NUCLEOTIDE METABOLISM

III. MONO-, DI-, AND TRIPHOSPHATES OF CYTIDINE, GUANOSINE, AND URIDINE*

BY HANNS SCHMITZ,t ROBERT B. HURLBERTJ AND VAN R. POTTER

WITH THE ASSISTANCE OF ANNE F. BRUMM AND DWAIN M. WHITE

(From the McArdle Memorial Laboratory, Medical School, University of Wisconsin, Madison, Wisconsin)

(Received for publication, October 12, 1953)

In the study of the biosynthesis of nucleic acids the pool of the nucleo- tides that occur in the acid-soluble extract of animal tissues has received relatively little attention, although many studies on the adenosine nucleo- tides in this fraction have been carried out in connection with their r61e as coenzymes and as energy-transmitting systems, by means of barium fractionations (2).

Studies on the nucleotides of the acid-soluble fraction of rat tissues as possible precursors of the nucleic acids have been reported by Hurlbert and Potter (3, 4), using CY4-labeled erotic acid as a precursor for the pyrimidine moieties, by LePage (5) and Edmonds and LePage (6), using glycine-2-Cl4 as a precursor for the purine moieties, and by Schmitz et al., using glucose- l-Cl4 as a precursor for the pentose moieties (7, 8).

In connection with these studies, chromatographic methods (9) have been developed on the basis of the earlier studies by Cohn (10, 1 l), and a number of previously unrecognized nucleotides of the acid-soluble extract have been isolated. The occurrence and nature of these compounds are the subject of the present report.

Methods

The experimental conditions and analytical procedures employed were the same as those described previously (9), unless otherwise noted.

* A preliminary report was included in a paper given at the Forty-fourth annual meeting of the American Association for Cancer Research, April 9, 1953 (1). This work was supported in part by a grant (No. C-646) from the National Cancer Insti- tute, National Institutes of Health, United States Public Health Service, and in part by an institutional grant (No. 71) from the American Cancer Society.

Abbreviations as follows: PCA, perchloric acid; RNA, ribonucleic acid; DNA, desoxyribonucleic acid; MP, monophosphate; DP, diphosphate; TP, triphosphate; A, adenosine; G, guanosine; I, inosine; C, cytidine; U, uridine.

t Present address, Institut fiir experimentelle Krebsforschung der Universitiit, Heidelberg.

$ Present address, Kemiska Institutionen, Karolinska Institutet, Stockholm.

41

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42 NUCLEOTIDE METABOLISM. III

It is perhaps important to emphasize again that for the present studies the neutralized acid-soluble extracts of rat tissues were placed directly on the anion exchange column, without previous treatment with barium or other fractionation procedures. For the separation of the total acid-soluble extract, the “formic acid” or Type I system, in which elution is started with gradually increasing concentrations of formic acid and later continued with a mixture of formic acid and ammonium formate, the longer column (I cm. X 20 cm.), in combination with a 500 ml. mixing flask, was preferred throughout these experiments, since the improved resolution yields less heterogeneous fractions for rechromatography on the “ammonium formate” or Type II system, in which elution is carried out with gradually increas- ing concentrations of that buffer in the pH range 5 to 4.

Besides routine determination of the ultraviolet light absorption in acid and alkali of the isolated compounds on a Beckman DU spectrophotometer, representative samples of these compounds were analyzed for ultraviolet light absorption on a Cary recorder.’ The absorption spectra as well as the other properties of the isolated nucleotides were checked against known samples2

In order to assure the identity of the bases of the nucleotides isolated from the acid-soluble extract of tissue, the free bases were obtained by acid hydrolysis. Aliquots of the adenine, uracil, and cytosine nucleotides were separately hydrolyzed in 70 per cent PCA at 100” for 90 minutes (12, 13), and the free bases were separated from the accompanying breakdown pr0duct.s of the sugar moiety by paper chromatography by means of the disodium phosphate-isoamyl alcohol system of Carter (14). The free bases were eluted and their ultraviolet light absorption spectrum checked against the free bases derived from the respective nucleotides of RNA. Since that paper system does not move the guanine to a significant extent from the origin, this base was obtained by means of hydrolysis of the nucleotide (in 1.0 N HCl at 100” for 30 minutes) and subsequent isolation from a cation exchange column as employed by LePage (5).

The enzymatic assay for 5’-nucleotides according to Heppel and Hilmoe (15) was used for identification of the position of the esterified phosphorus.3

Results

In the discussion which follows, it will be helpful to refer to Paper II (9) in which Fig. 4 represents a diagram of the behavior of these nucleotides

1 We wish to express our gratitude to Dr. A. L. Wilds, Department of Chemistry, University of Wisconsin, for his instruction and help in the use of the Cary recorder.

2 We are greatly indebted to Dr. W. E. Cohn, Hiology Division, Oak Ridge Labo- ratory, Oak Ridge, Tennessee, for a gift of cytidine- and guanosine-5’-phosphates.

8 We wish to thank Dr. Thomas Singer, Institute for Enzyme Research, Univer- sity of Wisconsin, for providing the snake venom (Crotahs adamanteus) and for his advice regarding the enzymatic assay.

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H. SCHMITZ, R. B. HURLBERT, AND V. R. POTTER 43

on both chromatographic systems and Figs. 2 and 3 present the actual data from an extract of liver.

Isolation of Cytidined’-phosphates (CMP, CDP, and CTP)-In the pool of total free nucleotides in the acid-soluble extract of liver, adenine, uracil, and, to a lesser degree, guanine, nucleotides predominate, and the amount of cytosine nucleotides is small in comparison with the former compounds. This is shown in the chromatogram obtained from liver (9). Although the presence of CMP seemed probable in the liver extracts, CTP was first isolated in connection with s&dies on nucleotide metabolism in Flexner-

.6-

E2f3 ML.5. /

CMP AMPccfJ CTP ADP

.4-

.3-

0 50

FIG. 1. Rechromatogram of CTP, CDP, and CMP on Type II column, with super- imposed rechromatogram of ATP, ADP, and AMP. The following applies to both samples: column, 1 cm. X 10 cm., Dowex 1 (formate); mixer, 250 ml.; reservoir, am- monium formate alone or with formic acid as indicated; fractions, 100 drops per tube; rate, 7 to 8 drops per minute. Sample followed by 250 ml. of Hz0 before ammonium formate was started; source, CTP and ATP fractions from Type I chromatograms of acid-soluble fraction of Flexner-Jobling rat carcinomas (8).

Jobling rat carcinoma (8). The isolation was greatly favored by three factors: (1) the proportionally greater amount of nucleotides that are pres- ent as the triphosphates in the tumor compared with liver; (2) the total amount of cytidine phosphates present in the acid-soluble extract of tumor is about 2 times higher than the amount found in liver; and (3) the high ratio of Ezr6: Et60 for the cytidine moiety made the presence of CTP appa- rent on the Type I chromatogram. Suitable fractions from Type I sepa- ration were rechromatographed, whereupon the CTP was clearly resolved and could be identified by chemical analysis. Subsequently, CMP and CDP of tumor extracts, as well as CMP, CDP, and CTP, from the acid- soluble extracts of liver, brain, and muscle were isolated by chromatog- raphy on the two types of columns.

Fig. 1 shows a rechromatogram of a CTP peak which had been well sepa-

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44 NUCLEOTIDE METABOLISM. III

rated from preceding or following compounds on the Type I column. This chromatogram is presented for purposes of illustration, since the pooled fractions from this peak were partially hydrolyzed by the use of heat during drying, before being placed on the Type II column. Thus in Fig. 1 the peaks marked CDP and CMP are breakdown products of the CTP. The positions occupied by CDP and CMP in this rechromatogram coincide exactly with the positions taken when CDP and CMP from Type I chromat- ograms of acid-soluble extracts are rechromatographed on the Type II column. The elution patterns obtained from Type I and Type II columns were presented in Fig. 4 of Paper II (9), which also shows how the rechro- matogram separates the various compounds of Figs. 1 and 2, and are dis- cussed throughout this paper.

In order to provide reference points, the chromatographic behavior of the well known adenine nucleotides on this system is shown in Fig. 1. A Type II rechromatogram of a partially hydrolyzed section of the ATP fraction taken from the Type I chromatogram from the same tumor sample has been superimposed on the cytidine phosphate chromatogram in Fig. 1.

The purity and recovery of the CTP, CDP, and CMP from the Type II columns depend on the degree of resolution obtained with the total extract on the formic acid column. If the CTP from the Type I chromatogram is not sufficiently separated from UDP-X3 (4), UDP, and ATP, the re- chromatography on a 1 cm. X 10 cm. Type II column is not likely to yield all of the CTP as a pure peak, and the latter part of the CTP peak will be contaminated with UDP-XI, and, if the ATP has been partially broken down to ADP, the latter can also interfere. However, the use of a 1 cm. X 20 cm. Type II column with a 500 ml. mixer will give a complete reso- lution of all three compounds.

The rechromatography of the CDP fractions from the formic acid col- umns is also dependent on the original resolution. On the formic acid column the CDP comes off within the early fractions of the double peak that is made up mainly of IMP followed by UMP. However, inorganic phosphate and at least one other unidentified nucleotide that is present in small amounts come off in that region. Although on rechromatography of this double peak the order of appearance of these compounds is different (Le., inorganic phosphate, CMP, if there is CDP breakdown, UMP, IMP, and CDP), their positions are still relatively close. For maximal recovery the 1 cm. X 20 cm. column is advantageous, although there is some benefit by cutting the fraction volumes to 4.0 ml. with the 1 cm. X 10 cm. column.

The rechromatography of CMP as taken from the Type I columns usu- ally requires special precautions because of heavy contamination with DPN and other compounds. The simplest way to obtain the CMP in pure form is to hydrolyze the impurities with 1.0 N HCl at 100” for 1 hour. The

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H. SCHMITZ, R. B. HURLBERT, AND V. R. POTTER 45

desiccated and neutralized hydrolysate can then be used for rechroma- tography on a Type II column or on a Type I column with 1 N formic acid in the reservoir and 4 ml. fractions. If a 1 cm. X 10 cm. column is used, the CMP will come off in Fractions 16 to 20. If the hydrolysis is not carried out, it is still possible to get adequate separation and retain the accompanying compounds by using the Type II column, with 4 ml. frac- tions, in order to separate inorganic phosphate from the CMP.

Isolation of Uridine-5’-phosphates (UMP, UDP, and UTP)-The data on the original isolation of the UMP, the radioactive labeling of these uri- dine compounds by the precursor, erotic acid, and the nature of three derivatives of UDP (UDP-X1, UDP-X2, and UDP-XJ are reported else-

.5. UMP GM= W-X’ UTP GDP

1 I5 0

FIG. 2. Rechromatogram of GTP, GDP, GMP, UTP, UDP, and UMP on Type II column. Column details as in Fig. 1. Source, combined GTP and UTP fractions from Type I chromntogram of acid-soluble fraction of livers from tumor-bearing rats (8).

where (4), while the recent analytical data on the UMP, UDP, and UTP are reported here as part of the 5’-nucleotide series.

The UDP is closely associated on the Type I chromatograms with one of its derivatives, UDP-X8, which on mild hydrolysis liberates both UDP and a reducing sugar. Several lines of evidence indicate that free UDP is a separate component of this peak, rather than arising as a breakdown product: (1) A known mixture of UDP and ATP gave the same elution pattern as the peak obtained from tissues; (2) after careful lyophilization of the UDP and UDP-X3 peak, similar amounts of UDP and UDP-X8 were resolved by the ammonium formate system, in preparations in which the absence of UMP in the chromatogram indicated that very little hydroly- sis occurred; and (3) in experiments in which equilibration of the radio- activity (from erotic acid) of the uridines was not complete, t,he UDP and the UDP-XI had significantly different specific activities (4). UMP, UDP, and UTP, as well as the UDP derivatives mentioned, have been found to

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46 NUCLEOTIDE METABOLISM. III

occur in the acid-soluble extract of brain and Flexner-Jobling rat carcinoma (8). Muscle, however, did not contain the UDP derivatives in sufficient amounts for identification, but probably does contain UTP (8).

On Type I chromatograms UTP is more or less closely associated with the preceding GTP (see liver (8, 9) and brain and tumor (8)). Neither the original nor the rechromatographed UTP peaks contained reducing sub- stances. Fig. 2 shows a rechromatogram of a pool of the GTP fractions and a part of the following UTP fractions which were rechromatographed together on the Type II column after partial hydrolysis. In addition t,o UTP and GTP, Fig. 2 also shows the positions of the mono- and diphos- phates of uridine and guanosine which correspond with the positions also taken by UMP, UDP, GMP, and GDP when the proper fractions from the Type I column are rechromatographed on the Type II column (see the next section).

Isolation of Guanosine-5’-phosphates (GMP, GDP, and GTP)-The prox- imity of the GTP and UTP on the Type I chromatogram has been pointed out, and Fig. 2 shows that the ammonium formate system is quite capable of separating these compounds. It is indicated in Fig. 2 that at Tube 100 the pH of the eluent in the reservoir was changed from about pH 5 to 4 by the addition of formic acid. This step was taken in order to obtain the GTP in a reasonably sharp peak; i.e., eluted with the minimum of eluent. In rechromatograms of GTP in which the pH was not decreased, the GTP was recovered as a rat.her broad peak. The peaks marked GDP and GMP resulted from a partial hydrolysis of the pooled GTP (and UTP) samples, as previously noted. Their positions on the rechromatogram coincide with the positions of GDP and GMP fractions taken from the Type I chromato- gram as such.

The rechromatography of the fractions containing UTP and GTP can be carried out on a 1 cm. X 10 cm. Type II column, since they are widely sepa- rated by this system. However, if partial hydrolysis has taken place, UTP and GDP n-ill not be entirely separated from each other (see Fig. 2). In this case, complete resolution is obtained with the 1 cm. X 20 cm. Type II column. Thus far the third nucleotide which has been encountered in the GTP-UTP peak from liver has not interfered with the GTP on the Type II chromatogram.

Rechromatography of the GDP fractions from the Type I columns will yield pure GDP if sufficiently resolved by the first separation. However, if the GDP was not sufficiently resolved from the preceding UDP-X2 (8,9), and, as is possible in the case of liver, from the following Ad-X (8, 9), and if there is some breakdown of GDP to GMP and of UDP-X2 to UDP, the resulting GMP will be closely associated with the UDP (see Fig. 2), and the GDP will be contaminated with one of the components of the Ad-X peak.

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H. SCHMITZ, R. B. HURLBERT, AND V. R. POTTER 47

Rechromatography of the GMP fractions yields pure GMP and is not interfered with by the TPN or another as yet unidentified compound which comes off the Type I column in the GMP fraction.

Spectrophotometric and Other Analytical Data on Isolated Nucleotides

The ultraviolet absorption spectra of the cytidine, uridine, and guano- sine phosphates as isolated from the acid-soluble fractions of tissues were compared with the respective authentic samples of the monophosphates and were identical with them. Table I summarizes some of the spectro- photometric characteristics of the isolated compounds in terms of absorp- tion maxima and minima in acid and in alkali and their molar extinction coefficients in acid. The spectra are indicated by ratios of the optical densities, calculated from the extinction at several characteristic wave- lengths, divided by the extinction of the compound at its maximum in acid. The spectrophotometric data for the di- and triphosphates fol- lowed that obtained for the monophosphates very closely.

The phosphorus analyses in Table I show that the nucleotides listed as di- or triphosphates contain, respectively, 1 or 2 PM of acid-labile phosphate and 2 or 3 PM, respectively, of total phosphorus.

All compounds were analyzed for ribose by the orcinol test, modified as described (9). The results obtained are expressed as micromoles of ap- parent ribose, calculated by use of adenosine-5’-phosphate as a ribose stand- ard. The orcinol reactions were carried on for longer periods of time in addition to that listed in Table I, and readings were taken at different time points up to 2 hours. While the E GGO for the purines decreases on additional heating, the pyrimidines show characteristic increases. Thus the optical density at 660 rnp for the uracil nucleotides in the orcinol reac- tion corresponds to an optical density of 0.16 PM of ribose per micromole of uracil after 15 minutes at loo’, and the optical density after 60 and 120 minutes additional heating corresponds to about 0.47 and 0.64 PM of ribose. The corresponding data for the cytosine nucleotides were 0,0.024, and 0.05 PM of apparent ribose per micromole of cytosine. This extended heating procedure was applied to both authentic and isolated nucleotides, and the results were in good agreement. In this procedure the Es00 of the blank does not change appreciably over the heating period, and the results are calculated in terms of the optical density given by the adenosinedl-phos- phate standard at 15 minutes. The fact that the tissue guanosine phos- phates contain 1 .O equivalent of ribose per molecule, when A-5’-P is used as standard, indicates these compounds to be the 5’-nucleotides, since under these conditions guanosine-2’(3’)-phosphate gives 20 per cent less apparent ribose.

It has been stated earlier that the monophosphates derived from the di- and triphosphates are identical with the original monophosphates on the

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TABL

E I

$ Da

ta

on

Abso

rptio

n Sp

ectra

, Ph

osph

ate,

an

d Ap

pare

nt

Ribo

se

Cont

ent

of

Puri$

ed

Nucle

otid

es

Obta

ined

by

Rech

rom

afog

raph

y of

Fr

actio

ns

from

Ac

id-S

olub

le

Extra

ct of

Ra

t Ti

ssue

s

The

ultra

viole

t ab

sorp

tion

data

re

pres

ent

the

sele

cted

be

st

sam

ples

an

d ar

e ex

pres

sed

in

rela

tion

to

the

optic

al

dens

ity

at

the

max

imum

in

acid

, wi

th

the

follo

wing

va

lues

as

the

milli

mol

ar

extin

ctio

n co

effic

ient

s:

cytid

ine,

13

.7 a

t 27

8 m

; ur

idin

e,

9.9

at

260

mp;

gu

anos

ine,

13

.3 a

t 25

6 rn

p;

and

aden

osin

e,

14.1

at

258

mp.

Th

e ch

emica

l da

ta

are

expr

esse

d in

te

rms

of t

he

micr

omol

es

of t

he

com

pone

nt

per

micr

omol

e of

bas

e,

as d

eter

min

ed

spec

tro-

phot

omet

rical

ly.

The

aver

age

valu

es

are

give

n on

the

up

per

line

(the

num

ber

of s

ampl

es

in

pare

nthe

ses)

an

d th

e ra

nge

of v

alue

s on

the

se

cond

lin

e.

Each

sa

mpl

e th

at

was

anal

yzed

fo

r ph

osph

ate

and

ribos

e re

pres

ents

a

sepa

rate

ov

er-a

ll iso

latio

n fro

m

a tis

sue

sam

ple.

Al

l of

the

co

mpo

unds

re

pres

ent

seve

ral

sam

ples

fro

m

both

liv

er

and

Flex

ner-J

oblin

g ra

t ca

rcin

oma.

In

ad

ditio

n,

som

e 2

of t

he

anal

yses

re

pres

ent

com

poun

ds

isola

ted

from

br

ain

or

mus

cle.

G

Chem

ical

analy

ses

s

I

8

abile

phos

ha

te (15

min.

3

- L -

-

__

0

Appa

rent

ribos

e is Y $ 0 E

0

1.00

(7

.0)

0.91

-1.1

5 1.

96

(9)

1.83

-2.1

1 0.

02

(5)

0.02

0.

95

(9)

0.83

-l .ll

1.

80

(4)

1.69

-2.0

5

0 5

0 0.16

(6

) 0.

16

0.15

(6

) 0.

12-0

.16

0.13

(4

) 0.

06-0

.16

- - Ul

travio

let

abso

rption

- I

Nucle

otide

PH

’ W

ave-l

ength

Mini-

Optic

al de

nsity

at

given

wa

ve-le

ngths

Op

tical

dens

ity

max

imum

in

acid

rota1

ph

osph

ate

Maxi-

m

um

Maxi-

Mi

ni-

mum

m

um

230

III/I

245

m,.

260

mp

fw

mP

280

241

273

251

280

241

1.00

0.

12

0.26

0.

13

0.50

0.

70

0.48

0.

62

0.50

0.

56

1.00

0.

12

0.26

0.

13

0.50

280

241

1.00

0.

12

0.27

0.

14

261

230

1.00

0.

22

0.22

0.

56

261

242

0.78

0.

55

0.74

0.

57

261

231

1.00

0.

23

0.23

0.

55

261

231

1.00

0.

23

0.23

0.

56

0.50

0.99

0.

77

0.99

0.99

-

215

In&

290

mp

-

Acid

Al

kalin

e Ac

id

“ ‘I

Alka

line

Acid

“

0.97

0.

67

0.97

0.97

0.61

0.

45

0.60

0.62

0.74

0.

21

0.75

0.74

0.03

0.

03

0.04

0.03

1.00

(1

3)

0.89

-1.1

4 2.

00

(7)

1.87

-2.1

2 2.

96

(11)

2.

75-3

.20

1.03

(5

) 1.

0-1.

06

1.96

(9

) 1.

77-2

.09

2.90

(4

) 2.

67-3

.11

CMP

CDP

CTP

UMP

UDP

UTP

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GMP

GDP

GTP

AMP

ADP

ATP

‘I

Alka

line

Acid

“

258

260

258

258

223

230

228

228

1.00

0.

93

1.00

1.00

1.00

1.

04

1.06

1.

00

0.22

0.

35

0.23

0.22

0.21

0.21

0.

21

0.24

0.

63

0.98

0.

40

0.03

0.

24

0.60

1.

04

0.38

0.

02

0.24

0.

63

0.97

0.

40

0.04

0.

24

0.64

0.

98

0.41

0.

03

1.97

2.

91

0.97

2.

00

1.00

(1

1)

1.00

(1

1)

0.93

-1.1

3 0.

93-1

.13

1.02

(7

) 1.

02

(7)

0.96

-1.0

6 0.

96-1

.06

0.98

(4

) 0.

98

(4)

0.95

-1.0

2 9

0.95

-1.0

2 9

1.00

1.

00

I+ I+

B B

2 2 .!N

.!N

* Th

e sp

ectru

m

read

ings

in

al

kali

were

de

term

ined

in

al

l ca

ses,

alth

ough

on

ly

repr

esen

tativ

e va

lues

ar

e pr

esen

ted

here

. So

me

m

fluct

uatio

ns

in t

he

valu

es

were

no

ted,

wh

ich

may

ha

ve

resu

lted

in p

art

from

th

e ac

tion

of a

lkali

on t

he

com

poun

ds

and

poss

ibly

a

F la

ck

of r

igid

co

ntro

l of

the

pH

. 2 z

Acid

25

6 22

8 Al

kalin

e 26

5 23

1 Ac

id

256

228

“ 25

6 22

8

- 0.

24

0.73

0.

96

0.69

0.

47

0.36

0.

65

0.93

0.

80

0.11

0.

24

0.74

0.

95

0.68

0.

45

0.24

0.

74

0.96

0.

69

0.46

1.02

(1

1)

0.82

-l .2

8 2.

01

(7)

1.93

-2.1

2 3.

01

(4)

2.81

-3.1

9 1.

01

0 1.00

(7

) 0.

94-l

.lO

2.07

(4

) 1.

88-2

.15

0.01

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50 NUCLEOTIDE METABOLISM. III

basis of chromatographic behavior. These monophosphates are also in- distinguishable from the authentic 5’-mononucleotides (obtained by enzy- matic hydrolysis of RNA) by the same chromatographic procedures and are readily distinguished from the 2’- and 3’-monophosphates, which are obtained by the alkaline hydrolysis of RNA (see Fig. 1 and “Standardi- zation of chromatograms” of Paper II (9)). These chromatographic com- parisons were facilitated by mixture of a radioactive compound from the acid-soluble fraction with the corresponding non-radioactive, authentic 5’-

TABLE II

Hydrolysis of Nucleotides by C’-Nucleotidase

The enzyme used was lyophilized snake venom (Crofalus adamanteus) (15). The reaction mixture was 0.18 M tris(hydroxymethyl)aminomethane buffer at pH 8.4, 0.09 M MgSOI, and contained between 0.5 and 1.0 PM of substrate per ml. Inorganic phosphate was determined by the Fiske-Subbarow method without removal of the venom protein.

Compound SOUPX

3’-AMP 3’-UMP 5’-AMP 5’-UMP 5’-GMP

“ “ “

5’-CMP “ ‘I

.- RNA

“

Pabst Laboratories Liver UDP RNA Liver GMP

‘I GDP ‘I GTP ‘( CMP I‘ CDP + CTP

RNA

Per cent hydrolysis per 15 min. at 380

!3 y “encnn per ml.

0 0

62

46 71 65 75 55 60 47

0 0

100 96 85

100 96

102 97 98 99

nucleotide. In the cases examined in this way (UMP on Type I and Type II, and CMP on Type II, columns), the resulting peaks containing the 5’- nucleotides were completely homogeneous with regard to both radioactivity and spectrum. Further evidence that the nucleotides of the acid-soluble fraction are nucleoside-5’-phosphates was provided by their lability to 5’- nucleotidase action. Table II summarizes the enzymatic assay with 5’- nucleotidase. The results show that the tissue nucleotides, as well as the authentic mononucleotides, were almost completely dephosphorylated by the enzyme, which was inactive towards adenosine- and uridine-2’(3’)-phos- phates.

All nucleotides reported in this paper were tested for reducing sugar (16) with negative results.

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H. SCHMITZ, R. B. HURLBERT, AND V. R. POTTER 51

A preliminary study has indicated that the higher phosphorylated nucleo- tides of all four bases mentioned in this report are also present in the acid- soluble extract of actively growing yeast.

DISCUSSION

There have been a number of indications recently that free nucleotides other than the adenine series may be present in living organisms. Leloir and coworkers recently isolated the coenzyme of the glucose-l-phosphate- galactose-l-phosphate transformation and showed that it was a glucose derivative of UDP. Coenzyme activity was reported for several animal tissues. Additional UDP derivatives have been reported by Park (17), and UDP has been enzymatically converted to UTP by Kornberg (18) (see reviews by Leloir (19, 20)). Dutton and Storey (21) have obtained a glucuronic acid derivative of UDP from rat liver. Lipton et al. (22) have isolated UTP in fairly large quantities from certain commercial fermenta- tion mixtures.4 Cytidine-5’-phosphate was tentatively identified by Stro- minger (23) as one of the products occurring in extracts of penicillin- inhibited Staphylococcus aureus. That guanine nucleotides might be present in acid-soluble fractions of animal tissue was indicated by LePage who obtained free guanine from hydrolysates of such extracts (5).

The occurrence of the 5’-mono-, di-, and triphosphates of adenosine (AMP, ADP, and ATP) in the acid-soluble extracts of animal tissues is well known, and it is thus of considerable interest to find that analogous com- pounds of guanosine, cytidine, and uridine also occur. It is emphasized that these compounds were obtained as the free nucleotides and not as products from hydrolysates of nucleic acids.

As far as we are aware, the occurrence of GDP, GTP, CDP, and CTP has not been previously reported, and free GMP, CMP, UDP, and UTP, in addition to the above compounds, do not appear to have been observed in animal tissues. In earlier studies on the biosynthesis of the pyrimidines Hurlbert isolated UMP and several derivatives from rat liver (24), and in concurrent studies the presence of UDP, UTP, and three derivatives of UDP was noted in liver (4). All of the above compounds were found in acid-soluble extracts of liver, brain, and Flexner-Jobling rat carcinoma (8).

Until recently the nucleoside-5’-monophosphates had never been ob- tained from nucleic acid hydrolysates, but Cohn and Volkin (25) have now shown that by use of a purified phosphodiesterase all four of the 5’-nucleo- side monophosphates could be obtained from RNA, and, in addition, the diphosphates of cytidine and uridine could be obtained. The diphosphates

4 We wish to express our thanks to Dr. S. A. Morel1 of the Pabst Laboratories for supplying us with limited quantities of their UTP, which appears to be identical with the UTP that we have isolated from acid-soluble extracts of animal tissues. Their product is now commercially available.

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52 NUCLEOTIDE METABOLISM. III

appeared to be the 2’) 5’ or 3’, 5’ variety and contained no pyrophosphates, judged by their stability to acid hydrolysis. The di- and triphosphates reported here are considered to be pyrophosphates on the basis of their proportion of easily hydrolyzed phosphate, which distinguishes them from the diphosphates obtained by Cohn and Volkin (25). In the present studies the labile phosphate has been determined on the basis of studies with the higher phosphates of uridine and adenine. Complete hydrolysis curves to justify the use of these conditions for the cytidine and guanosine phosphates have not been made because of lack of material.

Thus the compounds described in this report are apparently analogous to the “muscle” adenine nucleotides rather than to the earlier products from the nucleic acids. It is obvious that as this work progressed the analogy with the adenine nucleotides became increasingly clear and became of heuristic value. Thus, the occurrence of CDP was not evident in the early chromatograms, but was suspected by analogy, and its isolation was facili- tated by empirical prediction of its probable location on the chromatograms (see Fig. 4 of Paper II (9)). However, the identification of the compounds is not based upon analogy, but rests on several lines of evidence, including enzymatic hydrolysis of the 5’-monophosphates, lability of the phosphates in the proper ratios, positions on the chromatograms, absorption spectra, and chromatography of mixtures of labeled and unlabeled compounds. Nevertheless, further studies will be required to establish whether the pyri- midine compounds contain ribose in the theoretical amount and whether the di- and triphosphates are split by appropriate pyrophosphatases. In addition it is not rigorously established that additional substituents are not present in the case of the di- and triphosphates dealt with in this paper. At the same time it may be pointed out that the acid-soluble extracts con- tain, in addition to the compounds described in this report, a number of derivatives that have been separated and distinguished from them and in some cases tentatively identified (9). In the case of the compounds de- scribed in the present report the presence of reducing compounds (8, 9), or of other materials which absorb in the ultraviolet region, has been ex- eluded .

The chief purpose of the present report is to call attention to the exis- tence of these nucleotides and to give sufficient details to permit their iso- lation. During further isolation work it is quite possible that additional compounds such as 5’-pyrophosphates with additional phosphate in the 2’ or 3’ position, or other unknown nucleotides, may be found to occur in the present preparations in small amounts. The occurrence of the more obvious unidentified nucleotides that have been separated has been de- scribed in Paper II (9).

While some of the compounds mentioned in this report may serve as co- enzymes or coenzyme precursors, they may also be considered as possible

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II. SCHMITZ, R. B. HURLBERT, AND V. R. POTTER 53

building blocks for the nucleic acids. Although such pyrophosphates have never been obtained among the breakdown products of the nucleic acids, their function as nucleic acid precursors is a possibility that is under in- vestigation (4, 9, 26). The fact that they do not occur among the break- down products of nucleic acids might be explained by assuming that the terminal phosphates (or pyrophosphates) (27) are lost when the mononu- cleotides are converted to nucleic acid, as in the case of so many established biosyntheses that utilize phosphate bonds. These studies, together with those of LePage (5) and Edmonds and LePage (6) on the biosynthesis of the purine nucleotides, suggest the possibility that some of the nucleotides may serve dual rales as coenzymes and as precursors of nucleic acids, and thereby serve to coordinate function with growth by an extension of the mechanism proposed in 1944 (28).

The present work establishes that all of the bases that occur in poly- merized form as ribonucleic acid exist in the acid-soluble fraction of animal tissues as mononucleotides at the three levels of .phosphorylation previ- ously established for the adenine nucleotides. With knowledge that these compounds exist and with methods for their isolation, further work on their possible rBles as coenzymes, as energy transmitters, and as building blocks for the nucleic acids is being carried out in vitro.

SUMMARY

1. The application of ion exchange methods has resulted in the isolation of the 5’-mono-, di-, and triphosphates of cytidine, uridine, and guanosine from the acid-soluble extract of rat tissues.

2. These compounds have been characterized on the basis of their spec- trum and their ribose and phosphorus contents, and by identification of the free bases after hydrolysis. The monophosphates (including those de- rived from the di- and triphosphates) have been distinguished from the cor- responding 2’- and 3’-monophosphates by chromatography and are identi- cal with corresponding authentic 5’-mononucleotides as judged by a number of chemical and chromatographic criteria.

3. Hydrolysis of these compounds by a specific snake venom 5’-nucleo- tidase provides further evidence that they are 5’-nucleotides.

4. The di- and triphosphates of cytidine, uridine, and guanosine con- tain, respectively, 1 and 2 moles of acid-labile phosphate per mole of base, which indicates that they are pyrophosphates analogous to the known adenosine-5’-mono-, di-, and triphosphates.

BIBLIOGRAPHY

1. Schmitz, H., Potter, V. R., and Hurlbert, R. B., Proc. Am. Assn. Cancer Res., 1, 47 (1953).

2. LePage, G. A., in Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and tissue metabolism, Minneapolis, 185 (1949).

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54 NUCLEOTIDE METABOLISM. III

3. Hurlbert, R. B., and Potter, V. R., J. Biol. Chem., 196,257 (1952). 4. Hurlbert, R. B., and Potter, V. R., J. Biol. Chem., 209, 1 (1954). 5. LePage, G. A., Cancer Res., 13, 178 (1953). 6. Edmonds, M., and LePage, G. A., Federation Proc., 12, 199 (1953). 7. Schmitz, H., Potter, V. R., and Hurlbert, R. B., Cancer Res., 14, 58 (1954). 8. Schmitt, H., Potter, V. R., Hurlbert, R. B., and White, D. M., Cancer Res., 14,

66 (1954). 9. Hurlbert, R. B., Schmitz, H., Brumm, A. F., and Potter, V. R., J. Biol. Chem..

209, 23 (1954). 10. Cohn, W. E., J. Am. Chem. Sot., 73, 1471 (1950). 11. Cohn, W. E., J. Cell and Comp. Physiol., !I& suppl. 1, 21 (1951). 12. Marshak, A., and Vogel, H. J., J. Biol. Chem., 189, 597 (1951). 13. Wyatt, G. R., Biochem. J., 48, 584 (1951). 14. Carter, C. E., J. Am. Chem. Sot., 72, 1466 (1950). 15. Heppel, L. A., and Hilmoe, R. J., J. BioZ. Chem., 199, 665 (1951). 16. Park, J. T., and Johnson, M. J., J. Biol. Chem., 179, 585 (1949). 17. Park, J. T., in McElroy, W. D., and Glass, B., Phosphorus metabolism, Balti-

more,l, 93 (1951). 18. Kornberg, A., in McElroy, W. D., and Glass, B., Phosphorus metabolism,

Baltimore, 1, 392 (1951). 19. Leloir, L. F., in McElroy, W. D., and Glass, B., Phosphorus metabolism, Balti-

more, 1, 67 (1951). 20. Leloir, L. F., Advances in Enzymol., 14, 193 (1953). 21. Dutton, G. J., and Storey, I. D. E., Biochem. J., 63, p. xxxvii (1953). 22. Lipton, S. H., Morell, S. A., Frieden, A., and Bock, R. M., J. Am. Chem. Sot.,

76, 5449 (1953). 23. Strominger, J. L., Federation PTOC., 12, 277 (1953). 24. Hurlbert, R. B., Federation PTOC., 12,222 (1953). 25. Cohn, W. E., and Volkin, E., Arch. Biochem. and Biophys., 36, 465 (1952). 26. Dounce, A. L., and Kay, E. R. M., PTOC. Sot. Exp. BioZ. and Med., 83,321 (1953). 27. Jones, M. E., Lipmann, F., Hilz, H., and Lynen, F., J. Am. Chem. Sot., 76, 3235

(1953). 28. Potter, V. R., Advances in Enzymol., 4, 201 (1944).

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Brumm and Dwain M. WhitePotter and With the assistance of Anne F.

Hanns Schmitz, Robert B. Hurlbert, Van R.CYTIDINE, GUANOSINE, AND URIDINEMONO-, DI-, AND TRIPHOSPHATES OF

NUCLEOTIDE METABOLISM: III.

1954, 209:41-54.J. Biol. Chem. 

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