COMMUNICATIONS TO THE EDITOR
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Vol. 81
particularly Zamecnik and his collaborators.1 TABLE I1 Relative specific T h a t additional reactions may be involved in activity in amino acid incorporation has been suggested by microsomal Protein Lipid Treatment Hendler2 upon noting a substantial incorporation lOOb 1006 None (control) of labeled amino acid by minced hen oviduct into Ribonuclease (0.1 mg./ml.) 42 107 lipoidal material similar to “pr~teolipids.”~ ATP and 3-phosphoglycerate omitted 21 115 We have examined the incorporation of labeled p-Chloromercuribenzoate ( M) 15 SG amino acids into lipid soluble material by cell-free 13 393d CrotoxinC(20 pg./nil.) preparations of rat liver. Phenylalanine was found Conditions as in Table I. Microsomes were analyzed to be incorporated readily into such material in after incubation of whole supernate. * Measured specific both microsomes and microsome-free soluble cyto- activity of the two fractions separately set equal to 100. plasmic supernate alone or together (Table I). Kindly furnished by Dr. 11. Fraenkel-Conrat. This stimulation has been verified repeatedly.
TABLE I R a t liver homogenates prepared according to Zamecnik and Keller‘ with medium Ah were centrifuged a t 10,000 X g for 10 min. t o yield the whole supernate, 3.5 ml. of which was incubated a t 37” for 2 hr. in SZ:COZ(95:5) with 5 pmoles of ATP, 50 pmoles of 3-phosplioglycerate and 1.15 prnoles ( 5 pc.) of ~~-plienj.lalanine-3-C‘~; total volume, 5 nil. Microsomes and soluble supernate were prepared before or after incubation by centrifuging the whole supernate a t 105,000 X g. Both the microsomes obtained after 30 min. centrifugation. suspended in cold medium A , and the first supernate were centrifuged an additional 60 min. Protein and lipid were prepared from the trichloroacetate-precipitated material essentially as described by Siekevitz.6 Fraction incubated
Whole supernate
C.p.m./rng.e hZicroson~al Protein Lipid
390 21s (14,200)b (3860) 136 714
Soluble supernate Protein Lipid
36 (5130)
Tryptophan and leucine were also incorporated into lipid while glycine, lysine and valine were incorporated to a much smaller degree. Support of this work by a grant from the National Science Foundation (G-5685) is gratefully acknowledged. PAPERNo. 1371 FROM THE JOSEPHL. HAINISG AGRICULTURAL EXPERIMENT STATION DEPARTMENT OF BIOCHEMISTRY, PURDUE USIVERSITY TOSHIO FUKVI WEST LAFAYETTE, INDIASA BERNARD L4XELROD RECEIVEDDECEMBER15, 195s JOURNAL
334 (1002) Microsomes .. .. Soluble supernate .. .. 39 567 e Net incorporation. Blanks containing trichloroacetic acid initially and incubated for zero and 2 hours were less than 10% as radioactive. * Figures in parentlieses indicate total c.p.m. in each fraction.
Sir:
P. C. Znmecnik, M. L. Stephenson and L. I. Hecht, Proc. Nall. Sci., U .S., 44, 73 (195s). R. W. Hendler, Science, 128, 143 (1958). J. Folch and M. Lees, J. Bid. Chrm.. 191, 807 (1951). (4) P. C. Zamecnik and E. R. Keller, ibid., 209, 337 (1954). ( 5 ) E.B.Keller and P. C. Zamecnik, i b i d . , 221, 45 (1956). (0) P. Siekevitz, i b i d . , 196, 549 (3952). (7) J. Hirsch and E. H. Ahrens, Jr., ibid., 233, 311 (1958). ( 8 ) W. Stepka, in Corcoran, “Methods in Medical Research,” The Year B u d l’iillishers, Chicdgu, 1052, Vul. 5, p . 25.
Upton, ibid., 224, 47 (1957). (3) I. E. Bush, J . Endocrinol., 9,95 (1953). (4) P. K. Bondy, G. V. Upton and G. E. Pickford, Nofurc, 179, 1354 (1957). (5) G. Pincus and E . B. Romanoff,“Ciba Colloquia on Endocrinol..” J. & A. Churchill, Ltd., London, 8,97 (1955). ( 6 ) J. Tamm, 1. Beckmanu and K. D. Voigt, A c f a Endocrinol., 97, 403 (1958). (7) C. 5. 0 . R . Morris a n d D. C. \Tilliams, “Ciba Colloquia on Eililucrinul..” 8, 157 (1955j.
ISOLATION O F CORTISONE AND CORTISOL FROM THE PLASMA OF PACIFIC SALMON
(0NCO RH YNCH US NE RK A )
Cortisol (I) has been identified as the principal adrenal-cortical hormone in the peripheral plasma of the normal human’ where it occurs at a concentration of approximately 10 pg. per 100 m1.2 It A major portion of the radioactive lipid-soluble is also the principal steroid in the blood of several material was separated from other lipids by silicic animalsa and has be.en tentatively identified in acid c h r ~ m a t o g r a p h y . ~This fraction, which was carp p l a ~ m a . ~ httempts to find cortisone (11) free of phosphorus, yielded on hydrolysis (6 hr in adrenal vein5 and peripheral human blood’ HC1, l l O o , 18 hr.) a number of amino acids includ- have been unsuccessful. Occasional blood samples ing radioactive phenylalanine, which was identi- have been reported to contain small amounts of a fied by co-chromatography on paper (butanol- “cortisone-like” substance,lJ but the evidence is acetic acid-water8) and radioautography with inadequate to establish identity. authentic phenylalanine. Milder conditions ( 2 We wish to report the isolation of I and I1 from V : HCI, looo, 2 hr.) released negligible amounts of Fraser River sockeye salmon (0. ?zerka) just prior phenylalanine. A mixture of the labeled lipoidal to their arrival on the Adams River spawning fraction with C14-phenylalanine was resolved by grounds. Blood was obtained from (ca). 100 fish paper chromatography. The radioactivity of the by severing the caudal artery. Ethyl acetate lipoidal substance was not diminished by extensive extractable steroids were obtained from 1760 ml. washing with a solution of non-radioactive phenyl- of plasma by the usual procedures. Chromatogalanine. raphy on purified Whatman no. 1 paper was perT h a t the incorporation of the amino acid into formed in three solvent systems known to resolve the lipid did not occur via the “conventional” I and I1 from closelyre lated steroids; (CHCla: pathway of incorporation into protein was proven C6Hal:1-forniamide (111), toluene-propylene glycol by the following: Microsomes were not required, (IV) and butyl acetate:ethylene glyco1:HzO 20:l :l. as noted above. Conditions which interfered with The corresponding U.S.P. reference standards and the “conventional” pathway did not curtail in- I and 11 were located on chromatograms both by corporation into the lipid-soluble material (Table (1) I. E. Bush and A. A. Sandberg, J. Biol. Chcm., ZO6, 783 (1953). 11). (2) P. K. Bondy, D. Abelson. J. Scheucr, T. K. L. Tseu and V. (1) Acad. (2) (3)
March 5, 1959
COMMUNICATIONS TO THE EDITOR
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alkali fluorescence and the reduction of blue tetra- has been investigated and the results clearly indizoleum. When substance I was treated with cate that this electrophilic reaction proceeds stereosulfuric acid a t 22' for 2 hr. i t exhibited the same specifically with retention of configuration. This spectra as U.S.P. I with Xmar. at 237, 282, 395 example indicates that the steric course of electroand 478 mp and O.D. ratios of 0.98:1:0.47:0.55 philic cleavage of organometallic compounds is and I1 gave the same spectra as cortisone with retention even in the absence of structural features Amax. a t 284, 343 and 420 and O.D. ratios of 1: which might alter the course of, or participate in, 0.38:0.31. The concentration of I and I1 was the reactions.2 determined by the quantitative Porter-Silber8 A mixture of the isomeric 4-methylcyclohexyltest for the dihydroxyacetone side-chain and each mercuric bromides was prepared by treating 4gave typical spectra with Xmax. a t 410 mp. Sub- methylcyclohexylmagnesium bromide with merstance I gave e242 mp 15.8 X lo3 (MeOH) and curic bromide. The less soluble trans-isomer was purified by recrystallization, m.p. 159.1-159.6'. I1 gave e237 mp 15.8 X lo3. The dihydroxyacetone side chain of I and I1 was Anal. Calcd. for C,Hl3HgBr: C, 22.26; H, 3.47; oxidatively removed with NaBiO3.l The ring Br, 21.16. Found: C, 22.10; H, 3.65; Br, 20.99. oxidation products of I and I1 were inseparable The cis-isomer was purified by chromatography, from authentic 110-hydroxy-A4-androstene-3,17-m.p. 130.8-131.2'. Anal. Found: C, 22.14; H, dione and A4-androstene-3,ll,l7-trione respectively 3.34. in both benzene-hexane :methanol :water1 and hepThe tentative assignment of the configurations of tane:benzene 1:l-fonnamide, each of which gives the cis- and trans-4-methylcyclohexylmercuric excellent resolution from closely related 17-keto- bromides based on melting points, relative stability, steroids. The yield of both oxidation products and product distribution from the Grignard was comparable t o that obtained from U.S.P. reaction, was confirmed by preparing the corsteroids and both gave positive tests for the A4-3 responding cis- and trans-1-deutero-compounds. The C-D stretching frequencies for equatorial ketone and 17-keto groups. Homogeneous I1 was obtained after a single and axial deuterium atoms in cyclohexane systems chromatogram in solvent 111, whereas I was re- have been determined previously.a trans-1-Deusolved further in solvent IV. The recovery of tero-4-methylcyclohexylmercuricbenzoate was precortisol (I) was 17 pg. and cortisone (11) 37 pg./lOO pared from the corresponding l-deutero-alkylmerml. of plasma, with an over-all recovery of ca. curic bromide, m.p. 99.4-99.8' ; Anal. Calcd. for 60-7075. Ci4HiTD02Hg: C, 40.04; H, 4.56; Hg, 47.78. At full sexual maturity Pacific salmon show ex- Found: C, 40.12; H, 4.38; Hg, 47.65. The intensive degeneration of the pituitary and a marked frared spectrum showed characteristic C-D stretchhyperphasia of the adrenal gland, and this has ing absorptions a t 2127 cm.-' (weak), 2148 cm.-l been postulated to play a dominant role in the (strong) and 2168 cni.-l (medium) ; therefore the degenerative changes and death of the fish after deuterium is predominantly axial. cis-1-Deutero~ p a w n i n g . ~The high levels of I and I1 lend sup- 4-methylcyclohexylmercuric benzoate was preport to the suggested high secretory activity of the pared similarly, m.p. 44.2-44.9' ; Anal. Found: gland. The plasma levels of I and I1 a t various C, 40.48; H, 4.38; Hg, 47.60. The infrared specstages of sexual maturity will be reported elsewhere. trum showed characteristic C-D stretching abCompounds I and I1 exhibited infrared spectra sorptions a t 2141 cm.-l (medium), 2166 cm.-l identical with cortisol and cortisone, respectively. (strong), and 2191 cm.-l (shoulder) ; therefore Acknowledgment.-We are grateful to Beckman the deuterium is predominantly equatorial. Our Instruments, Inc., for recording the infrared results indicate that the benzoxymercuri group , as compared to the methyl group, has a relatively spectra of the micro size samples. small preference for an equatorial over an axial (8) R. H. Silber and C. C. Porter, J . B i d . Chem., 210, 923 (1954). conformation. (9) 0. H. Robertson, Science, 126, 1295 (1957). The isomeric 4-methylcyclohexylmercuric broFISHERIES RESEARCH BOARDOF CANADA D. R. IDLER CHEMISTRY SECTION OF THE A. P. RONALD mides were cleaved with a variety of brominating TECHNOLOGICAL STATION P. J. SCHMIDTagents in several solvents. The competing free VANCOUVER, B. C. radical and polar processes, which were earlier RECEIVED DECEMBER 27, 1958 noted in the iodine cleavage of 4-camphylmercuric iodide,2C were encountered here. By varying the reaction conditions, either process could be ELECTROPHILIC ALIPHATIC SUBSTITUTION. I. made to predominate. When either isomeric ELECTROPHILIC SUBSTITUTION STUDIES WITH cis- AND ~~u~s-~-METR~CYCLOHEXYLMERCURIC organo-mercury compound was cleaved by bromine BROMIDES' in carbon tetrachloride solution under nitrogen, the Sir: free radical cleavage predominated and the prodWe wish to report the synthesis of a pair of uct distribution was 47.5% cis- and 52.5% transsimple isomeric organo-mercury compounds, cis- 4-methylcyclohexyl bromides. When more polar and trans-4-methylcyclohexylmercuric bromides, solvents, or hypobromous acid and other sources of which are suitable for studying the stereochemistry (2) For examples using stereochemically complex compounds see of electrophilic substitution a t a saturated carbon (a) A. N . Nesmeyanov, 0. A. Rustov, and S. S. Poddupnaya, laocst. Akad. Nauk,S.S.S.R. Otel, Khim. N a u k , 649 (1953): and (b) S. Winatom. Furthermore the bromine cleavage of the T. G. Traylor and C. S. Garner, TBISJOURNAL,77, 3741 (1955); cis- and trans-4-methylcyclohexylmercuric bromides stein, (c) S. Winstein and T. G. Traylor, ibid., '78, 2597 (1956). (1) This work was supported in part by a grant from the Research Corporation.
(3) E. J. Corey. M. G. Howell and A. Boston, ibid., '78, 5036 (1956).
