Dec. 20, 19.59
COMMUNICATIONS TO THE EDITOR
It is not easy to anticipate whether the balance between quantum-mechanical delocalization energy and the compression energy necessary to force classical structures (like the “Kekule” structures of type X) into the same geometry will indeed place species VI-VI11 in the category of homo-aromatic mesomeric compounds. While quantum-mechanical delocalization energies in cases VI-VI11 will be much less than in cases 111-V, we probably can expect compression energies to be less also. The balance between compression energy and quantummechanical delocalization energy in cases VI-VI11 appears to be sufficiently analogous to the one for I, t h a t the observed results with I warrant experiments to test for the occurrence of homo-aromatic structures VI-VIII. These are being undertaken. Examples of homoconjugation have been discussed previously, such as the h ~ m o a l l y l i ccation, ~~ the anti-7-norbornenyl (termed a bishomocyclopropenyl cation by Roberts3‘) and the “planar pseudo-aromatic structure” for tropilidene visualized by Doering.? The latter example could be called monohomobenzene. Cases VI-VI11 discussed above differ from these by having complete equivalence of the classical contributing structures. Perhaps “perhomo-aromatic” is a useful term for VI-VIII. (3) (a) E . g . , RI. Simonetta and S. Winstein, THISJOURNAL, 76, 18 (1954); (b) S. Winstein, M. Shatavsky, C. Norton and R. B. Woodward, ibid., 7 7 , 4183 (1955): (c) W. G . Woods, R. A. Carboni and J. D. Roberts, ibid., 78, 5653 (1956). (4) W. E. Doering, et al., ibid., 78, 5448 (1956). DEPARTMENT OF CHEMISTRY UNIVERSITY OF CALIFORNIA S. WINSTEIN
for growth: (1) serine hydroxymethylase, (2) a vitamin Blz-containing enzymea,*and (3) a fraction partially purified by ammonium sulfate precipitation and chromatography on adsorbents. The vitamin Bln-containing enzyme was purified about GO-fold b y means of ammonium sulfate fractionation and then by adsorption and elution from calcium phosphate gel and by chromatography on diethylaminoethyl cellulose and hydroxylapatite. The enzymatic system carried out methionine synthesis in the presence of the protein fractions and all of the indicated cofactors (Table I). The addition of reduced flavin compounds to incubation mixtures obviated the requirement for DPNH when incubation was carried out under hydrogen. TABLI
Expt.
Additions
X
D P S H , oxidized F.4D DPiYH None Reduced FMN Reduced FAD Filtered catalyst suspension (without flavin compounds) Reduced FMN Reduced FMX
B
C
Sir: Several investigators ha\-e studied the biosynthesis of the methyl group of methionine in cell-free systems with serine or iornmaldehyde as donors and homocysteine as acceptor of the one-carbon fragn1cnt.l-6 In extracts of ccrtaiii mutants oi Eschcrichici coli, cofactor requirements were shown previously fur pyricl~xal phosphste, tetrahydrofolic acid, adenosine triphosphate, DPSI-1: a n d vitainin B1:.’.3.5 Iieceritly iTl this laboratory a requircnicrit for F-ID or F N S has bccn demonstrated. The effect o i the flavin coenzymes has been shown i v i t h a system consisting of these three purified enzyme iractions obtained from E. coli mutant 1 l:j-3,g which requires vitamin €3,. or methinnine (1) This work was supported in part by grants from t h e National Science Foundation and t h e Greater Boston Chapter of the Massachusetts Heart Association. ( 2 ) C . W. Helleiner and D. D. Woods, Biochem. J.. 63,26P (1956). (31 F. T. Hatch, S. Takeyama and J. &I. Buchanan, Federation Pmc., 18, 243 (1959). Doctor, T. L. P a t t o n and J. Airapara, A r c h . Biochem. (4) \’. ?. I. Bzophys., 67, 401 (1957). ( 5 ) A. Kakao and D. hI. Greenbrrg, J . B i d . C h e m , ‘230, 603 (1958). ( l i ) A. Stevens and \V. Sakami, ibid., 234, 2063 (1959). ( i ) Abbreviations used are: DPI\‘H, reduced diphosphopyridine nucleotide: F A D , flavin adenine dinucleotide; F h l S , riboflavin-5phosphate. (8) R. L. Kisliuk and D . D . \Voods. Fcderation Proc., 18, 261 (1959). (9) B. D. Davis and E. S. Mingioli, J . Bactcviol., 6 0 , 17 (1950).
I
All vessels contained per ml. : potassium phosphate buffer, pH 7.2, 50-100 pmoles; id-serine, 5-10 pmoles; Lhomocysteine, 10 pmoles; pyridoxal phosphate, 0.25 pmole; adenosine triphosphate, 5 pmoles; Mg+’, 10 pmoles; tetrahydrofolic acid, 0.5 pmole; serine hydroxymethylase; BIZcontaining enzyme; and third enzyme fraction. To this basic system these additions were made when indicated : DPXH, 2 pmoles; oxidized FAD, 0.16 pmole; reduced FAD or FMX (catalytic hydrogenation with 307, palladium on charcoal), 0.2 pmole. Incubation was for 2 or 3 hours a t 37”. Methionine was assayed microbiologically with Leuconostoc mesentemides P 60.
Los ANCELES24, CALIFORSIA RECEIVED NOVEMBER 16, 1959 REQUIREMENT FOR FLAVIN COENZYME IN THE ENZYMATIC SYNTHESlS OF METHIONINE 1N VITRO’
6325
Gas phase T’.TZ
N*
hlethionine synthesized mpmoles
71 11
i\’Z
7
Hz Hz
592 388
HZ Hz He
42 236 34
It is believed t h a t the high values for methionine synthesis obtained were due to regeneration of reduced flavin by hydrogen gas catalyzed by traces of palladium which escaped filtration. Incubation under helium resulted in very little methionine formation. These preliminary results suggest that the requirement for pyridine dinucleotide in methionine biosynthesis can be explained by its role in reducing the flavin component of the system. (10) American Heart Association Advanced Research Fellow. (11) Karl Taylor Compton Fellow of the h‘utrition Foundation in Biochemistry. (12) United States Public Health Service Predoctoral Fellow. (13) Iiational Science Foundation Predoctoral Fellow. DIVISIONO F BIOCHEMISTRY FREDERICK T. HATCH'^ DEPARTMENT OF BIOLOGY SHIGEYUKI TAKEYAMA~’
MASSACHUSETTS INSTITUTE RENATAE. CATHOU’~ OF TECHNOLOGY ALLANR . LARRABEEI3 CANBRIDGE 39, hfASS.4CHUSETTS JOHN M. BUCHANAN RECEIVED OCTOBER 26, 1959 IDENTlFICATION OF A STEROL WITH ACRASIN ACTIVITY IN A SLlME MOLD Sir :
hcrasin is a chemotactic hormone produced by individual amoeboid cells of the slime mold, Dictyostelium discoitieum.‘sYp3 In response to a E . H . Rungan. Collectiug S e t . lVoods H o l e , 17, 88 (1942) (2) J . T . Bonner, J. l i w p t l . Zool., 106, 1 (1947). (3) B. %I.Shaffer, ,l-ature, 171, 957 (1R53). (1)
6526
COMMUNICATIONS TO THE EDITOR
diffusion gradient of this hormone, the cells stream together to form a multicellular unit, which then undergoes further differentiation. Earlier work has indicated t h a t acrasin is stable toward acid and alkali4J and that certain steroids could simulate the hormone to some extent.6 \Ye have isolated a sterol with weak acrasin activity in the Shaffer test3 from Dtctyostelium discozdeum, grown on a complex agar medium in the presence of E . coli.2 At early aggregation, the cells were harvested with cold water, then boiled for 15 minutes to stop enzymatic activity. To each 1-1 liter portion of cell suspension 150 ml. of concentrated hydrochloric acid was added and the material was boiled for 10 minutes. After cooling, the hydrolysate was extracted with CH2C12. The extract was fractionated according to the procedure previously used for the isolation of fecal steroids The alcohol fraction, isolated via the hemiphthalates, was chromatographed on alumina grade 111, and crystalline material was eluted by petroleum ether-benzene (1:1). No biological activity was detected in the acidic, ketonic, and non-alcoholic fractions and apparently all of the activity was recovered in the crystalline alcohol eluted from alumina. Since the infrared spectrum of the recrystallized sterol (I) suggested its identity with A%tigmasten3d-01 (11), an authentic sample of this sterol was synthesizedYby reduction of A 4 '?-stigmastadien-3one to A%tigmasten-3-one (111) with lithium in liquid ammoniag and further reduction of I11 to I1 with LiXlH4.'O The synthetic I1 showed the same biological activity and infrared spectrum as I. Oxidation of I with chromic acidll gave a product with the same infrared spectrum as I11 and the acetates of the synthetic I1 and of the isolated I showed identical infrared spectra. Confirmatory evidence for the identity of I, its oxidation product TABLEI hIp
( a ) ~ l ~ C,
, I 2OC
+ + +
I 1.56 0-157 0 5 2 I1 163 0-163 5 5 1 159 0 2 Liter,itureI4 173 0-174 0 +2U Ilelijdro I I11 170 0-171 0 +22 166 0-167.0 f2O Literature14 156 0 - 4 9 -1cetate of I -1cetate (if I1 146 0-146 5 - 5 0 144 0-144 5 - 6 Literatweld
S3 98 84 00 83 94
H, d 12.09
12 15 11 40
81 39 81 29
11 74
S1 52
11 18
11 65
(4) M. Sussman, F. Lee and N. S.R e r r , Science, 123, 1171 (l95G). ( 5 ) B. M. Shaffer, Science, 123, 1172 (1950). (6) B. E. Wright and 51. L. Anderson in "The Chemical Basis of
Development," edited by \V, D. lIcElroy and B. Glzrss, Johns Hopkins Press, Baltimore. M d . , 1958, p. 2Citi. (7) E. H c f t m a n n , E. Weiss, H . K. Miller and Iz. llosettig, .Ii.ch. Biochc?ia. uizd Bioplzyr., 8 4 , 321 (1939). ( 8 ) We thank hI. J. Thompstm for his cooperation in the iyntheais of reference substances. (9) D. H. R . Barton, D. A . j. Ives and B. R. Thomas, ( ' I i e o z i d r y $ gpee aiid
( > . 11.
I< Summcrs, .1.
('lii,iii
.Sot , ti87
VOl.
s1
(dehydro I) and its acetate with 11, 111 and the acetate of I, respectively, is summarized in Table I. Whereas the melting points of the isolated I and its derivatives differed from those of the corresponding authentic samples, presumably owing to polymorphism, they showed no depression upon admixture. The possible relationship of I to the in vivo form of acrasin is under investigation. I is not as active as certain crude unhydrolyzed preparations; it may exist naturally as a conjugate, or act ~ y n e r g i s t i c a l l y . ~Details ~ of the isolation procedure and identification will be presented elsewhere. (15) K. R. Sussman, 31 Sussman, and F. Lee Fu, Bacleiiof Pioc
THE ENZYMATIC CARBOXYLATION OF BUTYRYL COENZYME A
Sir: The enzymatic ATP-dependent Carboxylation of acetyl Co-I,',' propionyl COX,^-' and /3-methylcrotonyl CoX8has been investigated. The present report describes the identification of the product of butyryl C o h carboxylation catalyzed by purified propionyl c a r b o ~ y l a s e . ~ J The carboxylase was prepared from dilute Trisg (0.002 Ai', fiH 7.3j extracts of bovine liver mitochondrial acetone powder. After aging the extract for 20 hours at 30", the protein precipitating between 45 and 35y0saturated ammonium sulfate was dissolved in and dialyzed for 12 hours against 0.002 iV1 Tris, p H 7.3. Enzyme prepared in this manner had a specific activity of 22 carboxylase units (Flavin, et d4) per mg. of protein. The relative rates of carboxylation of acetyl CoAl,propionyl CoA and butyryl Co-1 catalyzed by ammonium sulfate-purified propionyl carboxylase are summarized in Table I. It is apparent that butyryl CoA and acetyl COX are carboxylated a t significant rates although the rate is much greater with propionyl Co-I. The reaction product of butyryl CoA carboxylation was investigated. Eight units of the purified carboxylase were incubated for 45 minutes a t 37' with 200 pmoles of Tris, PH 8.3, 8 pmoles of -ITP, S ,umoles of IlgCl,, 10 pmoles of glutathione, 2 pmoles of butyryl Co-4 and 2.5 pmoles (5 pc.) in a total volume of 3.0 ml. Following of KHC1400a a 4S-minute incubation a t 3 i " , 1.0 m1. of 4:V sodium hydroxide was added; 30 minutes later the mixture was acidified with hydrochloric acid and continuously extracted with diethyl ether for 21 (1) S.J. \Vakil, THISJ O U R N A L , 80, G-l(i5 (19581. ( 2 ) J. V. I'nrmica and I
