2022
COMMUNICATIONS TO THE EDITOR
Vol. 80
Calcd. for C12H18BrN02S: C, 46.45; H, 2.60. m.p. 191', in 957, yield. The formic acid was disFound: C, 46.41; H, 2.70.) Reduction furnished tilled and isolated as the sodium salt in 100yo 2-amino-2'-bromodiphenyl sulfide, m.p. 62-63'. yield. The glyoxylate was obtained in S 3 ~ yield o (Anal. Calcd. for ClzHloBrNS: C, 51.44; H , 3.60. and was characterized as described earlier3 and by Found: C, 51.00; H, 3.63. Hydrochloride, m.p. its 2,4-dinitrophenylhydrazone,m.p. 192O. Xli132-134". '4nal. Calcd. for ClzHllBrCINS: C, quots of these fractions were plated and counted in 45.51; H, 3.50. Found: C, 45.57; H, 3.79.) a gas flow counter. More than 98.57, of the C14 of Benz-o-chloroanilide6 and o-chlorophenylacetone7 the degraded gulonolactones were recovered in the were made by standard methods. Acetoacet-o- glyoxylate fraction. The radioactive glyoxylates chloroanilide was the product of Union Carbide were further oxidized with HI04 to equimolar Chemicals Co. Products were identified by mixed amounts of COz and f ~ r m a t e . This ~ formate conmelting points with authentic samples or by com- tained all the CI4 of the glyoxylate. These results parison of melting points of the product and a t demonstrate that the C-2 of inositol becomes C-5 least one derivative thereof with literature values. of the racemic as well as of the D-glucuronate. The yields reported are not considered to be Thus, inositol cleavage by the purified enzyme optimum. system occurs between C-1 and C-6 to form exclusively D-ghcuronate. The formation of L( 6 ) F. D. Chattaway and K. J. P. Orton, Ber., 33, 2396 (1900). (7) I. B. Johns and J. M. Birch, THISJ O U R N A L , 60, 919 (1938). glucuronate may result from cleavage of inositol between C-3 and C-4, assuming that no racemizaVENABLECHEMICAL LABORATORY tion occurs of a possible intermediate between UNIVERSITY O F NORTH C A R O L I ~ A BJORS F. EXRUTFORD CHAPELHILL,N. C. J. F. BUN NET^ inositol and D-glucuronate. Further studies arc' RECEIVED MARCH21, 1958 needed to confirm this latter mechanism. (3) F. C. Charalampous, J . Biol. C h e w , 226, 585 (1957).
THE SITE OF CLEAVAGE OF MYO-INOSITOL BY PURIFIED ENZYMES OF RAT KIDNEY Sir:
DEPARTMENT O F BIOCHEMISTRY SCHOOL OF MEDICINE FRIXOS C. CHARALAMPOUS USIVERSITYOF PENNSYLVANIA SYLVIA BUMILLER SUEGRAHAM PHILADELPHIA, PENNSYLVANIA RECEIVED MARCH1, 19%
We have reported previously' the cleavage and conversion of inositol to glucuronic acid (racemic mixture) by rat kidney extracts. The enzyme OF CYANOACETIC ACID AS A preparation has now been purified, and the system IDENTIFICATION METABOLITE OF 8-AMINOPROPIONITRILE (BAPN) that forms D-glucuronate has been obtained free of AND OTHER NITRILES' the one that forms the L-isomer. Resolution was Sir: obtained by treatment with calcium phosphate gel The administration of P-aminopropionitrile (B which adsorbs only the system that forms the Lisomer. The enzyme system forming the D-isomer APN) to rats or rabbits has been shown to result was further purified 200-fold. Attempts to elute in the formation of a metabolite which could be dethe adsorbed enzyme from the gel produced inac- tected in the urine or blood serum by an orangetive preparations. Employing inositol-2-C14 as pink spot on paper chromatograms developed with the substrate and incubating separately with either diazotized sulfanilic acid.2 Efforts to isolate the the crude enzyme system or the purified enzyme metabolite from ethyl acetate extracts of acidified capable of yielding only the D-isomer, we isolated urine led t o the separation of a colorless, crystalline, 8 to 10 mg. of the respective radioactive glucuronic nitrogenous solid which was very soluble in water, acids. They were converted to their lactones with highly acidic, distillable in vacuo, and exhibited the m.p. of 176-178" for the racemic lactone' and 180" characteristic chromatographic behavior.2 Furfor the D-glucuronolactone having a n [CY]'~D ther examination of this solid showed that i t +18.6" (c 1 in H20, 1 = 1 dm.). The radioactive was not pure, but the metabolite was obtained glucuronolactones were diluted fivefold with the from i t in substantially pure form by ion-exchange respective non-radioactive glucuronolactones and chromatography on Dowex-1 resin (acetate form) crystallized ta a constant specific activity of ap- and has now been identified as cyanoacetic acid. proximately 1755 c.p.m. per micromole. The dis- The isolated substance melted a t 67-68', undetribution of C'4 within each of the glucuronates pressed by admixture with an authentic sample, was obtained as follows: the uronic acids were re- and corresponded closely with known cyanoacetic duced with NaBH4 to gulonic acids which were con- acid in its infrared spectrum and chromatographic verted to their lactones. The lactone derived behavior on paper. Anal. Calcd. for C3H302N: from the racemic glucuronate was optically inactive C, 42.36; H, 3.56; N, 16.47. Found: C, 42.33; and could not be crystallized. The lactone of the H, 3.65; N, 15.42. D-isomer had a m.p. of 184-185' and an [ c Y ] ~ ~ D Cyanoacetic acid was also detected in the urine +56" as expected for L-gulonolactone (c 1 in HzO, of rats following administration of 3,3'-iminodipro1 = 1 dm.). The gulonolactones were titrated pionitrile, 3.4 ethylene cyanohydrin and valerowith NaOH and were oxidized with HI04 to 1 mole nitrile. Administered cyanoacetic acid was exof formaldehyde, 1 mole of glyoxylate and 3 moles (1) Supported in part by grants A538(C8) and A1498 from the of formate per mole of gulonolactone. The form- National Institutes of Health, U. S. Public Heaith Service. (2) J. T. Garbutt, J. J. Lalich, S. H. Lipton and F. M. Strong, aldehyde was isolated as the dimedon derivative, Federation Proc., in press. (1) F.C. Charalampous and C. yras, J . B i d . Chem., 228, 1 (1957). (2) We are grateful to Dr. Laurens Anderson for a generous gift of inositol-2-C'd.
(3) J. Delay, P. Pichot, J. Thuillier and J. P. Marquiset, Compt. rend. soc. biol., 146, 533 (1952). ( 4 ) H. A . II.irtinaun and H. F. Stich, Science, 126, 445 (1927).
April 20, 1958
COMMUNICATIONS TO THE EDITOR
2023
creted unaltered. Aminoacetonitrile, 2,2'-imino- b y hydrogen reduction of the U308 to UOZ,nor by diacetonitrile and y-aminobutyronitrile failed to hydrogen firing of mixtures produced by evaporagive rise to cyanoacetic acid. It appears that in tion of various solutions containing U and Pu. the metabolism of BAPN and certain other ali- Coprecipitation of the U and Pu as peroxide was phatic nitriles by the rat the organic cyano group is also tried and was not successful. After the samples were esamined by X-ray diflargely unaffected and that deamination and/or oxidation occur a t the other end of the molecule. fraction, a radiochemical Pu assay was done to Results to date tend to indicate that aliphatic check the composition. The Pu assay agreed with nitriles are metabolized in the animal body to form the compositions as made in each case. X-Ray powder patterns were taken in a 114.6 w-carboxynitriles which then undergo cleavage as proposed for the fatty acids5 The color reaction mm. diameter powder camera using filtered copper with diazotized sulfanilic acid affords a very sensi- K-radiation. (Wave lengths, a1 = 1.54051, a2 = tive method for detecting cyanoacetic acid and may 1.54433, mean a = 1.5418 A.) The lattice parambe useful for studying the metabolism of other or- eter was determined in most cases by graphical ganic cyano derivatives. A detoxication product extrapolation of the values found for the high-angle of inorganic cyanide, 2-imino-4-thiazolidine car- lines on each pattern. Results are presented in boxylic acid,6 gives a somewhat similar color with Table I. The error limits on the compositions are this reagent but is easily distinguished from cyano- estimated, those on the lattice parameters are estiacetic acid. Neither cyanoacetic acid or cyanacet- mated uncertainties in the graphical extrapolation. amide produced any of the symptoms of BAPN The solid solution has the fluorite structure, the toxicity when fed to weanling rats a t the rate of 2 same as PuOz and UOz. mg. per ml. of drinking water for seven weeks.' TABLE I Cyanoacetic acid may be a detoxication product of U02-Pu02 SOLID SOLUTION FLUORITE STRUCTURE BAPN. Its relationship, if any, to the connective Compn. (mole % PuOz) Lattice parameter (A,, 25') tissue degeneration characteristic of BAPN toxicity 0 5.4700 zk 0.0001 is not known. (5) D. E. Green, B i d . Reus., 29, 330 (1954). (6) J. L. Wood and S. L. Cooley, J. B i d . Chem., 118, 449 (1056) (7) J. J. Lalich, unpublished experiments.
DEPARTMENTS OF BIOCHEMISTRY AND PATHOLOGY OF WISCONSIN UNIVERSITY MADISON, WISCONSIN RECEIVEDMARCH 6, 1958
S. H. LIPTON J. J. LALICH F. M. STRONG
UOt-Pu02 SOLID SOLUTIONS*
Sir: An X-ray diffraction investigation has been made of the solid phase relationships in the UO2-PuO2 system. The results show that a continuous solid solution exists, and that the lattice parameter varies essentially linearly with composition. A slight negative deviation from linearity may exist. Some trouble was experienced in preparing satisfactory samples of the mixed oxide. Plutonia is an extremely inert substance, and a t 1000° the reaction rate between PUOZand UOz is very slow. Various ways of producing an intimate mixture of plutonium and uranium compounds that could be converted to oxide were tried. The method which seemed best was to co-precipitate Pu(0H)d and (NH&UzO, by dropping a Pu(1V)-U(V1) solution (total metal concentration cu. 14 g./l.) into hot ammonium hydroxide, taking care to keep the pH always above 8. The mixed precipitate was dried in air a t 70°, then fired in hydrogen by raising the temperature to 100OO over 6 or 7 hours and holding a t 1000O overnight. Samples were furnace-cooled in hydrogen. It appears to be quite important that the firing be done in such a way as to avoid the production of well crystallized PuOz as a separate phase. For example, a solid solution was not produced by initial firing of the hydroxide precipitate in air followed (1) Work done under the auspices of the Atomic Energy Commisdon.
12 6 f 0.3
20.0 f . 3 35.9 f .3 45.1 f .3
63.1 f . 3 73.8 =I= .3 79.4 i .3 100.0
5.449 5.4544 5.441 5.4345 5.420 5.414 5.407 5.3960
f .003 f .0006 f ,003 ,0005 .000 f ,002 f ,003 f .0003 =I= f
Thanks are due the Misses Marian Gibbs and Gladys Sturdy for measuring the films and preparing the samples, respectively; and Mr. A. Zerwekh for the Plutonium assays. UNIVERSITY O F CALIFORNIA ROBERTN. R. MULFORD Los ALAMOSSCIENTIFIC LABORATORY Los ALAMOS,NEW MEXICO F. H. ELLINGER RECEIVED MARCH1, 1958 S-ADENOSYLMETHIONINE AND ERGOSTEROL SYNTHESIS Sir :
The side-chain C-28 methyl group of ergosterol have been shown to arise intact from m e t h i ~ n i n e . ~ . ~ This transfer of a methyl group was of interest since this was the first demonstration that a carbon atom may act as the acceptor of a methyl group. I n order to participate in certain methyl additions, methionine must first react with ATP4 to form an adenosylsulfonium compound, S-adenosylmethionine,bwhich may then act as the methyl donor to the appropriate acceptor. Involvement of S-aclenosylmethionine as the actual donor in the transfer of methyl groups to sulfurs and nitrogen7 has been de(1) This work was performed under the auspices of the U.s. Atomic Energy Commission. (2) G. J. Alexander, A. M. Gold and E. Schwenk, THIS JOURNAL, 79, 2967 (1957). (3) G. J. Alexander and E. Schwenk, ibid., 79, 4554 (1957). (4) H.Borsook and J. W. Dubnoff,J . B i d . Chem., 171, 863 (1947). (5) G. L. Cantoni, THISJOURNAL, 74, 2942 (1952). (6) S. K. Shapiro, Bacferiol. Proc. A m . Bacleriologisfs, 116 (1957). (7) G. L. Cantoni. Phosphorus Mcfabolirm, 2 , 129 (1952).
