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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 that 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 Literatweld 144 0-144 5 - 6
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 ~ynergistically.~~ 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 Coh carboxylation catalyzed by purified propionyl carbo~ylase.~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 3i", 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
