8 14
VOl. 77
COMMUNICATIONS TO THE EDITOR
-
2.5 x
4c3 M
. I‘-
EDTA
0 . 2 5 M EDTA pH -4.2
p H = 40.9
cs
-
Li
4 10
9g
\\
8 7
6
5 25
30
35
40
VOLUME OF E F F L U E N T (ml.).
Fig. 1.-Separation
of alkali metals by anion exchange.
which are analogous to the acid uptake described earlier .8 Acknowledgment.-The author is indebted to Dr. K. A. Kraus for helpful criticisms and advice
and t o Mrs. L. W. Magnusson, Jr., for valuable technical assistance.
oAgR~~~~N~~~~~~~L~~~~~~~~~ OAKRIDGE,TENNESSEE
COMMUNICATIONS T O T H E E D I T O R THE ACTIVATION OF CHYMOTRYPSINOGEN
10-30 fold molar excess with respect to active chymotrypsin of diisopropylphosphofluoridate (DFP). Free peptides were isolated by applying tion2 of chymotrypsinogen is accompanied by the to an ion exchange column (0.9 X 15 cm.) of XE-64, formation of several peptides, probably arising 150-400 mesh, Na+ form’ either the activation from a common precursor, containing 8 or 9 mixture in the presence of DFP, or the fraction different amino acid residues. We now wish to soluble in 10% trichloroacetic acid (TCX). Elureport the isolation and structure of a dipeptide tion with 0.3M sodium citrate buffer, PH 5.3, which is probably the only peptide liberated in yielded a sharp peptide peak which emerged with stoichiometrically significant amounts during the the same effluent volume as arginine. The yield of peptide was approximately 0.8 mole per mole of activation of chymotrypsinogen. Electrophoretic analysis, end group a n a l y ~ i s , ~chymotrypsinogen activated (molecular weight specificity requirements of the activating enzymes 23,000). Acid hydrolysis of the peptide yielded and other considerations1 suggested that a basic approximately equal amounts of serine and arginpeptide was liberated during the conversion of ine. Analysis of the acid hydrolyzate of the entire n--chymotrypsin to the 6-form. Hence activation TCA-soluble fraction yielded the same two amino mixtures were prepared under conditions yielding acids in stoichiometrically significant amounts, preponderantly 6 - c h y m o t r y p ~ i n . ~The ~ ~ products in addition to much smaller amounts of numerous amino acids. When the peptide was converted of activation were characterized by activity to the dinitrophenyl derivatives and subsequently measurements (esterase6) and by electrophoresis in hydrolyzed, only DXP-serine was obtainedg on acetate buffer, pH 4.97, usually in the presence of a paper chromatograms, indicating that the peptide (1) H . Keurath, J. A. Gladner and E. W. Davie, in W. D . McElroy had the structure seryl-arginine. The same pepand B. Glass, “The Mechanism of Enzyme Action,” John Hopkins tide was observed by paper chromatography as Press, Baltimore, Md., 1954. product of the slow activation of chymotrypsino( 2 ) M. Kunitz and J. H. riorthrop, J . Gen. Physiol.. 18, 433 (1935). gen, whereas it was absent under conditions yielding (3) R D . Wade and W. J . Dreyer, unpublished experiments. (4) F. R. Bettelheim and H. Xeurath, J . Bid. Chem., 2 1 2 , 241 predominantly a-chymotrypsin, thus indicating (19.56). that i t could only have arisen during the n--6
Sir : It was previously reported’ that the slow activa-
3v4
( 5 ) C. F. Jacohsen, Compt. rend. Irav. Lab. Carisberg, Serie chim., 26, 325 (1Y47). (6) S. Kaufman, H. Iieurath :%nd G. W. Schwert, J . B i d . Chem., 177, 493 (1949).
(7) C.H W. Him, S Moore and W H Stein, rbrd , 200, 493 (1953) (8) F Sauger, Bmchem J , 39, 607 (1945), 46, 126, 563 (1949) (9) S. Blackburn and A F Lowther, rbrd , 48, 126 (1951)
Feb. 5, 1955
COMMUNICATIONS TO THE EDITOR
815
conversion but that its liberation is not a pre- ase, there was still some remaining reaction with cyanide.3 requisite for chymotryptic activity. On the basis of these data, and others previously It was suggested that this cyanide reacting reported, it now appears possible t o identify the residue might be due to the presence of nicotinamino acid sequence in chymotrypsinogen which is amide mononucleotide or riboside. We have primarily involved in the activation process. The recently reinvestigated this residue, and have following considerations summarize the pertinent found that DPN with high purity prepared from points of evidence: (1) n-chymotrypsin differs either liver or yeast still contains material which from chymotrypsinogen in possessing an N- reacts with cyanide and is resistant to the action of terminal isoleucyl-valine s e q u e n ~ e , ~both * ‘ ~ proteins the Neurospora enzyme. However, the residue being devoid of a C-terminal group reactive toward is not nicotinamide mononucleotide or riboside carboxypeptidase4; (2) the conversion of n- to since the compound does not promote the growth &chymotrypsin yields a C-terminal leucine group, of Hemophilus parainfluenzae, which will grow on no new N-terminal group, and the dipeptide seryl- either the riboside or nucleotide as well as on arginine; (3) the action of a-chymotrypsin on DPN.4 We have now been able to isolate a chymotrypsinogen yields neither enzymatic activ- compound from a number of highly purified ity nor the dipeptide, l1 suggesting that seryl- commercial DPN preparations, which give the arginine is not a C-terminal sequence in chymo- same analysis for adenine, nicotinamide, ribose trypsinogen. Omitting from consideration factors and phosphate as DPN, but has no alcohol dehydroarising from the presence of the N-terminal half- genase activity. The compound was obtained by cystine group found in chymotrypsinogen12 i t seems first treating the DPN preparation with the Neuromost likely that the amino acid sequence involved spora DPNase, and then separating the residual in activation is leucyl-seryl-arginyl-isoleucyl-valine, cyanide reacting material from the adenosineand that the arginyl-isoleucine bond is opened in diphosphate ribose by column chromatography. the trypsin-catalyzed formation of n-chymotrypsin. The compound was precipitated from acid acetone. The subsequent, chymotrypsin-catalyzed4 con- We have tentatively named this compound “DPN version of n- to &chymotrypsin involves the hy- isomer” because of its identical analysis with drolysis of the leucyl-serine bond, giving rise to the DPN. From 5 g. of Pabst DPN5 300 mg. of the dipeptide, a C-terminal leucine group and an N- “isomer” was isolated. We have estimated the terminal isoleucyl-valine sequence. It is worthy of concentration of the “isomer” t o be from 10 to 15% note that, as in the activation of trypsinogen,la in the purified DPN. the splitting of a single bond suffices t o produce The isomer moves a t the same Rp as DPN on enzymatic activity and that all of these active paper in a large number of solvents. Likewise it enzymes have the same N-terminal dipeptide cannot be separated from DPN by column chrosequence. l 4 The splitting of the leucyl-serine bond matography. The isomer can be distinguished from of n-chymotrypsin is without effect on the specific DPN not only by its inactivity in the alcohol esterase activity of the activation mixture. De- dehydrogenase (yeast or liver), the muscle triosetails of this work will be published elsewhere. phosphate dehydrogenase and muscle lactic dehydrogenase systems, but also by its inability to (10) M. Rovery and P. Desnuelle, Biochim. et. Bioghys. A d a , 18, s e n e as substrate for the Neurospora DPNase and 300 (1954); M. Rovery, M. Toilroux and P. Desnuelle, ibid., 14, 145 (1954). as a growth factor for Hemophilus parainjluenzae. (11) W. J. Dreyer, unpublished experiments. The compound reacts with hydrosulfite to give a (12) F. R . Bettelheim, J . Bioi. Chem., 912, 235 (1955). product of reduction showing an absorption peak (13) E. W. Davie and H. Neurath, J . B i d . Chcm., in press. (14) P. Desnuelle and C. Fabre, Bull. Soc. Chim. Bio1.,36,181 (1954). a t 348 mp; furthermore, the cyanide addition product of the isomer has a maximum absorption DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF WASHINGTON WILLIAM J. DREYER a t about 332 mp as compared to the 325 maximum 5, WASHINGTON HANSNEURATH for DPN. SEATTLE RECEIVED DECEMBER 4, 1954 DPN can serve as the nicotinamide source for a nicotinamide requiring Neurospora mutant. However, the isomer does not promote the growth of the mutant (Table I). On heating the isomer for ISOLATION OF A DPN ISOMER CONTAINING 30’ a t 100’ (PH 5)) the nicotinamide ribosidic NICOTINAMIDE RIBOSIDE‘ IN THE a LINKAGE‘ bond is cleaved; and this results in growth activity Sir: for the mutant. This and other evidence indicate It has been reported previously that treatment that the pyridine component of the isomer is of diphosphopyridine nucleotide (DPN) with the DPNase from Neurospora trassa results in the nicotinamide. Treatment of the isomer with the snake venom cleavage of the nicotinamide riboside bond.2 Although the DPNase completely destroyed all pyrophosphatase results in the liberation of all the activity of the D P N for yeast alcohol dehydrogen- adenine as 5‘-adenylic acid. The nicotinamide mononucleotide moiety of the isomer has a similar (1) Contribution No. 108 of the McCollum-Pratt Institute. Aided cyanide addition product as the parent compound by Grant No. C-2374 C, from the National Cancer Institute of the National Institute of Health, The American Cancer Society as recommended by the Committee on Growth of the National Research Council and the American Trudeau Society Medical Section of the National Tuberculosis Association. ( 2 ) N . 0. Kaplan, S. P. Colowick and A. Nason, J . B i d . Chcm., 191, 473 (1951).
(3) S. P. Colowick, N. 0. Rapfan and M. M. Ciotti, i b i d . , 191, 447 (1951). (4) W. Gingrich and F. Schlenk, J . Bacl., 41, 536 (1944); N. R. Bachur and N. 0. Kaplan, in preparation. ( 5 ) We wish to thank the Pabst Laboratories for generously supplying some of the D P N used in these experiments.
