COMMITNICZTIONS TO THE EDITOR
4056
Vol. 73
nen-3@-01-20-one,'~which represents one of the int e r m e d i a t e ~in~our synthesis of cortisone.
Frozen beef adrenals were thawed a t 2', minced, suspended in buffer solution or 0.88 M sucrose solution and homogenized with a Potter-Elvehjem (18) €I B MacPhillamy arid C K Scllolr J Bzol C h r ~ r, 178, 37 type homogenizer. The resulting homogenates (1949) RESEARCH LABORATORIES, STNTEX, S. A. G. ROSENKRANZwere differentially centrifuged according to the I.AGUNA MAYRAN 41 3 J . PATAKI methods of Schneider and Hogeboom4 and the MEXICOCITY17, D. F. CARL1)JERASSI various supernatant and sedimented layers were tested for enzyme activity. RECEIVED JVNI:22, 19.51 T o test for enzyme activity, portions of the stdinients or supernatants in amounts equivalent to 1 g. of original tissue were incubated from one ENZYMATIC SYNTHESIS OF 17-HYDROXYCORTICOto five hours in 5 ml. of buffer containing 400 STERONE micrograms of 17-hydroxy-11-desoxycorticosterone. Sir: After incubation, the mixture was extracted with The biosynthesis of 17-hydroxycorticosterone chloroform, the chloroform evaporated to dryness has been demonstrated in intact adrenal glands, and the residue partitioned between petroleum extirpated adrenal glands perfused with a medium ether and 70% ethanol. The 70% ethanol phase containing 17-hydroxy-11desoxycorticosterone ace- was evaporated to dryness and the 17-hydroxyPate2 and adrenal gland hoinogenates to which 17- corticosterone formed was determined by the hydroxy-11-desoxycorticosterone had been added fluorescence and by chromogens produced with as the ~ u b s t r a t e . ~We wish to report the oxidation sulfuric acid. For the identification of the prodof 17-hydroxy-11-desoxycorticosterone to 17-hy- ucts, the residues of several incubations were coindroxycorticosterone by an enzyme system associ- bined, dissolved in chloroform and chromatoated with the insoluble cellular constituents of the graphed on silica gel with 7-10% ethanol in chloroadrenal cells. form. The figure shows the amount of 17-hydroxyCH20H CH2OH corticosterone formed by fractions separated by I 1 C--(> C=O differential centrifugation between 2000 g for ten minutes and 19000 g for 30 minutes. It will be &I3 1 1 0 ,,F,t/\OH seen that essentially all of the activity is in the I ' I granules while only traces are present in the super/\A. natant layer or the loosely packed layer above the Enzyme granules. The reaction proceeds best a t a pH near the neutral point and a t temperatures be(y+"V tween 35 and 40'. Above 40' there is a rapid de17-Hydroxy-1l-desoxy17-Hydroxycorticosterone cline in activity. It is inhibited by 0.01 M diethyl corticosterone dithiocarbamate, partially inhibited with 0.01 M HCN and disodium versonate and slightly I I I inhibited with 0.01 M sodium azide. With the -Granules buffer used, fumarate ion is necessary for the reaction. Adenosinetriphosphate does not enhance the rate of conversion. The product of the reaction has been isolated in crystalline form. The crystals separated in small striated cylinders and cruciform aggregates and melted a t 199-205'. A second crystallization yielded crystals which melted a t 204-208' and gave no depression of melting point with an authentic i00r sample of 17-hydroxycorticosterone obtained by saponification of 17-hydroxycorticosterone acetate. The product gave a distinct green fluorescence when treated with sulfuric acid and when chromatographed according to the methods of ZaffaronP 0 GO 120 180 positioned itself in the regions characteristic of T I M E IN M I N U T E S , 17-hydroxycorticosterone. Both the specific rotation and infrared spectrum were shown to conFig. 1.-17-Hydroxycorticosterone formed in differentially centrifuged fractions of a beef adrenal homogenate incubated form to those of 17-hydrosycorticosterone.
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with 400 micrograms of 17-hydroxy-11-desoxycorticosterone in a buffer composed of 0.01 BI glucose, 0.062 M NaCI, 0.04 M(NaHPO4-h.aHzPO4, PH 7.0), 0.025 A l KCI, 0.01 ill sodium fumarate and 0.004 AI MgSOn. (1) D. H. Nelson, H. Reich and L. T.S;irniicls. Science, 111, 578 (1950). 12) 0. Hechter, R.P. Jacobseli, R. Jennloz, H. Levy, C. W. Marshall, G. Pincus and V. Schrnkrr, A r r h f?inchcm., 25, 457 (1950). (:1) L) A. McGinty, (;. S . Sniitli, M. I,. W'ilson and C. S. Worrel, Science, 112, 806 ( 1 9 5 0 ~ .
NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES,NATIONAL INSTITUTES OF HEALTH, PUBLICHEALTH SECURITY AGENCY SERVICE,FEDERAL BETHESDA 14,MARYLAND MAXL. SWEAT RECEIVED JUNE 20, 1951 (4)
n'
C Schnelder and 0 11 Hogeboom, J . Bid Chem., 183, 123
(1950)
(5) R B Burton, A Zafironi and E €I Keutmann, rbrd , 188, 763 (1951).
