Separation of nucleotides, nucleosides, and bases from ribonucleic acid on Dowex-1 X 8

275

PRELIMINARY NOTES

the R N A biosynthetic pathway, possibly at the IMP stage, is more readily accessible to M A P administered as the free base than to adenine. Such a conclusion implies the existence of an efficientsalvage pathway for MAP. Preliminary results indicate that certain of the yeast fractious active with respect to A M P and I M P pyrophosphorylases x° can condense 5-phosphoribosylpyrophosphate with MAP. Studies directed toward the isolation of the resulting nucleotide and its possible subsequent conversion by deaminases have been undertaken.

Laboratory o/ Chemical Ph~[~acology, National Heart Institute, .Bethesda, Md. (U.S,A.)

D. E. DUGGAN E. TITUS

t j . W. LITTLI~FIELD AND D. B. DUsN, Siochem. ]., 7° (x958) 642. t D. B. DUNN, Biochim. Bioph~/s. Acta, 34 (z959) 286. t F. F. DAvm, A. F. CASLUCCX.aND I. F. ROUBEXN, J. Biol. Chem., 234 (x959) x525. * D. B. I)UNN, J. D. SMXTX~AND P. F. SPAHR, J. Molec. Biol., 2 (x96o) xx 3. i C. N. ] ~ M v , Federatio~ Pro¢., x9 (I96O) 3x3. * D. E. DUCa3AN AND E. O. TXTUS, J. Pharmacol. Exptl. Therap:, x3o (I96O) 375T D. E., DUC~AN, M. G. WSmZRT ASP E. O. Txzus, Cancer Research, 2x (x96I) xo47. s L. E. GAUDETTE AND B. B. BRODIE, B~ochsm. Pha~,ma¢ol., 2 (x959) 89. • C. G. MACK~NZm AND W, R. FmSZLL, J. Biol. Chem., 232 (x958) 4x7 • l• A. KORNBERG, I. Lme~RUAN AnD E. S. SXuMS, J. Biol. Chem., 2x 5 (x955) 4x7 •

Received November

2nd, x96I Biochim. Biophys. Acta, 55 (1962) 273-275

Separation of nucleotides, nucleosides, and bases from ribonucleic acid on Dowex-I X 8 No single method for the chromatographic separation of the bases, nucleosides, and nucleotides obtainable from KNA has been described. In previous studies incomplete separation of members of these three classes of compounds have been obtained on an anion-exchange column employing buffers ranging from pH Io.2 to 2..75 (ref. I). During the chromatographic "analysis of acid-soluble nucleotides from liver, a number of closely spaced peaks were observed very early in the run. Subsequent work suggested that these were nucleosides and bases. Extended studies have shown that excellent: separation of these substances, in addition to nucleotides, may be obtained on Dowex-I resin by adjustment of temperature, flow rate, resin particle size, column length, pH and ionic composition. A representative chromatogram obtained with an artificial mixture is shown in Fig. I. To maintain the relative order of all components, the entire elution was carried out at one pH, in this instance pH 4.4- Data on buffers, temperature and flow rates are given in the figure and legend. Thymine and inosine were added to indicate their position on the chromatogram. The technique allows the breakdown products obtained from RNA by either alkaline or acid hydrolysis or by irradiation to be analyzed on one column. A complete description of the automatic analytical system and operaBiochim. Biophys. Acta, 55 (x962) 275-276

e76

PRELIMINARY NOTES

i cY~o~

0.5

I

,

1.0

l URACIL

-71

~1 _o.,,.,~!1 !11 20"

! i

1111

0.2

I

~ | ~NOS~NE

,

~

,n

~

I|ll

[.t

i111

n A o.,= I I

r

-'~

°~

II

1.5"

Ikl r,v/

I

CYTIOYLIC3'

I

I!1

i!1

,

CYTIDYLICl 2 'I7' ]

I1

O! 0

I

~ \, 2

3

4

ri_U.. 5

6

- - f~l t

I III

k

< 03

,7

L_ I ....... / LJ I

8

?

9

tO

H

~P

TIME (h)

i

URIDYLIC 3'7

~.0

I

n

ADENYLIC 5h

>- O.5 U Z< nO

II

GUANYLIC13'-'l'v- - "

,

ADENYLIC 2'--~

A

<

ue©YUC 2'- I I;

.... t ..... t3

:~' i~z...... 14

;I

^/

l 0.1

_ _

45

J/

\7

,.

f 46

17

~8.

It00m[--~ '19

20

24

22

23

24

TIME ( h )

Fig. I. Separation of a mixture ofribonucleotides, nucleosides, and bases by anion exchange. Exchanger: Dowex-I X 8 acetate, hydraulically si~ed from through 4oo-mesh material, o.9 × zSo cm. Eluting solution: o.6 M sodium acetate grading linearly to 2 M sodium acetate, all at p H 4-4; flow and temperature: o.48 ml/min a t 20 ° changing to 0.82 ml/min at 45°; recording: absorbancy at 26o (solid line) and 280 nap (dashed line) recorded a t 5-sec intervals using I-cm quartz flow cells and a recording system developed a t ORNL (ref. 2). • "

t ional procedures together with results obtained with DNA degradation products and tissue acid-soluble nucleotides will be published elsewhere.

Biology Division, Oak Ridge National Laboratory,* N . G . ANDERSON Oak Ridge, Tenn. and The Catkolic SISTER FRANCIS CLARE LADD University of America, Washington, D. C. (U.S.A.) 1 W. E. ColiN, in E. CI..IARGAFFAND J. N. DAVIDSON, The Nucleic Acids, Vol. x, Academic Press, Inc., New York, p. 2 i i . s N. G. ANDERSON, Anal. Chem., 33 (x96x) 97 o.

Received October 5th, 1961 * Operated b y Union Carbide Corporation for the U.S. Atomic Energy Commission.

Biochim. Biopkys. Acta, 55 (z962) 275-276