines and the pathways of de novo synthesis is unknown, although both pathways lead to the purines of the polynucleotides. Studies with glycine, a specific precursor involved in the synthesis of purines, have shown (5, 6, 7) relative uptakes in the pentose nucleic acid (PNA) and desoxypentose nucleic acid (DNA) fractions and in various organs, which are somewhat different from those obtained with adenine (8). A simultaneous administration of U4-adenine and N15glycine has substantiated the different relative incorporations into the DNA and PNA of liver (9), and has suggested the existence of two mechanisms of synthesis for both types of nucleic acids. An analogous differential between the incorporations of N16-adenine and C14-formate, another precursor of purines (10, II), has recently been demonstrated in liver by Goldthwait and Bendich (12). The relative incorporation of adenine into a series of organs has been surveyed (8) and data are available for the incorporation of glycine into the nucleic acids of some organs (6). This report deals with a comparison 1 This investigation was supported by grants from the National Cancer Institute, of the National Institutes of Health, Public Health Service; and from the U. S. Atomic Energy Commission, Contract AT(30-l-910. 2 Present address: Jules Bordet Institut, Brussels, Belgium. 3 Present address: Department of Radiotherapeutics, Cambridge, England. 310

RENEWAL OF PNA IN VARIOUS ORGANS

311

of the incorporation of formate into the PNA of a number of organs with the available data from other precursors. EXPERIMENTAL

AND RESULTS

Four male Sherman strain (Rockland Farms) rats, weighing approximately 300 g. each, were fed a diet of Purina chow. Each received an intraperitoneal injection of C4-labeled formate (obtained from the Oak Ridge National Laboratory) three times at 24hr. intervals (total 10 pmoles representing 4 X 10’ counts/min./rat). The animals were sacrificed 24 hr. after the last injection and the livers, spleens, kidneys, intestines, pancreas, and testes were separately pooled. A section of each organ was removed and the histological slides were examined to confirm the normal morphological picture. The sodium nucleates were isolated (1,2) and were hydrolyzed with N NaOH (4 ml./100 mg.) at 21” for 21 hr. The DNA was removed by acidification with HCl to pH 5-6 and addition of 2 vol. alcohol. After centrifugation, the supernatant was diluted to a concentration of less than 0.01 M chloride ion, and six ribonucleotides mere separated by t,he ion-exchange procedure of Cohn (13). Further purification and concentration of some of the nucleotide fractions was accomplished by rechromatographing them on the same type of column. This isolation procedure permitted calculation of the approximate ribonucleotide pattern for the PNA of each organ, and the results were similar to those available for mammalian (14, 15) PNA in that the cytidylic and guanylic acids predominated in each organ. The quantities of the b-isomers of the purine nucleotides were slightly greater than of the a-nucleotides, with the exception of those cases where the majority of the guanylic acid b was lost through bacterial action 06). Infinitely thin samples of each nucleotide, including the a- and bisomers of the purine nucleotides, were prepared on 10 sq. cm. aluminum planchets from aliquots of more than one column fraction, the concentrations and character of which were determined from the ultraviolet absorption data. Radioactivity determinations were carried out with an Internal Geiger-Mtiller Flow Counter, Radiation Counter Laboratories Mark 12, Model 1, helium-isobutane gas. Planchets were caounted to a standard error of 35?&. There was an extensive incorporation of the formate into the purine nucleotides and but a trace incorporation into the pyrimidine nucleotides (Table I). As in previous results with formate (10) and with adenine

312

DROCHMANS,

MARRIAN

AND

BROWN

(2) the renewal of the a- and b-isomers was the same within experimental error. Without confirmation, no significance is attached to t’he apparent difference between the adenylic acids from liver. There arc large differences in the renewal of t>he PNA purincs of various organs, which do not fully parallel t,he relative differences found with adeninc.

The relative incorporation of formate into the PNA-adenine and guanine in most tissues ranges from 1.4 to 2.4 (Table I), and is compatible with the ratio of 1.7 found for pooled viscera (10). However, this ratio is 0.8 for the spleen and may suggest that, in that organ, there is a proportionately greater utilization of guanine which has arisen de novo. TABLE Incorporation

of Cl4-Formate

Activity

into

I

Purine

and Pyrimidine

expressed in counts/min./r

Nucleotidcs

mole

Adenylic acids Ratio A/C’ b

-

Intestine Kidney Spleen Liver Pancreas Testis

a’b

1.4 2.1

1

400 ~ 360 130 ~

490 350 120

320 350 150 : 80 90

15 6 10

i /

21 7 18

0.8 1.4 2.4

1.6

u A: average values for the adenylic acids; G: for guanylic acids.

The incorporat’ion of formate int,o the PNA of intestine is greater than that into any of the other organs studied and is analogous to the rapid incorporation of Y5-arginine (17) and S35-methionine (18) int,o the proteins of that organ. The sevenfold difference between formatc incorporation into intestine and pancreas, tissues of similar high metabolic activit’y and similar embryologic origin, indicates that it is still impractical to attempt to correlate such relative renewals with any particular type or function of the tissues involved. These results JCth formate, like Reichard’s with X1”-glycine (6) and Abrams’ wit,h CL4-glycine (19), showed a great’er incorporation into the PNA of intestine than into that, of normal liver. In the intestinal PNA, the ratio of the iworporation of NL5-glycine ((i) into the ring nitrogens

RENEWAL

OF PNA

IN

VARIOUS

313

ORGANS

of the adenine and guanine was 1.25, which is comparable to the 1.35 found here with formate, so that the result,s with N’“-glycine and formate are approximately parallel, although results with carbon-labeled glycine (10) showed essentially equal incorporation into t,he two purines. The survey (8) of the incorporation of orally administered adenine int.o the total nucleic acids of five organs resulted in the sequence: liver, kidney, intestine, spleen, testis. Adjustment of those values for incorporaCon into t’he PKA alone does not change the sequence, although it brings the liver and kidney values to more nearly the same level. The most striking difference between the incorporation of the small precursors and the preformed purine lies in the markedly greater incorporation of adenine into liver PNA and t,he small incorporat,ion of formate into that, nucleic acid fraction. The ratio of the incorporation of the intraperitoneTABLE Comparisons

of the Incorporation PRXUWXS

administered

1. Formate 2. Formate and Adenine 3. Adenine

II

of Formate and Adenine of Liver and Intesfine” Liver PN.4. adenine

410 63 0.205 (0.73)

Intestinal Ph‘A-adenine

2780 35-1 0.126 (0.43)

into the PNA-Adenine Ratio liv./intest.

0.16 0.25

(::;I

a These values are counts/min/p mole for formate? and atom per cent adenine. Values in parentheses are adjust,ed; see text.

-X’j for

ally administered formate (Table I) into the adenine of the liver PNL4 and of the intestinal PNA was 0.16. That of adenine into the same organs was 1.9 and, with approximate adjustment based upon the relative proportions of PNA and DNA (20) and relative label in the Dn’A’s (8), this leads to an adjust)ed rat#io of 1.6-l .‘i. In connection with another experiment, Goldthwait and Bendirh (I 2) have recently administered C”-formate alld W5-adenine simultaneously (intraperitoncal administrat,ion, 1 day) and, in confirmation of the individual experiments, they have found t’hat these ratios were 0.23 for the formate and 1.G for the adenine. The three experiments are summarized in Table II, and from these data, the conclusion was drawn (21) that the utilization of purine derivatives synthesized de novo predominates in the intestine, while in the liver there is a preferential utilization of preformed purine. Abrams’ (IO) recent studies of the differential incorporat’ion of glycine

314

DROCHMANS,

MARRIAN

AND BROWN

and adenine into liver and intestine have reaffirmed the existence of two mechanisms (7, 9) for the synthesis of PNA (and DNA) and have suggested that the purine-utilizing system is relatively inefficient in the intestine. His data also indicate a proportionately better utilization of adenine by the liver, in that adenine “spared” the utilization of glycine in the liver to a greater extent than it did in the intestine [liver to intestine ratio 0.11 with adenine and 0.38 without, Expt. 39-111 (19)]. The greater absolute incorporation of adenine into the liver PNA found in our laboratory was not paralleled by Abram& finding that the relative incorporation of it into liver and into intestine was 0.84. This difference remains unexplained, but it should be noted that Abrams’ results involved the administration of adenine at a level (1.9 mmoles/kg./day), 9.5 times that used in our experiments (0.2 mmole/kg./day). Int’raperitoneally injected adenine persists (22) in the blood for many hours and leads to deposition of 2,8-dioxyadenine in the kidneys (23) and the deposition of the crystals increases with increasing dosage (22).4 Even the smaller dose does exert a “sparing” effect on the incorporation of formate (12). However, these results andothers (1, 2) at widely varying levels are complementary and suggest that administered adenine does not bring about major modifications of the mode of nucleic acid synthesis, with the exception of the possible influence of the higher dose on the incorporation into intestinal PNA (19). The quantity of adenine administered in any of these experiments probably has a greater influence than that of the administered formate or glycine on the sizes of the available ‘Lpools,” and consequently on the balances between alternative metabolic pathways. It is possible that the existence of more than one distinct pathway of incorporation of purines or purine derivatives into nucleic acids, and a preferential utilization in the liver of a route from intact purines, may be involved in the extremely large difference between the incorporation of administered adenine into the PKA and into the DNA in nongrowing 4 Despite the fact that the deposition of crystals in the renal tubules is minimal at the lower dosage (0.2 mmole/kg./day) the longer period of administration (3 days) still results in appreciable morphologic renal damage. The diuretic action of adenine may- operate to reduce the accumulation of the crystals, particularly at the lower dosage. This action might also influence the over-all retention of an administered sample of adenine. A comparison has now been made of the Wistar (22), Sherman, and Sprague-Dawley rats and has not indicated any significant strain differences in this fate of adenine, at the two dosage levels under discussion (Dr. Stephen Sternberg, personal communication).

RENEWAL

OF PNA IN VARIOUS

ORGANS

315

liver. It should also be noted that these pathways are not always utilized to the same relative extent, as is evidenced by the fact that during liver regeneration after partial hepatectomy (9) the increase in the incorporation of adenine into the DNA is much greater than is the increase in the incorporation of the small precursor, glycine. Adequate interpretation of the reasons for the large differences in the incorporation of formate into various organs, and of the divergent results for the incorporation of different precursors into the same final products, is not now possible. They emphasize again (24, al) the general caution necessary in the interpretation of simple “renewal” data in terms of “turnover.” SUMMARY

The relative incorporation of formate into the purine ribonucleotides of various organs of the rat was greatest in the intestine, followed in order by kidney, spleen, liver, pancreas, and testis. The renewal of the PNA-adenine was greater than that of the guanine in all organs except spleen, and again there was no differentiation between the isomeric purine nucleotides. It is emphasized that, in the intestine, formate can be utilized relatively more effectively for PNA-purine synthesis than is adenine, while the reverse is true in the liver. REFERENCES 1. BROWN, G. B., ROLL, P. M., PLEKTL, A. A., AND CAVALIERI, L. F., J. Bid. Chem. 172, 469 (1948). 2. MARRIAN, D. H., SPICER, V. L., BALIS, M. E., AND BROWN, G. B., J. Biol. Chem. 189, 533 (1951). 3. GREENBERG, G. R., Federation Proc. 10, 192 (1951); J. Biol. Chem. 190, 611 (1951). 4. BUCHANAN, J. M., Abstracts 119th Meeting, p. 13~. Am. Chem. Sot., Boston, 1951. 5. BERGSTRAND, A.,ELIASSON, N. A., HAMMARSTEN, E., NORBERG, B., REICHARD, P., AND voirl UBISCH, H., Cold Spring Harbor Symposia Quant. Biol. 13, 22 (1948). 6. REICHARD, P., J. Biol. Chem. 179, 773 (1949). 7. LEPAGE, G. A., AND HEIDELBERGER, C., J. Biol. Chem. 188, 593 (1951). 8. FURST, S. S., ROLL, P. M., AND BROWN, G. B., J. Biol. Chem. 183, 251 (1950). 9. FURST, S. S., AND BROWN, G. B., J. Bid. Chem. 191, 239 (1951). 10. TOTTER, J. R., VOLKIN, E., AND CARTER, C. E., J. Am. Chem. Sot. 73, 1521 (1951). 11. MARSH, W. H., J. Biol. Chem. 190, 633 (1951).

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12. GOLDTHWAIT, D. A., AMI BENUIIICH, A., J. Bid. Chem., in press (l!)jz). 13. COHN, W. E., J. L,lm. Chem. Sot. ‘71, 2275 (1949). 14. CHAROAYF, E., iVfauasax~~, B., FISCHER, E., GRJS:EN, C., DOSIC;ER, It., .~ND ELSOS, T>., J. Hid. Chem. 166, 51 (1950). 15, ABRAMS, It., B&z. Uiochem. 30, 44 (1951). 16. CAvhLIERI, I,. F., J. d j/z. Chetrk. ,%c. 73, 4899 (1951). 17. ISLOCK, Ei., J. Biol. Che,,k. 165, 469 (1916). 18. TARVER, H., ASL) EEIIIHARUT, N. O., .I. tiiol. Chew 167, 395 (1947). 33, 4.76 (1951). 19. ABRAM, I<., Arch. Riocheuk. Hiophys. 20. DA~IDSOS, J. N., cold Spring lick&or Syrkposiu Quarkt. Viol. 12, 50 (1917). 21. BROWN, G. B., Ciba Foundat.ion Conferences, Isotopes in Biochemistry, p. 164. Churchill, T~ondon, 1951. 22. PHILIPS, F., THIERSUI, J. II., ANI) BENI)ICH, A., J. I’hnrtrkacd. Exptl. ‘I’herup. 104, 20 (1952). 23. BESDICH, A., BROW.U, G. B., PHILIPS, F., AXD THIERSCH, J. B., J. Bid. (‘hem. 163, 267 (1950). 24. VOLKIN, J<., ANI) CARTER, C. E., J. .lnz. Chwk. Sot. 73, 1519 (1951).