COMNUNICATIONS TO THE EDITOR
3928
VOl. 79
hIr. Joseph F. Alicino (Squibb Institute for Medical Research, New Brunswick, New Jersey).
slopes, 0.45 and 0.65, respectively, are typical of randomly coiled chains: we conclude that this is the configuration under these conditions. DEPARTMENT OF CHEMISTRY CARLDJERASSI However, below p H 5 in 0.15 d P salt and below WAYNESTATEUNIVERSITY DETROIT,MICHIGAN 0. H A L P E R N $H 6.5 in water the p0ly-~4samples displayed a RECEIVED JUNE 3, 1957 different spectrum having a maximum a t 252 ml.c (El% 255) and had values of so and [ q ] that dePOLYNUCLEOTIDES. 1, MOLECULAR PROPERTIES pended On the concentration and the temperature AND CONFIGURATIONS OF POLYRIBOADENYLIC a t which the transition had been brought about ACID I N SOLUTION’ by lowering the pH. By varying the concentration of poly-A (0.2 to Sir: We wish to report the discovery of two confipra- 1.5 g./100 CC. for the sample having a molecular : one a weight of 126,000) prior to lowering the PH to 4.9 tions of polyriboadenylic acid (PO~Y-A) flexible, randomly coiled, molecularly dispersed (acetate buffer of ionic strength 0.15) the solutions form and the other a rigid, presumably helical were found to exhibit systematic variations in so form composed of variable numbers of poly-^ and [VI that were clearly indicative of the formamolecules. tion of stable aggregates. The weights of these Using polynucleotide phosphorylase from Azote- aggregates increased with the concentration Of bactar vinelandii,2 samples of po1y-A were prepared poly-24 a t which the transition was induced. at various enzyme-substrate ratios and collected Calculating the molecular weights (or particle a t different extents of reaction in order to provide weights) as before, it was found that the so and [ q ] varied with weight as shown in the right side of Fig. covering a Ride molecular weight These samples in solution above p~ 6.5 in water 2. The linear relations that result and the values or above PH 5.7 in 0.15 Izd salt, exhibited a char- of the slopes, 0.36 and 0.92, respectively, indicate acteristic absorption spectrumhaving a maximum that the species are homologous in structure and 295), were not birefringent, and that the differences in weight are the result of difat 257 ml.c displayed sedimentation constants, and intrinsic ferent extents of linear growth. Unlike the ranviscosities, [?]$ that depended on molecular weight domly coiled configuration, this form does not show in the manner shown at the left side of ~ i 1.~ a .marked increase in specific viscosity upon removal The molecular u7eights Were calculated from the of salt. This and the behavior in flow birefringence Flory-Mandelkern equation3 using 2.3 x 106 for lead to the conclusion that the structure is rela/p and 0.55 for partial specific volume. ~h~ tively rigid and rod-like, but not quite to the extent found for deoxyribonucleic acid (DNA). The v7-1 stability of this structure appears to result from the lowering of the electrostatic energy brought about by titrating about half of the adenine groups. The failure to find spectra intermediate in charI NT E 3 RU PTED acter between the two described further suggests that the transition is abrupt and complete and that practically all the chromophoric groups (adenine) undergo the same change in environment. Moreover, a t p H 4.9 in 0.15 Ad acetate, the transition to the randomly coiled form occurs in a very narrow temperature range near ‘7;io, only a few - COIL degrees below the transition temperature observed for DNA. This cooperative behavior indicates a highly ordered arrangement of secondary bonds for the acid-stable form. 02 These observations are compatible with a strucoil I 1 I I L A ture in which poly-A molecules are associated IO& 5 ~ 1 0 45 5 t ~ io6 ~ 5 5 x io6 through hydrogen-bonded base-pairing in a double stranded helix,4 each strand of which has gaps where Molecular weight. one poly-A molecule ends and another begins. Fig. 1.-Molecular weight dependence of sedimentation This would resemble the interrupted helical model constant, $0 in svedbergs aiid intrinsic viscosity [7]25, in once proposed for DNA,5 but the gaps in our case dl /g., of t h e two configurations of poly-A. For the curves may be larger. The increased rotational freedom on the left, the solvent was 0.15 NaC1, 0.015 M citrate, at the gaps would account for the smaller space $11 7.1; for those on the right it was 0.15 Macetate, PH 4.9. filling properties exhibited by this configuration
1
I
1
,
The open circle ( 0 ) represents measurements a t pH 3.7 with no added salt present. (1) This investigation was supported b y a research grant (C-2170) from the National Cancer Institute, Public Health Service. (2) 11. Grunberg-Manago and S. Ochoa, THISJOURNAL, 77, 3165 (1055): M. Grunberg-hlanago, P. J. Ortiz and S. Ochoa, Biochim. Bioghyr. Acta, 20, 169 (1056). W e are very grateful t o Professor Ochoa for making. available t o us unpublished information on the purification of the enzyme. (3) P. J. Flory and L. Mandelkern, J . Chcm. Phys., 2 0 , 212 (1952).
(4) A two-stranded structure of this type has been proposed by F. H. C. Crick, D. Davies, A. Rich and J. D. Watson and is discussed by J. D. Watson in “The Chemical Basis of Heredity,” W. D. McElroy and B. Glass, editors, the Johns Hopkins Press, Baltimore, 1957 and by A. Rich in the Proc. Acad. Sci., Ncw Y o r k , in press. It is not necessary t h a t the detailed atomic arrangements of the helical configuration in solution be the same as t h a t which may ultimately be established in t h e solid state. (5) C.A. Dekker and H . Schachman. Proc. N a f l . A c a d . Sci., 40, 894 (1954).
July 20, 1 0 5 i
CONXUNICATIONS TO THE EDITOR
3929
cinate, from the four carbon chain, was isolated by chromatography on Dowex 1*and on pa.per using aqueous pheno1.I The succinate isolated from the oxidation product of synthetic spermidine,8 labeled in the four carbon chain, contained about 33y0 of the radioactivity u t on the Dowex 1 (6) We are very much indebted t o Mrs. Elizabeth Klemperer who column, while less than 0 . 3 h of the radioactivity was reponsible for many of the measurements in this investigation. HARVARD UNIVERSITY JACQUES R. FRESCO was found in the succinate from the oxidation CAMBRIDGE, MASSACHUSETTS PAUL DOTY products of isolated spermidine and synthetic spermidine8 labeled in the three carbon chain. RECEIVED ,MAY 23, 1957 The low level of activity in the succinate shows that the isolated spermidine is not. labeled in the INCORPORATION OF T H E CARBON CHAIN OF four carbon chain and is consistent with labeling METHIONINE INTO SPERMIDINE'J in the three carbon chain. Sir: TABLE I1 The cumulative work of several laboratories has Total radioactivity in spermidine fractions, c.p.m. indicated the metabolic importance of the polyfiH f ATP - ATF amines putrescine, spermidine and spermine in 7.2 2 . 1 x 104 520 diverse biological systems. Tabor, et ~ l . have , ~ 8.2 2 . 5 X IO3 795 shown that putrescine is the source of the fourIncubation, four hours a t 37"; incubation mixture, 100 carbon chain of spermidine and spermine. Recent Mg. 2 c " - ~ ~ - m e t h i o n i n(3.8 e X lo6 c.p.m.) per vessel, 0.01 kf results obtained by the author show that the side putrescine, 0.008 M glutathione, 0.003 M MgClZ, 0.~30001,I1 chain of methionine is an efficient precursor of the pyridoxal phosphate, 0.1 M buffer, 2.5 ml. 1 : 1 extract of 74A-3b, 0.02 M KrATP as indicated, total volume 5 ml.; three carbon chain of spermidine in Neurospora buffers are p H 7.2, potassium phosphate + Tris hydrochlocrassa. ride (1 : l ) , pH 8.2, Tris hydrochloride. Neurospora crassa strains 74A-3b (wild type) and A possible mechanism for this reaction is the 38706 (blocked in the conversion of homocysteine to methionine) were grown a t room temperature transfer of the methionine side chain from S-adenoon Fries minimal medium supplemented with 50 syl methionine to putrescine in a manner similar pg./ml. of ~ ~ - m e t h i o n i n e - 2 C ' ~ The . spermidine to the transfer of the methyl group of thi.s com~ h e almost was isolated by grinding the mold with sand and pound first observed by C a n t ~ n i . T 5% TCA4 and chromatography of the extract on absolute ATP requirement for the incorporation of 0.6 X 20 cm. columns of Dowex 50-X2 using an HCI C14 methionine into spermidine by a cell free exgradient. The spermidine containing fractions tract of 74A-3b as shown in Table I1 is corisistent were combined and dried; spermidine trihydrochlo- with this hypothesis. Further evidence for the ride was crystallized from methanolic hydro- presence of radioactivity in the spermidine from gen chloride and ethyl acetate. The specific activi- the PH 7.2 incubation mixture was obtained by ties of the added methionine and the isolated paper ionophoresis in PH 8.2 borate buffer. Tabor, spermidine are compared in Table I. Amine et aZ.,1° have extended these studies in extracts of concentrations were determined as described by E. coli and have found further evidence that SRosenthal and Tabor.6 "Radiopurity of the iso- adenosylmethionine is an intermediate. ( 6 ) J. K . Palmer, Bull. 589 Conn. Agric. Expt. Station (1955). lated spermidines was checked by paper ionophore(7) H. K.Berry, H. E. Sutton, L. Cain and J. S. Berry, Biochemical sis in pH 8.2 borate buffer and by descending paper Institute Studies IV. No. 5109, Univ. of Texas, Austin, Texas, 1951, chromatography (3 parts 1-propanol :1 part 0.2M p. 22. sodium acetate buffer). I n all cases the radioac(8) Kindly supplied by Dr. E. Jackson of the National I m t i t u t e of tivity moved in one spot and was in the same Arthritis and Metabolic Diseases. (9) G. L. Cantoni, "Phosphorus Metabolism," Vol. 11, Johns Hopposition as added authentic spermidine trihydro- kins Press, Baltimore, Md., 1952, p. 129. chloride. (10) H. Tabor, S. M. Rosenthal and C. W. Tabor, personal communication. TABLE I
relative to DNA. If this structural proposal is the correct one, it may be possible for ribonucleic acid, also, t o assume a double-stranded configuration in solution through pairing of only the adenine nucleotides.6
Strain
74A-3b 38706
Specific Activities Methionine Spermidine (initial) (isolated) c.p.m./pmole c.p.m./smole
6.36 X lo4 1 1 . 4 X lo'
3 . 3 8 X 104 6.34 X 10'
Ratio Spermidine/ Methionine
0.53 0.56
Spermidine was degraded by oxidation with alkaline permanganate. The small yield of suc(1) A preliminary report of this work was presented before the American Society of Biological Chemists: Federation Proc., 16, 189 (1957). (2) This work was supported in part by Research Grant C-3436(A) from the National Cancer Institute, National Institutes of Health, Bethesda 14, Md. (3) H. Tabor, S. M. Rosenthal and C. W. Tabor, Federation P70C.. 15, 367 (1956). (4) Abbreviations used are: TCA, trichloroacetic rcid: ATP, adenosine triphosphate; Tris, tris-hydroxymethylaminomethane. (5) S. M. Rosenthal and C. W. Tabor, J. Pharmacol. and E x p t l . Thrr., 116, 131 (1956).
RADIOISOTOPE SERVICE,VETERANSADMINISTRATICIN HosPITAL, AND DEPARTMENT O F BIOCHEMISTRY, DUKE~JXIVERSITY, SCHOOL OF MEDICINE,DURHAM,NORTH CAROLINA, AND NATIONAL INSTITUTE OF ARTHRITISAND METABOLIC INSTITUTES O F HEALTH, BETHICSDA 14, DISEASES, NATIONAL MARYLAND RONALD C. GREENE RECEIVED MAY9, 1957 T H E HETEROGENEITY OF POLYSACCHARIDES A S REVEALED BY ELECTROPHORESIS O N GLASS-FIBER PAPER
Sir: Glass-fiber paper' has been used in conjunction with a borate buffer p H 9-10 for the electrophoretic separation2of a wide variety of organic substances (1) M. J. O'Leary, R . B. Hobbs, J. K. Missimer and J. J Erving, T a p p i , 87, 446 (1954). (2) D. R. Briggs, E. F. Garner, R . Montgomery and F. Smith, A d . Chcm., 28, 1333 (1956).
