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VOL. 21 (I956) SHORT COMMUNICATIONS 387 L'hypoxanthine est un pr6curseur important des nucl6otides ad6nyliques chez la levure; dans une 6tude sur la synth~se de l'acide nncl6ique chez divers embryons, STEINERT7 6tait arriv6 une conclusion semblable, il avait montr6 que dans les premiers stades du d6veloppement la synth~se des nucl6otides ad6nyliques se fait largement aux d6pens d'hypoxanthine. Laboratoire de Chimie biologique de la Faculti des Sciences, Universitd libre de Bruxelles (Belgique) H. CHANTRENNE S. DEVREUX l S. KERR, K. SERAIDARIAN ET G. B. BROWN, J. Biol. Chem., 188 (1951) 207. 2 H. CHANTRENNE, zJ rch. Biochem. Biophys. (sous presse). g B. EPHRUSSI, a . I~IoTTINGUER ET A.-M. CHIM~kNES, Ann. Inst. Pasteur, 76 (]949) 351. 4 H. CHANTRENNE ETC. COURTOIS, Biochim. Biophys. Acta, 14 (1954) 397. 5 R. ABRAMS, ~4rch. Biochem., 3 ° (I951) 44. 6 S. KERR, K. SERAIDARIAN ET M. VVTARGON, J. Biol. Chem., 181 (I949) 76I. 7 M. STEINERT, Biochim. Biophys. Acta, 18 (1955) 511. Re~u le 31 mai 1956 The presence of uracil, uridine and hypoxanthine in ether-ethanol extracts of Tumor 755 and other biological material During a study of the ultraviolet-light-absorbing compounds present in ether extracts of trans- plantable adenocarcinoma 755 and in the organs of C57BL mice carrying these tumors as well as in those of normal rabbits paper chromatograms consistently showed the presence of uracil. When larger quantities of tissue were used uridine was also found. Subsequent shaking with ethanol extracted hypoxanthine from these tissues, from the milk of RIII mice carrying the agent of spontaneous mammary tumor and from frogs' eggs. Neither uracil nor uridine was detected in extracts from the last two sources. The organs were dropped into liquid nitrogen immediately after sacrificing the animals. Later they were rapidly disintegrated at low temperature and dried from the frozen state. The intact eggs and milk were freeze-dried, the latter after centrifugation to remove fat. The desiccated materials were extracted with anhydrous ether in a Soxhlet apparatus for 5 clays, the extract was concentrated, an equal volume of water and 0.2 volume, or more, of lO% trichloroacetic acid added and the mixture stirred mechanically for i to 1 ~2 hours. The water layer was removed, concentrated to a very small volume under reduced pressure and subjected to chromatography. The ether-extracted materials were shaken with absolute ethanol for 6 to 7 hours a day for 5 days using fresh solvent daily. The pooled extracts were centrifuged, the supernatant fluid taken to dryness under reduced pressure and the residue dissolved in a little water and analyzed. In some cases, e.g. liver extracts, the residue contained lipides which had escaped removal by the ether. They were dissolved in ether; at the same time the hypoxanthine went into solution as shown by its presence in the water layer when this ether solution was treated as the initial extract with this solvent had been. It is not known why this purine dissolved in ether at this point and failed to do so (luring the initial extraction with this solvent. Uracil was sometinles found in the alcohol extracts in addition to being present in the original ether solution. After the chromatograms were developed the dark areas seen under a n.y. lamp were cut out, eluted, and the solution concentrated and again analyzed, a different solvent being used for the new chromatogram. Light absorption measurements were then made on eluates of the dark areas seen under the lamp using eluates of equal areas from unused parts of the sheet for control solutions in the cuvette. Uracil. This compound, found in ether extracts of mouse tumor 755, spleen, kidney, liver and lung as well as in extracts of rabbit liver, kidney and lung (mixed) had RF values of 0.28, o.37 and 0.85, respectively, on chromatograms developed with n-butanol and ammonia (60, io of 0. 7 M); n-butanol, formic acid and water (77, io, 13) ; and 72 % phenol (solvents i, 2 and 3). These values were in agreement with those of an authentic sample of uracil as were the wave lengths at which maximal and minimal absorption occurred by solutions adjusted to pH 2, 7 and 13. Uridine. This compound was present in the ether extracts of tumor, in those of mouse spleen and kidney (mixed) and in those of rabbit liver and lung (mixed). The R/; values, when solvents z and 2 were used, were o.13 and o.2o. They were the same for a known sample of uridine. Light absorption measurements were made at pH 2 and 13. The positions of maximal and minimal absorption were the same for the tissue compound and the known sample. On heating the tissue compound with HCIO 4 at IOO° for 2 hours uracil was formed. 25

The presence of uracil, uridine and hypoxanthine in ether-ethanol extracts of Tumor 755 and other biological material

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Page 1: The presence of uracil, uridine and hypoxanthine in ether-ethanol extracts of Tumor 755 and other biological material

VOL. 21 (I956) SHORT COMMUNICATIONS 387

L'hypoxan th ine est un pr6curseur impor t an t des nucl6otides ad6nyliques chez la levure; dans une 6tude sur la synth~se de l'acide nncl6ique chez divers embryons, STEINERT 7 6tait arriv6

une conclusion semblable, il avait montr6 que dans les premiers stades du d6veloppement la synth~se des nucl6otides ad6nyliques se fait largement aux d6pens d 'hypoxanth ine .

Laboratoire de Chimie biologique de la Faculti des Sciences, Universitd libre de Bruxelles (Belgique)

H. CHANTRENNE S. DEVREUX

l S. KERR, K. SERAIDARIAN ET G. B. BROWN, J . Biol. Chem., 188 (1951) 207. 2 H. CHANTRENNE, zJ rch. Biochem. Biophys. (sous presse). g B. EPHRUSSI, a . I~IoTTINGUER ET A.-M. CHIM~kNES, Ann. Inst. Pasteur, 76 (]949) 351. 4 H. CHANTRENNE ETC. COURTOIS, Biochim. Biophys. Acta, 14 (1954) 397. 5 R. ABRAMS, ~4rch. Biochem., 3 ° (I951) 44. 6 S. KERR, K. SERAIDARIAN ET M. VVTARGON, J . Biol. Chem., 181 (I949) 76I. 7 M. STEINERT, Biochim. Biophys. Acta, 18 (1955) 511.

Re~u le 31 mai 1956

The presence of uracil, uridine and hypoxanthine in ether-ethanol extracts of Tumor 755 and other biological material

During a s tudy of the ul traviolet- l ight-absorbing compounds present in ether extracts of t rans- plantable adenocarcinoma 755 and in the organs of C57BL mice carrying these tumors as well as in those of normal rabbi ts paper ch romatograms consistently showed the presence of uracil. When larger quant i t ies of tissue were used uridine was also found. Subsequent shaking with ethanol extracted hypoxan th ine from these tissues, from the milk of R I I I mice carrying the agent of spontaneous m a m m a r y t u m o r and from frogs' eggs. Neither uracil nor uridine was detected in extracts from the last two sources.

The organs were dropped into liquid nitrogen immediately after sacrificing the animals. Later they were rapidly disintegrated at low tempera tu re and dried from the frozen state. The intact eggs and milk were freeze-dried, the lat ter after centrifugation to remove fat. The desiccated materials were extracted with anhydrous ether in a Soxhlet appara tus for 5 clays, the extract was concentrated, an equal volume of water and 0.2 volume, or more, of lO% trichloroacetic acid added and the mixture stirred mechanically for i to 1 ~2 hours. The water layer was removed, concentra ted to a very small volume under reduced pressure and subjected to chromatography . The ether-extracted materials were shaken wi th absolute ethanol for 6 to 7 hours a day for 5 days using fresh solvent daily. The pooled extracts were centrifuged, the supe rna tan t fluid taken to dryness under reduced pressure and the residue dissolved in a little water and analyzed. In some cases, e.g. liver extracts , the residue contained lipides which had escaped removal by the ether. They were dissolved in ether; at the same t ime the hypoxan th ine went into solution as shown by its presence in the water layer when this ether solution was t reated as the initial ext rac t with this solvent had been. I t is not known why this purine dissolved in ether at this point and failed to do so (luring the initial extract ion with this solvent. Uracil was sometinles found in the alcohol extracts in addition to being present in the original ether solution.

After the ch romatograms were developed the dark areas seen under a n.y. l amp were cut out, eluted, and the solution concentrated and again analyzed, a different solvent being used for the new chromatogram. Light absorpt ion measurements were then made on eluates of the dark areas seen under the lamp using eluates of equal areas from unused par ts of the sheet for control solutions in the cuvette.

Uracil. This compound, found in ether extracts of mouse t u m o r 755, spleen, kidney, liver and lung as well as in extracts of rabbi t liver, kidney and lung (mixed) had RF values of 0.28, o.37 and 0.85, respectively, on ch romatograms developed with n-butanol and ammonia (60, io of 0. 7 M); n-butanol, formic acid and water (77, io, 13) ; and 72 % phenol (solvents i, 2 and 3). These values were in agreement with those of an authent ic sample of uracil as were the wave lengths at which maximal and minimal absorpt ion occurred by solutions adjusted to p H 2, 7 and 13.

Uridine. This compound was present in the ether extracts of tumor , in those of mouse spleen and kidney (mixed) and in those of rabbi t liver and lung (mixed). The R/; values, when solvents z and 2 were used, were o.13 and o.2o. They were the same for a known sample of uridine. Light absorpt ion measurements were made at p H 2 and 13. The posit ions of maximal and minimal absorpt ion were the same for the tissue compound and the known sample. On heat ing the tissue compound with HCIO 4 at IOO ° for 2 hours uracil was formed.

25

Page 2: The presence of uracil, uridine and hypoxanthine in ether-ethanol extracts of Tumor 755 and other biological material

388 SHORT COMMUNICATIONS VOL. 21 (1956)

Hypoxanlhine. This purine was present in the ethanol extracts of tumor, of kidney and lung (mixed) as well as of livers of mice and of rabbi ts and in those of mouse milk and frogs' eggs (stage 8, Shumway) . The RE values, using solvents 1 and 2, were o.15 and o.24 in agreement with those of a known specimen of hypoxanth ine . Light absorpt ion max ima and minima at pH 2, I i and 14 by the eluates of the dark areas produced on the chromatograms by the extracts aud by the authent ic sample of the purine were the same, at each of these pH values. A dark area having an RF corresponding to tha t of guanine was also seen on a chromatogram of the ether extract of frogs' eggs bu t the intensi ty was too slight to permi t making measurements of light absorption.

These compounds have also been found, chromatographical ly, among a nunlber of nucleic acid fragments, in N,N-dimethyl formamide extracts of normal and cancerous mouse tissues 1. Hypoxan th ine and guanine have been observed in cold HCIO 4 extracts of frogs' eggs 2.

The present data show tha t t r ea tment of desiccated tissues with alcotlol and ether will remove some nucleic acid fragments. If desired, this can serve as a fractionation procedure. The content of salts and other water-soluble compounds of tissues is minimal in this fraction, which is therefore par t icular ly adaptable to chromatography .

Department o/Biochemistry, College o/Physicians and Surgeons, Columbia University and Francis Delafield Hospital, New York, N .Y . (U.S.A.)

VICTOR ROSS

1 G-. D . DUROUGH AND D. L. SEA'ros!, J. Am. Chem. Sot., 76 (1954) 2873- ~' M. STEINERT, drch. intern, physiol., 60 (1952) 192.

Received May 31st, 1956

Stimulation de I'activit6 phosphatasique acide d'extraits de pomme de terre et de ma'is par I 'h6t6roauxine

R6cemment , nous avons 6t6 amend A postuler une interaction entre l 'h6t6roauxine et le syst6me p h o s p h a t a s e s - - a c i d e ribonucldique lors de la germinat ion des zygotes chez Allomyces 1.

Pour v6rifier in vitro une telle interaction h6 t6 roaux ine - -phospha tases , nous nous sommes adress6 ~ la phosphatase(s) acide(s) v6g6tale, pr6sentant un p H op t imum d'activit6 (pH 5 selon GLICK 2) compris dans la zone d 'action physiologique de l 'hdtSroauxine (pH 3-6 environ) a-

Le tubercule de p o m m e de terre est assez riche en phosphatase acide 4. Nous prdlevons 2o g de pa renchyme et les broyons dans 6o ml de NaC1 o.85 %. La bouillie obtenue est abandonn6e i h A la temp6ra ture du laboratoire et une nuit au frigidaire, en pr4sence de toluol. Apr6s deux centrifugations successives fl 4ooo t . /min, le surnageant est utilis6 sans autre comme extrai t phosphatas ique.

L'activit6 pbosphatas ique acide de cet extrai t est estim6e, en absence et en pr6sence d 'une dose d 'h6t6roauxine suppl6mentaire A celle contenue naturel lement dans le tissu de p o m m e de terre, laquelle est consid6rde ici comme une constante. L'intensit6 de l 'hydrolyse enzymat ique du glyc6rophosphate, utilis6 comme substrat , est mesur6e au p H 5.I.

Voici la composit ion du mdlange, incub6 A 37 °, en tubes /~ essais: I.O ml SO1. glyc6rophosphate de Na A I O.o, en t am p on ac6tate A p H 5.1 selon GLICKS; 0. 5 ml

d 'ext ra i t phosphatas ique ; i.o ml d'eau alcoolis6e comprenan t une proport ion variable de sol. lO-23//d 'acide fl-indolylac6tique. Les tubes t6moins ne contiennent que de l'eau alcoolis6e (20 %).

L 'eau alcoolis6e est remplac6e par 1 ml d'acide trichlorac6tique ~ IO o/ clans les tubes destin6s l 'es t imation de la quant i t6 initiale de phosphore inorganique.

Le dosage du phosphore inorganique initial ou lib6r6 par l 'action enzymique est r6alis6, sur une part ie aliquote du m61ange en incubation, selon la m6thode de BERENBLUM ET CHAIN 8 d6crite par DELSAL ET MANttOURIg: mesure de l 'extinction A 655 nl~ (colorim6tre de COLEMAN) du colorant bleu form6 par r6duction au SnCI~ du phosphomolybda te en solution rz-butanolique. Une courbe d '6talonnage est 6tablie, avec cette m6thode, pour des concentrat ions de 1-15 ~ de phosphore.

Voici les r6sultats obtenus avec l 'extrai t phosphatas ique de pomme de terre a. vari6t6 d 'hiver: