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 to 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 THE 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 Imtitute 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).
