polybenzoate derivative gave an elemental analysis and molecular weight which corresponds to a heptulosan tetrabenzoate. -4nal. Calcd for CJeH28010:C, 69.2; H, 4.62; mol. wt , 608. Found: C, 69.7; H,4.86; mol. wt. (Kast), (313.
homogeneous when an acid hydrolysate gave tiearly iiitegral molar ratios upon amino acid analysis'* : - & h a , Xrg,, 9sp*, Glus, Glyd, His, Leu, Lys4, Met, rhea, Prod, SerJ, Tyr?, Val3 One mole of tryptophan was found. i Its homogeneity was further confirmed by finding, upon tryptic digestion, thc TUFTS U N I V E R S I T Y S C H O O L O F MEDICINEIISIES-GIEH SIC number of peptides containing tryptophnii, histiAND THE NEW ENGLAND CENTER HOSPITAL YIJAI K NIGAM dine, methionine, tyrosine and arginine TTXS coii30 BENXETSTREET, BOSTON11, hIASS. WILLIAMI1 FISIIMAUsistent with the amino acid coniposition and ihc specificity of trypsin. RECEIVED SEPTEMBER 23, 1959 Carboxypeptidase digestion of the hormone and of the C-terminal octadecapeptide isolated from a tryptic digest indic3ted the C-terminal sequence to ISOLATION AND STRUCTURE OF HUMAN be Leu. Glu.Phc. The N-terminal sequence14 of CORTICOTROPIN (ACTH)' the horinone W ~ Sfound to be Scr.Tyr.Ser.Alet. Sir: GlU. Tryptic digestion split the hormone into four Preliminary structural work on a corticotropin isolated from acetone dehydrated human pitui- major and three minor fragments. They were T~-----H+Taa -+ separated by i6nophoresis In pyTz / ~ r . T y r . S e r . M e t . G l u ( H i s , P h e , A r ~ ! ~ ~ ~ ~ s ~ , P r o , V a ~ ) ~ ( L y s i ,A r ~ ~ridine-acetate ,l~ro.Val~~ buffer at p H 6.5 and - ( V a l . T y r , P r o ~ , A s p ~ , S e r ,TCIJ l y , G l u ~ , A l a ~ , P h e ) L e u+I, G l u . P h e . ~ purified further by paper chromaFig. 1.-Constituent amino acids and possible arrangement of fragtography' The positiolls of each peptide and their constitmeats from tryptic digestion of human ACTH. Three additional pepamino acids'G are shoW1l Fig' tides ( T 4 b , T5, T6) were obtained. Their composition indicated that they 1. Peptides with composition simiwere derived from Ta,. The relative positions of Ta and T4%have not lar to those isolated from human been ascertained. By analogy with ACT11 from other species, tlie material have been reported for porstructure shown seems t o be correct. cine,4 ovine3 and bovine3 ACTH: (12) S. Moore, D. H . Spackman and W.H. Stein, A t i d . Cirrm., 30, taries indicated that it is similar to corticotropins 1185 (1938). from other s p e ~ i e s . ~ ~ ~ ~ ~ (13) A. B. Lerner and C. P. Barnum, Arch. Biochetn., 10, 417 (1040). An oxycellulose purified concentrate of AISH (14) H. Fraenkel-Conrat, J . I. Harris and A. L. Levy, "Methods of and ACTH, from human g l a n d ~ , was ~ J ~adsorbed ~ Biochemical Analysis," edited by D . Glick, Interscience Publishers, Inc., New Pork, PIT. Y.,1955, pp. 383-3'37. on a diethylaminoethyl cellulose8 column a t 5'. T h e systems used were either 1-butanol-acetic acid-water Gradient elution was established to 0.2 ilf) pII ( 4 :(15) 1 : 5 ) or 1-butanol-acetic acid-pyridine-water (30:6:24:20). 5.5, through a mixing flask of 300 ml. of 0.005 M , (16) Amino acid composition was determined by ion-exchange p H 7.0 ammonium acetate buffer. Since ACTH chromatography12 or paper chromatography in the butanol-acetic possesses intrinsic MSH a c t i ~ i t y ,the ~ in vitro acid-water ( 4 : 1 : 5 ) system. T E I I 11. LEE frog skin bioassay'O was used to locate active MSH S E C T I O N O F DERMATOLOGY AAROX13. LERNER OF MEDICIKE and ACTH fractions. The major fraction was DEPARTMENT U S i V E R S I T Y S C H O O L O F MEDICISE purified further on a carboxymethyl celluloses YS 1A2 Ll VE IIAVEN 11, C O N N E C T I C U T V I N A B U E I . T S E R - ~ A U G S C l I column; stepwise elution was used with 0.05 M , R E C E I V E D AUCXST 25, 1959 p H 5.9, and 0.25 M , $H 6.9, ammonium acetate. Two active fractions were resolved. Fraction A possessed MSH but no ACTH activity. Fraction STRUCTURE OF MELATONIN' B possessed 26 USP units of ACTH" and 4 X 104 S i r : units of intrinsic MSH per mg. and was judged Melatonin, I, found in bovine pineal glands and in smaller amounts in peripheral nerves of man, ( 1 ) This investigation was supported by grants from the Amcrican Cancer Society a n d t h e United States Public Health Service. monkey and cattle, is the most effective known (2) W, F. White and W. A. Landmann, THISJ O U R N A L , 77, 1711 lightening factor of frog (Rana pipiens) (1055). Unfortunately, I exists in pineal glands in such (3) C. H. Li, I. I. Geschwind, R. D. Cole, I. D . Raacke, J. I. IXarris minute quantities t h a t conventio~~alapproaches and J. S. Dixon, Nalirre, 176, 687 (1955); C. IT. Li, J. S. Ilixon and D. Chung. THISJOURNAL, 80, 2587 (1059). to studying its structure were impossible. We (4) R . G. Shepherd, S. D. Willson, K . S. Howard, P. H. Bell, D. S . wish t o report experiments t h a t led to the concluDavies, S. I3 Davis, E. A. Eigner and N. E. Shakespeare, ibid., 78, 5067 sion that I is N-acetyl-5-methoxytryptamine. (1956). I and 5-methoxyindole-3-acetic acid, 11, also ( 5 ) We wish t o express o u r gratitude t o Dr. M. S. Raben for the generous donation of buman A C T H crude concentrate, t o Dr. J. D. Fisher present in pineal glands, were isolated by a profor ACTH assays, and t o D r . W. F. White for the generous donation of cedure previously described.b V5th 1.5y0methanol
If
a highly purified carboxypeptidase preparation. (6) R . W. Payne, M.S . Raben and E. B. Astrvwd, J . Dial. Ciwin , 187, 719 (1950). (7) E. B. Astwood, R l . S. Raben, R . W. Payrie and A. T3. Grady, THISJ O U R N A L , 73, 2969 (1951). (8) E. A. Peterson and 13. A. Sober, ibid., 78, 7 a l flQ5G). (9) P. H. Bell, ibid., 76, 5565 (1954). (10) K. Shizume, A . B. Ixrner anrl T. B. I;itxpntrick, End&crinoL., 5 4 , 533 (1854). (11) This prcpar:ttirifi was f r , u r i i l t o Iir l i i i ? t : i l j l e I>v I ) r I . 1) Fisher.
(1) This investigation was supported hy grants from t h e United Ytntes Public Health Service. 12) A. B. Lerner, J. D Case, I'. Takahashi, T.H . Lee a n d W. Xlori THISJOURNAL, 80, 2587 (1958). (3) A. B. Lerner, J. D. Case, W. Mori and M. R . Wright, .Vafw?, 183, 1821 (1859). ( 4 ) A B. Lerner a n d J. U. Case, J . I n w s f . Z l i ~ n n t . ,32, 211 (1959). (5) A . B. Lerner, J , I). Case, K. Diernann. I).
