VOL. 33 (1959) PRELIMINARY NOTES 281

cell walls of S. aureus was, therefore, also reinvestigated (Table I) and was also found o/ to be 33 /o L-alanine and 67 O/:o D-alanine. The data, therefore, are consonant with

the original interpretation. Elsewhere, data are being presented which have led to elucidation of the amino

acid sequence of the peptide as Ala-Glu-Lys . Ala. AlaS, ~. Two precursors of UDPAG- Lact-Ala. Glu. Lys .Ala .Ala are known, viz. : UDPAG-Lact-Ala 1 and UDPAG-Lact- Ala. Glu G, in each of which the alanine residue is IOO% L-alanine. If, as seems likely from these data, it is presumed that the first alanine residue in the peptide sequence is also L-alanine, the other two alanines must be D-alanine residues. The glutamic acid in the peptide is entirely D-glutamic acid and the lysine is entirely L-lysinO,4, 8. The sequence of the peptide is, therefore, L-Ala. D-Glu. L-Lys-D-Ala" D-Ala.

This work was supported by Grant 3981 from the U.S. National Science Foun- dation.

JACK L. STROMINGER

Department o/Pharmacology, ROBERT H. THRENN Washington University School o] Medicine,

St. Louis, Mo. (U.S.A.)

1 j . T. PARK, J. Biol. Chem., I94 (1952) 877, 885, 897. 2 j . L. STROMINGER, J. Biol. Chem., 224 (1957) 509. 3 j . L. STROMINGER AND R. H. THRENN, unpubl ished observat ions. 4 j . T. PARK AND J. L. STROMINGER, Science, 125 (1957) 99. 5 j . L. STROMINGER, Compt. rend. tray. lab. Carlsberg, Ser. Chim., in the press. 6 j . L. STROMINGER AND R. H. THRENN, J. Pharmacol. Exptl. Therap., 122 (1958) 73 A, and

Biochim. Biophys. Acta, in the press. 7 L. ~V. MCCAMAN AND O. H. LOWRY, unpubl ished observat ions. 8 H. B. BURCH, O. H. LOWRY, A. M. COMBS AND A. ~'~I. PADILLA, Abstracts, Div. Biol. Chem.,

Am. Chem. Soc. i29th meeting, Dallas, April (1956).

Received January 3oth, 1959

The exceptional resistance of certain oligoribonucleotides to alkaline degradation

Studies of the alkaline hydrolysis of RNA have indicated that, following digestion for 24 h in I M alkali at room temperature, a fraction is always found which is distinguished by the fact that it requires much stronger eluents for its removal from anion-exchange columns than are needed for the elution of the mononucleotides 1,2. We have verified DOUNCE'S finding s that the u.v. absorption of the fraction is due primarily to the presence of adenine and guanine. We regularly find that the fraction represents 5 % of the absorption of our I N KOH hydrolysates at 2600 Jk. RNA has been prepared by many different methods from yeast and mammalian tissue and has without exception yielded this fraction which is bound tightly to anion-exchange resins.

The isolation and fractionation procedures will be published in detail elsewhere but briefly they consisted of adsorbing the neutralized hydrolysate of RNA on

Abbreviat ions: RNA, ribonucleic acid; ApAp, ApUp, etc., see J. Biol. Chem., 233 (1958) 3.

282 PRELIMINARY NOTES VOL. 33 (I959)

Dowex-i-acetate, eluting the mononucleotides with I M acetic acid + o.15 M sodium acetate and then eluting the tightly bound material with aqueous solutions of acetic acid and Na2S04 (S04 = is a strong eluting agent which allows the removal of the fraction from the column to be carried out at a relatively high pH of 2.8). Preliminary separation of three sub-fractions was possible by varying tile concentrations of acetic acid and Na2SO 4 (Fig. I).

Single compounds can be obtained from each of these three sub-fractions by chromatography on DEAE-cellulose. Using paper chromatography with an iso- propanol-e thanol- tar t ra te buffer system developed by Drs. MOSCARELLO and HANES in this laboratory, nine different compounds have been detected in addition to five other spots which are believed to be mixtures of tri- and tetra-nucleotides.

One compound which is found in the first subfraction has persistently been the major non-mononucleotide component of the alkaline hydrolysates. I t was isolated in a pure state and found to contain equimolar amounts of adenine, ribose and phosphorus. The compound, although relatively stable in alkali, could be degraded to adenylic acid after a further 24-h incubation in I M KOH at 25 °. I t was suspected that the compound might be diadenylic acid (ApAp) and, as such, might appear in alkaline hydrolysates of polyadenylic acid. Polyadenylate was hydrolyzed in I M K 0 H at 25 ° for 2, 5, IO and 24 h. Chromatography of the hydrolysates in the iso- propanol-e thanol- tar t ra te buffer system showed that a compound with the same RE

as the compound suspected of being ApAp appeared in large amounts and reached a maximum of 30 % of the hydrolysate after IO h (Fig. 2). The remarkable feature of the hydrolysis of polyadenylate is that after 2 h there are only negligible amounts of mononucleotide liberated whereas good preparations of RNA from yeast prepared by the method of CRESTEIELD 6t al. a were converted to mononucleotide to the extent of 50-85°0 in the same period. This result is substantiated by the time-courses of hydrolysis of ApAp, ApUp and GpCp (Fig. 3) which clearly indicate the marked resistance of ApAp to hydrolysis in I N KOH relative to the other two compounds. This exceptional resistance of ApAp to alkaline hydrolysis would account for the

' O.SMHAc, c, I O.6MNao SOz.

SO4

- VC~LUM£ OF" E F F L L } E N T " ( L )

Fig. i . F r a c t i o n a l e l u t i o n of o l i g o n u c l e o t i d e s f r o m

D o w e x - I - a c e t a t e .

100 1 -- .

oj/

0 6 12 18 24h

Fig . 2. T i m e c o u r s e of t h e h y d r o l y s i s of p o l y a d e n y l a t e

in I 34 K O H a t 26 ° .

8°F

~ 2o

0 4 8 12 16 20 24 TIME (h)

Fig. 3- T i m e c o u r s e of h y d r o l y - sis of d i n u c l e o t i d e s in i ~,~I K O H

a t 26 °.

VOL. 33 (1959) PRELIMINARY NOTES 283

resistance of polyadenylate to alkaline degradation as well as explain why ApAp is the major non-mononucleotide constituent of alkaline hydrolysates of RNA.

The rate at which mononucleotides are liberated by alkaline hydrolysis from different preparations of RNA varies widely. For instance, the RNA fraction of DAVIS AND ALLEN which is particularly rich in their "fifth mononucleotide" ~ is much more resistant to alkaline hydrolysis (even after purification by the salmine-precipi- ration technique) than the RNA which forms a gel ill I M NaC1. This increased resistance to alkaline degradation may be at least partially attributable to a relatively high proportion of resistant inter-nucleotide linkages.

Using the methods developed by MARKHAM AND SMITH 5 for the identification of oligonucleotides, we have been able to identify ApAp, ApGp, GpCp, GpAp and ApUp in the 24-h, I M KOH hydrolysates of RNA*. Of the four remaining compounds which have been studied, base analyses indicate that two of the compounds contain only guanine, one contains equimolar amounts of adenine and guanine and the other contains equimolar amounts of guanine and uracil. These compounds are resistant to degradation by alkali and may be the same as those reported by ALLEN et al.~, 7.

It is interesting that all of the compounds which have been identified contain inter-nucleotide linkages which are resistant to RNase. This may be coincidental but it is possible that if RNase acts by allowing an OH- catalysis to occur near neutral pH's (similar to the idea of HELLERMAN for arginase action s) then the inherently greater resistance of various linkages to OH- catalysis may be of some significance in accounting for their resistance to enzymic hydrolysis.

The authors wish to express their gratitude to Dr. S. OCHOA for the polyadenylate used in this work and to Dr. G. SCHMIDT for the very generous supply of prostatic phosphatase used in the identification of the dinucleotides. The authors also wish to thank Professor C. S. HANES and Dr. M. MOSCARELLO for allowing us to use their paper-chromatographic system and to refer to its use in this paper prior to its publication.

This investigation was supported by the National Research Council, Canada.

Department o/Biochemistry, University o~ Toronto, Toronto (Canada)

t3. G. LANE*"

G. C. 13UTLER

1 L. COHEN,'Ph.D. thesis, Univers i ty of Toronto, 1954. 2 j . L. POTTER AND A. L. DOUNCE, J. Am. Chem. Sue., 78 (1956) 3o78. 3 A. M. CRESTFIELD, K. C. SMITH AND F. ~V. ALLEN, J. Biol. Chem., 216 (1955) 195. 4 F. F. DAVIS AND F. ~¥. ALLEN, J. Biol. Chem., 227 (1957) 907. 5 R. MARKHAM AND J. D. SMITH, Biochem. J., 52 (1952) 558.

K. C. SMITH AND I ~'. ~¥. ALLEN, J. z~n~. Chem. Sue., 75 (1953) 2131. 7 j . v¢. KEMP AND F. \¥. ALLEN, Biochim. Biophys. Aela, 28 (1958) 51. 8 A. LEHNINGER, Physiol. Revs., 3 ° (195o) 393.

Received December 23rd, 1958

* The dinucleotides formed in the alkaline hydrolysa tes are mix tures in which about 65 % of O / the secondary phosphory ls are 3' and 35 /o are 2'.

** Holder of a Research S tudentsh ip f rom the National Research Council, Canada.