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The metabolism of the nucleic acids of rat bone marrow
507
SHORT COMMUNICATIONS
SC 93046
The metabolism of the nucleic acids of rat bone m a r r o w I.
The stimulation
in vitro of nucleic acid biosynthesis by a rat-liver
extract and the identification of the stimulating factor It has been suggested that liver plays an important role in the biosynthesis of the purines of bone-marrow nucleic acidsl, 2. Experiments in vitro carried out by us 3 have shown that, in the rat, the biosynthesis of purines and pyrimidines of marrow nucleic acids is stimulated b y a particle-free extract of rat liver suggesting that synthesis of pyrimidines as well as of purines is under hepatic influence. The experiments described in this paper were undertaken to obtain information about the nature of the stimulating factor present in the rat liver. The biological material, the experimental procedure and the analytical methods were those already described3, 5. In attempts to determine which components of the particle-free rat-liver extract might be responsible for its stimulatory activities, the effects of dialysis and boiling were studied. The E14C]formate uptake was stimulated by the supernatant of boiled particle-free rat-liver extract and by the extract diffusate. These two fractions kept the activity of the original extract, and no activity remained with the fraction retained in the dialysis bag. No stimulatory action was detected in the 70 %, 50 % and 30 % protein fractions of the extract precipitated with (NH4)2SO~. These results indicate that the activity of the extract is dialysable and resists boiling. The effect of the boiled extract on rat marrow differs from that on rabbit marrow in which no stimulating effect is observed e. The non-protein nature of the stimulating substance prompted us to follow other separation steps. The extract was treated with HC104, and the perchloric acid supernatant was treated with charcoal as described in Table I. The perchloric acid supernatant and the fraction not absorbed on charcoal maintained their activity whereas, as shown in Table I, the eluate was not active. Ultraviolet absorbing material, pentoses and deoxypentoses were present in the active perchloric acid supernatant, but disappeared in the active charcoal fraction. This evidence excludes the possibility that known nucleotides (ribo- and deoxyribonucleotides), nucleosides, folic acids, vitamin BI,, cobamide coenzymes, NAD or N A D P can be the hepatic stimulant for rat marrow. Metals as cofactors can be eliminated too, because an ashed sample of the liver extract was inactive. SOSKA et alY have reported that the synthesis in vitro of DNA in guinea-pig marrow is increased in the presence of ATP, cytidylic and deoxycytidylic acids. This finding provides further evidence that the metabolism of the marrow nucleic acids is different in different animal species 3. HARRINGTON8 has shown that purine nucleotide synthesis is stimulated b y cytidine and glucose in Ehrlich ascites tumour cells in vitro. HENDERSON AND LE PAGE 9 have reported stimulation of purine synthesis in Ehrlich ascites tumour b y a rat-liver extract, suggesting that the stimulatory effect is due to its glutamine content. THOMSON" et al. 6 found that a mixture of glucose, glutamine, Biochim. Biophys. Acla, 95 (1965) 507-509
SHORT COMMUNICATIONS
jO8 TABLE
I
EFFECTS OF SEVERAL EXTRACTS OBTAINED FROM THE SAME BATCH OF PARTICLE-FREE RAT-LIVER EXTRACT THAT CONTAINED THE SAME AMOUNT OF GLUCOSE (IO pmoles) ON THE UPTAKE in vitro OF [W]FORMATE BY THE NUCLEIC ACIDS OF RAT BONE MARROW Abbreviation PFLE, particle-free rat-liver extract. The perchloric acid liver supernatant was prepared b} precipitating IO ml of cold PFLE with drops of cold 70 y0 HClO, until the pH reached I. After the centrifugatior the supernatant was neutralized with I N KOH. The precipitated perchlorate was separated by centrifugation IO ml of perchloric acid supernatant was stirred with I g of charcoal (Merck) that had been washed wit.1 5 N NaOH, H,O, and 5 N H,SO, and finally with H,O until the pH became 6.5. The mixture wa: centrifuged, and the supernatant (not the adsorbed fraction) was kept for testing. Elution was carried out wit1 50 y0 ethanol and concentrated ammonia (IOO : I) (adsorbed fraction). The fraction not adsorbed on charcoa was pipetted on to a column of Sephadex G-25 (Pharmacia, Uppsala) and fractions were collected using distillec water as eluant. Fractions lacking glucose that appeared at the beginning were pooled and lyophilized, ant designated as F-I. Fractions with glucose were eluted afterwards. They were combined and lyophilized, am designated as F-II. The conditions of the incubation were as previously described3.
Extracts
and additions
to incubation
medium
Specific
Control PFLE Perchloric acid supernatant Fraction not adsorbed on charcoal Sephadex G-25 (Fraction F-II) PFLE retentate PFLE retentate+ IO rmoles of glucose Glucose (IO pmoles)
activity (countslmin
Whole RNA adenine
DNA adenine
3 33 25 16
600 I5 400 9 700 3 200 3 100 800 6 500 5 300
300 200 500 400 12 800 4 300 ‘5 700 15 500
per pmole of bases) DNA thymine 4 400 21
800
18 800
Acid-soluble adenine 35 600
273 000 224 000
13 000 II 500
I I4 000
6 07.5 I9 7oo 18 200
36 7oo 103 500 72 000
aspartic acid and glycine produces a slight increase of the incorporation ilz vitro of [14C]formate into the RNA and DNA adenine of rabbit bone marrow. The behaviour of rat bone marrow differs radically from that of the tissues reported above because a mixture of 0.5 mM glutamine, 0.5 mM aspartic acid, 0.5 mM glycine and 0.5 mM sodium formate showed no effect. Since none of a great number acid synthesis showed an activity
of cofactors, comparable
precursors or substrates for nucleic with that of particle-free rat-liver
extract or its fractions, further experiments were carried out in a search for a chemical property common to the extract and the various active fractions obtained from it. One common property soon emerged: the presence of a high concentration of reducing substances as measured by the technique of Folin and Malmrose. Glucose, as measured with the glucose oxidase (EC 1.1.3.4) technique, was one of the major components of the reducing substances. The average glucose content of the extract, the perchloric acid supernatant, and the charcoal-treated fraction was 30 pmoles/ml. Table I shows the effect of several fractions separated from the same recentlyprepared batch of the extract. The amounts of each extract added to the maxrow were adjusted so that they contained the same amount of glucose (IO pmoles), and the activities of the extracts were compared with that of IO ,umoles of glucose alone. The response of the tissue to the different fractions and to glucose tended to follow similar patterns but showed some differences in the magnitude of the responses. The specific activity of RNA and DNA adenine was higher when bone marrow was incubated with the extract than when it was incubated with its perchloric acid supernatant or with the fraction obtained after treatment with charcoal or Sephadex G-25 or B&him.
Biophys.
Acta, g5 (1965)
507-509
509
SHORT COMMUNICATIONS
with glucose alone. The specific activity of thymine was nearly the same after it had been incubated with the extract or its fractions or with glucose alone. The stimulatory action on nucleic acid biosynthesis of the liver extract and its fractions, as well as of glucose, suggest that one of the substances present which stimulates the biosynthesis of purines and pyrimidines is glucose, but that apart from glucose, there is another substance(s) capable of stimulating formate incorporation into purines. This suggestion is supported by the fact that the activity of the original extract was lost when it was dialysed and it was not restored completely when the same amount of glucose as contained in the original extract was replaced. The finding that glucose stimulates both purine and pyrimidine biosynthesis in rat bone marrow points to another difference 3 in the behaviour in vitro of rat and rabbit bone marrow, for in the latter, addition of glucose 1° does not stimulate purine or pyrimidine biosynthesis. The specific activities of thymine of rat marrow and of rabbit marrow without glucose, are similar, but that of purine bases is much greater in rat marrow than in rabbit marrow. The effect of the extract not explained by glucose, is on the other hand much greater in r a b b i t marrow than in that of the rat 2,3. From the experiments discussed above we conclude that the stimulatory effect of a particle-free rat-liver extract on the nucleic acid biosynthesis in vitro is due mainly to the glucose content of the extract with the additional effect of another substance(s) that enhances the incorporation of formate into the purines. Glucose m a y act as a source of energy or as precursor of ribose, as suggested in a previous communication n. The other compound (s) that stimulate only the putine biosynthesis is (are) closely similar in behaviour to the factor present in mouse liver, which stimulates purine biosynthesis in rabbit bone marrow 2& We should like to thank Professor G. HODGSON for m a n y valuable discussions and suggestions. The able technical assistance of Mr. F. GARRIDO is grateful acknowledged. This investigation was supported in part by a grant (GA-MNS-6oI74) from the Rockefeller Foundation.
Instituto de Ciencias, Casilla 653, Universidad de Chile, Santiago (Chile)
MARCO P E R R E T T A MARIA P I E B E R - P E R R E T T A J o s l ~ MINGUELL*
I L. G. LAJTHA AND J. R. VANE, Nature, 182 (1958) 191. 2 R. M. S. SMELLIE, R. Y. THOMSON AND J. N. DAVIDSON, Biochim. Biophys. Acta, 45 (1958) 593 M. PERRETTA AND 1V~.PIEBER-PERRETTA,Biochim. Biophys. Acta, 61 (1962) 828. 4 J. w . GOODMAN AND C. C. CONGDON, Arch. Pathol., 72 (1961) 912. 5 1V[. PERRETTA, W. RUDOLPH, G. AGUIRRE AND G. HODGSON, Biochim. Biophys. Acta, 87 (1964) 157. 6 R. Y. THOMSON, G. RICCERI AND M. PERRETTA, Biochim. Biophys. Acta, 45 (196o) 87. 7 J. SOSKA, L. BENES, V. DRASIL, Z. KARPFEL, E. PALACEK AND M. SKALKA,The Initial E[/ects o[ Ionizing Radiations on Cells, A c a d e m i c Press, N e w York, L o n d o n , I96O, p. 153. 8 H. HARRINGTON,Biochim. Biophys. Acta, 68 (1963) 5o9. 9 J. F. HENDERSON AND C. A. LE PAGE, J. Biol. Chem., 234 (1959) 2364. IO R. Y. THOMSON, R. M. S. SMELLIE AND J. N. DAVIDSON, Biochim. Biophys. Acta, 29 (1958) 308. I I M. PI~RRETTA, M. PIEBER-PERRETTA AND J. MINGUELL, Arch. Biochem. Biophys., lO 5 (1964) 449-
Received October 5th, 1964 P r e s e n t a d d r e s s : C~.tedra de B i o q u f m i c a , E s c u e l a de M e d i c i n a V e t e r i n a r i a , U n i v e r s i d a d de Chile, S a n t i a g o (Chile).
Biochim. Biophys. Acta, 95 (1965) 5 o 7 - 5 o 9
SHORT COMMUNICATIONS
SC 93046
The metabolism of the nucleic acids of rat bone m a r r o w I.
The stimulation
in vitro of nucleic acid biosynthesis by a rat-liver
extract and the identification of the stimulating factor It has been suggested that liver plays an important role in the biosynthesis of the purines of bone-marrow nucleic acidsl, 2. Experiments in vitro carried out by us 3 have shown that, in the rat, the biosynthesis of purines and pyrimidines of marrow nucleic acids is stimulated b y a particle-free extract of rat liver suggesting that synthesis of pyrimidines as well as of purines is under hepatic influence. The experiments described in this paper were undertaken to obtain information about the nature of the stimulating factor present in the rat liver. The biological material, the experimental procedure and the analytical methods were those already described3, 5. In attempts to determine which components of the particle-free rat-liver extract might be responsible for its stimulatory activities, the effects of dialysis and boiling were studied. The E14C]formate uptake was stimulated by the supernatant of boiled particle-free rat-liver extract and by the extract diffusate. These two fractions kept the activity of the original extract, and no activity remained with the fraction retained in the dialysis bag. No stimulatory action was detected in the 70 %, 50 % and 30 % protein fractions of the extract precipitated with (NH4)2SO~. These results indicate that the activity of the extract is dialysable and resists boiling. The effect of the boiled extract on rat marrow differs from that on rabbit marrow in which no stimulating effect is observed e. The non-protein nature of the stimulating substance prompted us to follow other separation steps. The extract was treated with HC104, and the perchloric acid supernatant was treated with charcoal as described in Table I. The perchloric acid supernatant and the fraction not absorbed on charcoal maintained their activity whereas, as shown in Table I, the eluate was not active. Ultraviolet absorbing material, pentoses and deoxypentoses were present in the active perchloric acid supernatant, but disappeared in the active charcoal fraction. This evidence excludes the possibility that known nucleotides (ribo- and deoxyribonucleotides), nucleosides, folic acids, vitamin BI,, cobamide coenzymes, NAD or N A D P can be the hepatic stimulant for rat marrow. Metals as cofactors can be eliminated too, because an ashed sample of the liver extract was inactive. SOSKA et alY have reported that the synthesis in vitro of DNA in guinea-pig marrow is increased in the presence of ATP, cytidylic and deoxycytidylic acids. This finding provides further evidence that the metabolism of the marrow nucleic acids is different in different animal species 3. HARRINGTON8 has shown that purine nucleotide synthesis is stimulated b y cytidine and glucose in Ehrlich ascites tumour cells in vitro. HENDERSON AND LE PAGE 9 have reported stimulation of purine synthesis in Ehrlich ascites tumour b y a rat-liver extract, suggesting that the stimulatory effect is due to its glutamine content. THOMSON" et al. 6 found that a mixture of glucose, glutamine, Biochim. Biophys. Acla, 95 (1965) 507-509
SHORT COMMUNICATIONS
jO8 TABLE
I
EFFECTS OF SEVERAL EXTRACTS OBTAINED FROM THE SAME BATCH OF PARTICLE-FREE RAT-LIVER EXTRACT THAT CONTAINED THE SAME AMOUNT OF GLUCOSE (IO pmoles) ON THE UPTAKE in vitro OF [W]FORMATE BY THE NUCLEIC ACIDS OF RAT BONE MARROW Abbreviation PFLE, particle-free rat-liver extract. The perchloric acid liver supernatant was prepared b} precipitating IO ml of cold PFLE with drops of cold 70 y0 HClO, until the pH reached I. After the centrifugatior the supernatant was neutralized with I N KOH. The precipitated perchlorate was separated by centrifugation IO ml of perchloric acid supernatant was stirred with I g of charcoal (Merck) that had been washed wit.1 5 N NaOH, H,O, and 5 N H,SO, and finally with H,O until the pH became 6.5. The mixture wa: centrifuged, and the supernatant (not the adsorbed fraction) was kept for testing. Elution was carried out wit1 50 y0 ethanol and concentrated ammonia (IOO : I) (adsorbed fraction). The fraction not adsorbed on charcoa was pipetted on to a column of Sephadex G-25 (Pharmacia, Uppsala) and fractions were collected using distillec water as eluant. Fractions lacking glucose that appeared at the beginning were pooled and lyophilized, ant designated as F-I. Fractions with glucose were eluted afterwards. They were combined and lyophilized, am designated as F-II. The conditions of the incubation were as previously described3.
Extracts
and additions
to incubation
medium
Specific
Control PFLE Perchloric acid supernatant Fraction not adsorbed on charcoal Sephadex G-25 (Fraction F-II) PFLE retentate PFLE retentate+ IO rmoles of glucose Glucose (IO pmoles)
activity (countslmin
Whole RNA adenine
DNA adenine
3 33 25 16
600 I5 400 9 700 3 200 3 100 800 6 500 5 300
300 200 500 400 12 800 4 300 ‘5 700 15 500
per pmole of bases) DNA thymine 4 400 21
800
18 800
Acid-soluble adenine 35 600
273 000 224 000
13 000 II 500
I I4 000
6 07.5 I9 7oo 18 200
36 7oo 103 500 72 000
aspartic acid and glycine produces a slight increase of the incorporation ilz vitro of [14C]formate into the RNA and DNA adenine of rabbit bone marrow. The behaviour of rat bone marrow differs radically from that of the tissues reported above because a mixture of 0.5 mM glutamine, 0.5 mM aspartic acid, 0.5 mM glycine and 0.5 mM sodium formate showed no effect. Since none of a great number acid synthesis showed an activity
of cofactors, comparable
precursors or substrates for nucleic with that of particle-free rat-liver
extract or its fractions, further experiments were carried out in a search for a chemical property common to the extract and the various active fractions obtained from it. One common property soon emerged: the presence of a high concentration of reducing substances as measured by the technique of Folin and Malmrose. Glucose, as measured with the glucose oxidase (EC 1.1.3.4) technique, was one of the major components of the reducing substances. The average glucose content of the extract, the perchloric acid supernatant, and the charcoal-treated fraction was 30 pmoles/ml. Table I shows the effect of several fractions separated from the same recentlyprepared batch of the extract. The amounts of each extract added to the maxrow were adjusted so that they contained the same amount of glucose (IO pmoles), and the activities of the extracts were compared with that of IO ,umoles of glucose alone. The response of the tissue to the different fractions and to glucose tended to follow similar patterns but showed some differences in the magnitude of the responses. The specific activity of RNA and DNA adenine was higher when bone marrow was incubated with the extract than when it was incubated with its perchloric acid supernatant or with the fraction obtained after treatment with charcoal or Sephadex G-25 or B&him.
Biophys.
Acta, g5 (1965)
507-509
509
SHORT COMMUNICATIONS
with glucose alone. The specific activity of thymine was nearly the same after it had been incubated with the extract or its fractions or with glucose alone. The stimulatory action on nucleic acid biosynthesis of the liver extract and its fractions, as well as of glucose, suggest that one of the substances present which stimulates the biosynthesis of purines and pyrimidines is glucose, but that apart from glucose, there is another substance(s) capable of stimulating formate incorporation into purines. This suggestion is supported by the fact that the activity of the original extract was lost when it was dialysed and it was not restored completely when the same amount of glucose as contained in the original extract was replaced. The finding that glucose stimulates both purine and pyrimidine biosynthesis in rat bone marrow points to another difference 3 in the behaviour in vitro of rat and rabbit bone marrow, for in the latter, addition of glucose 1° does not stimulate purine or pyrimidine biosynthesis. The specific activities of thymine of rat marrow and of rabbit marrow without glucose, are similar, but that of purine bases is much greater in rat marrow than in rabbit marrow. The effect of the extract not explained by glucose, is on the other hand much greater in r a b b i t marrow than in that of the rat 2,3. From the experiments discussed above we conclude that the stimulatory effect of a particle-free rat-liver extract on the nucleic acid biosynthesis in vitro is due mainly to the glucose content of the extract with the additional effect of another substance(s) that enhances the incorporation of formate into the purines. Glucose m a y act as a source of energy or as precursor of ribose, as suggested in a previous communication n. The other compound (s) that stimulate only the putine biosynthesis is (are) closely similar in behaviour to the factor present in mouse liver, which stimulates purine biosynthesis in rabbit bone marrow 2& We should like to thank Professor G. HODGSON for m a n y valuable discussions and suggestions. The able technical assistance of Mr. F. GARRIDO is grateful acknowledged. This investigation was supported in part by a grant (GA-MNS-6oI74) from the Rockefeller Foundation.
Instituto de Ciencias, Casilla 653, Universidad de Chile, Santiago (Chile)
MARCO P E R R E T T A MARIA P I E B E R - P E R R E T T A J o s l ~ MINGUELL*
I L. G. LAJTHA AND J. R. VANE, Nature, 182 (1958) 191. 2 R. M. S. SMELLIE, R. Y. THOMSON AND J. N. DAVIDSON, Biochim. Biophys. Acta, 45 (1958) 593 M. PERRETTA AND 1V~.PIEBER-PERRETTA,Biochim. Biophys. Acta, 61 (1962) 828. 4 J. w . GOODMAN AND C. C. CONGDON, Arch. Pathol., 72 (1961) 912. 5 1V[. PERRETTA, W. RUDOLPH, G. AGUIRRE AND G. HODGSON, Biochim. Biophys. Acta, 87 (1964) 157. 6 R. Y. THOMSON, G. RICCERI AND M. PERRETTA, Biochim. Biophys. Acta, 45 (196o) 87. 7 J. SOSKA, L. BENES, V. DRASIL, Z. KARPFEL, E. PALACEK AND M. SKALKA,The Initial E[/ects o[ Ionizing Radiations on Cells, A c a d e m i c Press, N e w York, L o n d o n , I96O, p. 153. 8 H. HARRINGTON,Biochim. Biophys. Acta, 68 (1963) 5o9. 9 J. F. HENDERSON AND C. A. LE PAGE, J. Biol. Chem., 234 (1959) 2364. IO R. Y. THOMSON, R. M. S. SMELLIE AND J. N. DAVIDSON, Biochim. Biophys. Acta, 29 (1958) 308. I I M. PI~RRETTA, M. PIEBER-PERRETTA AND J. MINGUELL, Arch. Biochem. Biophys., lO 5 (1964) 449-
Received October 5th, 1964 P r e s e n t a d d r e s s : C~.tedra de B i o q u f m i c a , E s c u e l a de M e d i c i n a V e t e r i n a r i a , U n i v e r s i d a d de Chile, S a n t i a g o (Chile).
Biochim. Biophys. Acta, 95 (1965) 5 o 7 - 5 o 9