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VOl. 72
111, R = H IV, R = C&CO--
C, 77.81; H, 7.67). The smooth nitration furnished VII, identical with the product from I (pale yellow prisms, m. p. 142’. Anal. Calcd. for CBH1403N1:C, 63.41; H, 5.7. Found: C, 63.57; H,5.96). Further reactions and rearrangements in this series will be reported shortly.
present finding would favor the latter view. Since the folic acid antagonist leads to the accumulation of the same imidazole as do the $-aminobenzoic acid antimetabolites, the latter presumably act by creating a ddciency of folic acid, which in turn is responsible for the failure in purine formation. The demonstration was conducted as follows: E . coli was grown in the manner of Stetten and CONVERSE MEMORIAL LABORATORY HARVARD UNIVERSITY J. B. PATRICK* Fox’ except that sulfadiazine was omitted and 0.2 CAMBRIDGE, MASSACHUSETTS BERNHARD WITI(OP$ mg. per cc. of 4-aminopteroylglutamic acida was RECEIVED NOVEMBER 13, 1949. added. Judged colorimetrically, about the same amount of diazotizable amine accumulated as * Harvard University Graduate School. when sulfadiazine was the inhibitor. Isolation of $ Harvard University Faculty 194%. the base was accomplished as in1 except that 2.5 times as much mercury salt was used and the FORMATION OF 4-AMINO-5-CARBOXAMIDOIMI- ether extraction was omitted. Final separation DAZOLR DURING GROWTHOF ESCHERICHIA COLI was made on paper strips with butanol-diethylene IN THE -PRESENCE OF 4-AMINOPTEROYL__ ~-~ glycol-water solvent, in an atmosphere containing GLUTAMICACID ammonia,’ in which the imidazole showed Rp of sir: 0.5. Identity of the isolated substance with synWhen Escherichia coli is grown in the presence thetic 4-amino-5-carboxamidoimidazole8was esof amounts of 4-aminoptero~flglutamicacid just tablished by comparison of (a) the Rp in the sold c i e n t to inhibit multiplication slightly, 4- vent just mentioned, (b) the absorption spectra amino-5-carboxamidoimidazoleaccumulates in the in the ultraviolet region a t pH 2.0 and 11.0, and medium, and has been isolated from it. This is the (c) the melting points (with decomposition) of the same substance which was found by Stetten and picrate. In every case the behavior of the known Fox1 when this and other bacteria were grown in and the unknown was the same. the presence of sulfadiazine or sulfapyridine. It THEROCKEFELLER INSTITUTE D. W. WOOLLEY was identified by Shive, et a1.,2and recognized as FOR MEDICAL RESEARCH R. B. PRINGLEg N. Y . the probable precursor in the biosynthesis of hy- NEWYORK, poxanthine. RECEIVED DECEMBER 7.1949 The accumulation of the imidazole through the (6) Kindly made available by Dr. T H. Jukes of Lederle Laboraintervention of the antimetabolite of folic acid is tories. (7) E. Vischer and E. Chargaff, J . B i d . Chcm., 176, 703 (1948) of importance in consideration of the mode of ( 8 ) E. Shaw and D. W. Woolley, ibid., 181, 89 (1849). action of sulfonamide drugs and of folic acid. (9) Fellow of the National Institutes of Health Thus, inhibition analysis has led to the conclusion that p-aminobenzoic acid participates in several reactions, of which the first to be affected by SPECIFICITY OF UREASE ACTION sulfanilamide derivatives is the formation of Sir: methionine, the next is concerned with purine Urease has been repeatedly cited’ as a strictly formation,* and less sensitive processes, presumspecific enzyme which hydrolyzes only urea. In ably the synthesis of folic acid,4 are then retarded. the course experiments with substances related On the other hand, Woods6 has concluded that to urea weofhave hydrolysis of biuret the primary action of the sulfonamides is the in- HP,N-C-NH-C-NHZobservedbya urease preparations. hibition of folic acid formation, and that syntheIt 11 sis of purines and of methionine are secondary 0 0 events in which that vitamin participates. The As much as 33% of nitrogen initially contained (1) M. R. Stetten and C. L. Fox, J . Bioi. Chcm., 161, 333 (1948). in solutions of biuret was identified as ammonia (2) W. Shive, W. W. Ackermann, M. Gordon and M. E. Get(by Nessler technique2) after prolonged enzycendaner, Tms JOURNAL, 69,725 (1947). (3) W. Shive and E. C. Roberts, J . Biol. Chcm., 182, 462 (1946). (4) K. C Winlder and P. G. de Haan, Arch Biochcm.. 18, 97 (1948). (8) D.D.wwdr, ~ u l lm. . cbim. bid., ao, 730 ( l o w .
(1) Sumner and Sommas, “Enzymes,” Academic Press, New York, N. Y., p. 156. (9) Ambrose, P;istt.korrkr and Kridl. Tnri JOUBNAL, 99, a17
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matic hydrolysis; odor of ammonia was observed showing that the two substrates compete for the after adding sodium carbonate; in the absence of same enzyme. The other experiments described buffers the pH changed from 7 to 8. Carbon diox- above point in the same direction and we believe ide was quantitatively measured by absorption by that hydrolytic activity of urease toward biuret Ascarite on aeration; it was also qualitatively has been established with a high degree of probadetected by the barium hydroxide test. Micro- bility. combustion of the C. P. biuret, obtained from In 5 X M solution of urea the production Eimer and Amend, gave N, 34.86%, whereas CZ- of ammonia is faster by a factor of 9 than in an NsHbO2.H*Ogives N, 34.71%; the well-known equimolar solution of biuret. This is the partial biuret test was positive. Thus hydrolysis of bi- explanation of the failure of Takeuchia to detect uret is definitely established; it occurred only in hydrolysis of biuret by crude extracts from soya the presence of urease preparations as was shown beans. by negative blanks. To establish that urease is Preliminary determinations of both the ammoresponsible for this reaction the following experi- nia and the carbon dioxide produced by urease in ments were performed. The ratio of ammonia solutions of biuret indicate a ratio of 3: 1, rather produced in a 0.016 M solution of biuret to that in than 3 :2, which would be obtained on complete a 0.0019 M solution of urea by aliquot portions of hydrolysis, suggesting that some other products a crude extract from jackbean meal was 1.5 a t 20' are being formed. The enzyme appears to become and $H 7.0 in phosphate buffer. Crude extract rapidly inactivated in hydrolyzing biuret, which from soya beans gave for this ratio 1.6. Crystal- suggests inhibition by some products of hydrolyline enzyme prepared from jackbean meal by the sis. method of Sumner and having a specific activity Urease occurs not uncommonly in living tissue of 63,000 Sumner units gave a ratio of 1.6. On and the need for elimination of urea from such adding M solution of Agf to the enzyme its tissue appears to be a weak justification for the activity was reduced to that of the blank both for presence of urease. The finding of another suburea and biuret. In a solution containing 0.048 strate toward which urease is active is therefore of 1cz of sodium sulfite and 0.013 M sodium bisulfite interest as suggesting an additional biological reaper liter in which the activity of urease toward son for urease. We are therefore extending our urea is reduced2 to one-fdth, the above ratio was experiments to derivatives of biuret and its still found to be 1.6. These ratios were reproduci- homologs (carbonyl diurea, tetruret, etc.) as well ble to 10-15%. On adding to a 1.5% solution of as studying the mechanism of the biuret hydrolyurea 1.0% biuret the rate of production of ammo- sis. nia by another preparation of crystalline enzyme DEPARTMENT OF CHEMISTRY (about 30,000 S. u. activity) was reduced by 11%. HARVARD UNIVERSITY W. H. R. SHAW* According to the data of Sumner, the production CAMBRIDGE, MASS. G. B. KISTIAKOWSKYt of ammonia is fastest in the 1.5% solution of urea, RECEIVED NOVEMBER 23, 1949 and thus the addition of biuret acted in the same (3) T. Takeuchi, 1. Coll. Agr., Tokyo, Imp. Univ., 1, 1 (IQOBmanner as a further addition of urea, virtually 1913). eliminating the possibility that two different en* Harvard University Graduate School. zymes are responsible for the two reactions and t Harvard University Faculty 1930-.
N E W BOOKS Scientific and Technical Abbreviations, Signs and SymPh.D., Professor of Chemibols. By 0. T. ZIMMERMAN, cal Engineering, University of New Hampshire, and IRVINLAVINE,Ph.D., Formerly Professor of Chemical Engineering and Head of the Department, University of North Dakota. Industrial Research Service, 'Dover, N. H., 1948. xii 476 pp. Illustrated. 14 X 22 cm. Price, $7.50.
A (aeronautics) to Z (zoology), with chemistry, physics, biology, botany, astronomy, mathematics, engineering, radio, electronics, hydrography, topography, meteorology, medicine, communications, machine and tool shopcraft, commerce, banking, military, navy, even plumbing and heating, in between, all well arranged by subjectsand Wds, in neat and readable tables. The sources drawn from are many, in particular the American Standards Association.
graphs and special compilations such the one here considered. Zimmerman and Lavine have done an extensive job of collecting the abbreviations, signs and symbols from
T. or B. L. E. in the section devoted to railway terms. The authors discuss favorably in the Preface the growing practice of omitting periods after abbreviations. The
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