Studies on mouse liver 21-desoxy- and 21-hydroxy-20a-hydroxysteroid : NADP

Depmtment of Biochemistry, University of British Columbia, Vancouuer 8, B. C. Received February 19, 1968

K~EIBIEL, R., AND BARRACH, M. Studies on mouse liver 21 -desoxy- and 21 -Rydroxy-20u-hydroxy- steroid : NABP 20u-oxidoreductase. Can. J. Biochem. 46, 1075-1080 (1968).

Corticosterone, 1 1-dehydrocorticosterone, 1 1 B-hydroxyprogesterone, and 1 1-ketoprogesterone have been compared as substrates in the soluble mouse liver 21-hydroxy- and 21-desoxy-28u-hydroxysteroid : NADP 20a-oxidoreductase enzyme systems. Each enzyme showed a high specificity for catalyzing 20u reduction by NADPHz that depended on the structure of the C-21 group. A much higher conversion rate was given by 11-dehydrocorticosterone than by corticosterone in the 21-hydroxy-2Om-hydroxy- steroid 20u-dehydrogenase system whereas 1 LP-hydroxyprog~tero~e and 11-ketoprogesterone gave approximately the same rates in the 21-desoxy-2Ou-hydroxysteroid 2Qu-dehydrogenase system. Corti- costerone and 1 L P-hydroxyprogesterone proved to be competitive inhibitors of the 20u reduction of 11-8ehydrocorticosterone as substrate in the 21-hydroxy-20%-hydroxysteroid 20m-dehydrogenase system. Both corticssteroae and substrate, but not 1 1P-hydroxyprogesterone, gave 2Qu-hydroxy derivatives. Similarly, 11-dehydrocorticosterone and 11-ketoprogesterone were competitive inhibitors of the 20ee reduction of 11 fl-hydroxyprogesterone as substrate in the 21-desoxy-20u-hydroxysteroid 20u-dehydro- genase system. Both 1 1-ketoprogesterone and substrate, but not 1 1-dehydrocorticosterone, were con- verted to 20u-hydroxy derivatives. The new sterond 20~-hydroxy-4-pregnens3,l l-dione has been synthe- sized by chemical and enzymatic methods, and its properties and those of its acetate are recorded.

In a previous publication from these labora- tories (I), it was shown that the dialyzed high- speed supernatant fraction of male mouse liver contains at least two 20a-hydroxysteroid dehy- drogenase enzymes. One of these catalyzes the reduction by NADPH,' of the C-20 carbonyl group of corticosterone, and the other, the C-20 carbonyl group of 2 1 -desoxycorticosterone (1 1 P- hydroxyprogesterone). Thus the specificity of these enzymes is determined in part by the sub- stituent at C-21, and they are therefore desig- nated either 2 1 -desoxy- or 2 1 -hydroxy-20a-hy- droxysteroid 20a-dehydrogenase. A comparison of corticosterone and 1 1-dehydrocorticosterone as substrates in the 21 -hydroxy-20a-hydroxy- steroid 20a-dehydrogenase system revealed 11- dehydrocorticosterone to be a much more reactive substrate than corticosterone (2). In view of this enhancing effect on reaction velocity of the 11-ketone group, it k a m e of interest to

lThe following abbreviations and trivial names are used: o.r.d., optical rotatory dispersion; NADP, nico- tinamide-admine dinucleotide phosphate; NADPW2, nicotinarnide-adenine dinucleotide phosphate, reduced form; cortexone, 21-hydroxy-4-pre-ene-3,20-dione; cor- ticosterone, 11 P,21-dihydroxy-4- pregnene - 3,20 - dime ; progesterone, 4-pregnene-3,20-dione; 11 bhydroxypro- gesterone, 1 1 P-hydroxy-4pregnene-3,20-dione ; 1 l-keto- progesterone, 4-pregnene-3,11,20-trione ; 11 -dehydrocor- ticosterone, 21-hydroxy-4-pregnene-3,lB ,20-trione ; mu- dihydrscorticostersrae, 1 l fl,2Qcr,21 -trihydroxy-4-pregnen- 3-one.

compare 1 1-ketoprogesterone with 11 p-hydroxy- progesterone as substrates in the 21-desoxy-2Oa- hydroxysteroid 20a-dehydrogenase system. Also because these 2 1 -hydroxy- and 2 1 -desoxy-ste- roids of otherwise identical structure require different enzymes to catalyze 20a reduction, the corticoid and progestin substrates under study were examined as potential enzyme in- hibitors in each other's specific 20a-hydroxy- steroid dehydrogenase systems. The present communication describes these experiments. The product of one of the reactions was 20~-hydroxy- 4-pregnene-3,11 -dione, and since this steroid has not been described previously, chemical and enzymatic methods are given for its synthesis. Physical properties of this new steroid and its acetate are also recorded. The literature relating to the present study and particularly the dis- tribution and specificity of mammalian 20a- hydroxysteroid dehydrogenases has been re- viewed in a recent publication from these iabora- tories (1).

Experiment a1 Mouse Liver Fi.acrion DS3 - 20 h

The preparation of this crude enzyme mixture has been described (1). It contains the soluble 21-hydfoxy- and 21 -daoxy-20u-hydroxysteroid 20~-dehydrogenases of adult male mouse liver and is the clear supernatant solution obtained after dialysis for 20 R at 10' of the high- speed (BOO 000 g, 60 min) supernatant fluid from fresh homogenized liver.

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1 076 CANADIAN JOURNAL OF BIOCHEMISTRY. VOk. 46, I968

Mouse Liver 21-Hydrox~t-2Qu-&m~~droxysteroid 2001-Behy- drogenase (Fraction 0.05 P) and 26-Besoxy-20a-hy- droxysferoid 283e-Del1y~hge1zase (Facfion 0.4 P)

Both these enzymes were separated from each other in good yield from fresh preparations offraction DS3 - 20 h. The methods have been described previously (1). Briefly, they involve adsorption of the enzymes on cal- cium phosphate gel and elution of 21-kydroxy-20a-hy- droxystersid 2&-dehydrogenase with 8.05 M phosphate, and of 21-desoxy-20%-hydroxysQeroid 20r-dehydrogenase with 0.4 A4 phosphate. After readsorption and elution of fraction 0.4 P, the preparations were stored at -20" as lyophilized products.

Standard Reactioa Mixtures The standard reaction mixtures have been described in

detail (1). They contain either 40 mg fraction 0.05 P, 0.25 miUimit/mg, m 188 rng fraction 0.4 P, 0.1 1 miUi- unitlmg, in 4.75 m1 H 2 0 and 8 d Krebs-Eggleston phosphate saline containing 15 nag sodium fumarate and either 3 3 g N m P H 2 (for fraction 0.05 P) or 1 mg B\BADPHa (for fraction 0.4 P), and steroid or mixtures of stemids in 0.25 d ethanol (total volume, 13 ml ; pH, 6.8).

Deberminatis~ of Enzyme AcfiviPg, Incubations were carried out in 125-ml Erlenmeyer

flasks at 37.5" for 30 min (unless indicated otherwise) under nitrogen in a DubnoE apparatus set for 50 cycles/ min. Reaction velocity was determined, as described in earlier reports (1, 2), by isolation and quantitative measurement of the product, 'The reaction time con- f oms with points on the time-progress c u m of both enzymes (1, 2) where product formation is linear with time and does not exceed 20 % of the original substrate. Since substrate sand coenzyme were in excess throughout each reaction, the measurement of the amount of product formed under these conditions represents initial velocity. The paper-chromatography systems used for product isolation have been described for the 2k-hydroxy deriva- tives of corticosterone, (24), 1 1-dehydrocsrticosterone (5), and 11 fbhydroxyprogsterone (I). The 2b-hydroxy derivative of H 1 -ketoprogesterone was isolated on paper after 5 %a in the toluene - propylene glycol system, and rrppmed at ~COrtiCOBterOnC 2.6.

20a-PIydroxy-4-p1'egnene-3,1 I -&one A solution of 109 mg 4-pregnene-3,11,2O-trione in

15 ml ethanol was added to a mixture of 300 ml mouse liver fmcrim BS3 - 20 h and 4868 ml Krebs-Eggleton. phosphate saline buffer containing 111 mg NADPH2 and 909 rng sodium fumarate, pH 6.8. The mixture was subdivided into 16 x 125-ml Erlenmeyer flasks and incubated for 6 h under nitrogen at 37.5" in a Dubnoff apparatus set for 50 cycIes/min. The contents of each flask were then extracted with 5 x 180 ml chloroform, and the extracts were pooled, dehydrated over sodium sulfate, and evaporated to dryness in vacus at 40". Chromatography of approximately 10-mg portions on paper in B/ToI9 for 5 h gave zones of the produet at R ,,,, , ,,,,,,, 2.6. After elution with methanol, filtration, and evaporation to drymess in ieacuo at 40°, the residue was crystdlized from acetone-water. Recrystallization

*Zaffaroni systems : Tol, toluene ; P, propylene glycol ; Be, benzene; Cy, cyclohexane; F, formamide; Lig, ligroin @.p. 65-1 10°).

from acetone-water gave 5 I mg 2&-hydroxy-4-pregnene- 3,fl-dione; m.p. 208-209.5"; [aclqDas +165" (c, 1.01 in acetone), [a]D25 +17gc (c, 0.62 in methamoI); k,,,CH3QH 238 mp. (IS 300); h,,,"2s04 2 h, 288 mp (832), 391 I I I ~ (31); 2bWxKBP 2.95, 5.90, 6.03, 6.19, 7.82, 8.15, 8.40, 8.52, 9.18, 9.34 10.43, 10.52, 11.16, 11.51, 12.13, 12.4, 12.88, 14.61 ga; s.r.d. in methanol (c 0.62, from 589 to 300 mp; e 0 . ~ 6 , from 300 to 210 mp), 30"; i(b15as +6m0, [41500 +885", I31400 -t-171O0, +25w0, 6+1351 f 257oQs E413e +2630°, +25505, [4laa7 +2790°, bb1saa +2750°, [433063 f 4690°, +I1 1m". E4laez +396m0, [+laao 0°, E4clqz1% -57 84)6%Os E+la 10 -56 7W0. -per chromatography: &,,,I ,,,,,,,,, P/Tol 2.6, R ,,,,,,,, $/To1 0.44, P/Be-Cy 61 : 1) 0.26, F / k 0.83, F/Cy 0.25, F/Be-Cy (1 :1) 0.57, F/Be-CHC13 (1 :1) 1.0. Found: C, 76.25; H, 9.18. C21H3003 requires C, 76.33; H, 9-15.

20a-Acetoxy-4-pregnene-3, d l -dioae A. From 2001-Hydro~y-4-pregnene-3, d 1 -dime A solution of 33 mg 2801-hydroxy-4-p~mene-3,11-

dione, prepared as described above, in 1 ml pyridine and 0.5 rnl acetic anhydride was heated on the steam bath for 15 min. After evaporating the solvents in uacuo, recrystal- lization of the product from acetone-hexme (waxy platelets) and ethanol-water (fine needles) gave 25 mg 20%-acetoxy-4-pregnene- 3, B 1 -dione; m.p. 136-137"; [a],25 +147.8" (c, 0.912 in acetone), [%IDa5 +l62' (c, 0.531 in methanol); A& (2k-acetate - 2Oa-01) f 5' (acetone), +IS0 (methanol); h,,,cH~Qn 238 my. (15 300); hmaXHzS04 (El Zl Cd 2 h, 288 mp (714), 391 rnp (26) ; hmaXKBP 5.79, 5.92, 5.98, 6.19, 7.83, 8.05, 8.63, 9.53, 9.84, 10.62, 10.71, 11.41, 11.88, 12.43, 12,86, 14.64 p; 0.r.d. in methanol (c 0.50, from 589 to 300 mp; c O.OQ5, from 300 to 210 mp)% 30"; [+]589 +596O, [+lsoo +878", [ ( ~ I ~ c I o +f680°, E41~a-1 f 252O0, t+ljsa +25m0, [4134ii +25m0, [$I337 +2480"9 841327 f 2670°, f 26M0, 141aoo +4840°, 8+ls~s -I-10 3w0, E(bclqz42 +38 9(33', [+I230 00, [4lz1a -54 30Qat E$loao - 5 2 m 0 . Paper chromatography: Rp,,ge,,,,,,, P/Lig 0.54, F/Cy 0.63, P/To% 1.0, B/Be-Cy (1 :1) 1.0, F/BeGy (1 :1) 1.0. Found: C, 74.02; H, 9,W. C23H3aQ4 requires 6 , 74.15; H3 8.55.

B. From 1 I ~ - ~ Y ~ ~ o x ~ - ~ ~ ~ - Q C @ ~ O X Y - ~ - ~ ~ @ ~ ~ ~ P ~ - ~ - O ~ ~ C / 1 L l ( 1 % 01

A mixture s f 37 mg 11 fl-hydroxy-20a-acetoxy-4-preg- nen3-one prepared as described previously dl) and 37 mg chromic anhydride in 0.5 mE pHdine was left over- night at room temperature. After the addition s f 1 ml of water, the solution was extracted with 5 x 5 d ether. The pooled ether extracts were evaporated to a oily residue in uacuo at 443". Crystallization from acetone- hexane and recrystallization from acetone-water gave 253 mg 2061-mtoxy-4-preaene-3,111dione, m.p. 135.5- 136.5". A mixture of equal parts with the acetate from procedure A above gave mop. 135.5-136.5". No separa- tion occurred on paper chromatography in the P/Tol, P/B-Cy, PILig, F/Be-Q, or F/Q systems. The in- frared absorption spectrum was identical with that of preparation A above.

Results and Discussion Enzyme Speeifieity

Table I compares the enzyme activities given

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KWEWBPBE AND DARWACH: 20a-HYDROXYSTEROID DE-WQGENASES 1077

TABLE I Specificity sf 21-bydroxy- and 21-dessxy-2Qa-hykoxysteroid 20a-dehydrogenase

Milliunits (mu)* Fn~ction Fraction Fracliom

Substrate @-21 C-1 f BS3 - 26 h 0.05 91 0.4 Pt

1 1-Dehydrocorticosterme @H20H C - 0 13.2 8.10 <Q.3 (8.34) (< 0.3)

Csrticosterone CH20H C --OH @) 1.46 8.76 < 0 . 3 (0.99) (< 0.3)

I 1-Ketoprogestersne @& C=O 39. 82 < 0 . 3 11 . O (0.35) (8 . 8b

1 1 p-Hydrsxyprsgesterone CH3 C --OH (PI 40.94: 0.42 13.9 Q.=0.3) 611.01

*Ow unit (U) of enzyme is defined as the amount that catalyzes the conversion of 1 pmole of substrate par minute under standard reacxn conditionsas. Results ape recorded as rnilliunits ( m u 0).

?The duplicate enzyme aesays fm each substrate, as shown parentheses, were dsw cm preparations of fraction 8.05 P and fractbpt 0.4 P made from a diEerent batch sffraction BSs - 20 h.

$Incubated under standard conditions (see text) except for time. PIP the case of fmcbion DSS - 20 fi incubation mixtures with eitber I f - ketopaogesterone or f 10-hydroxyprogesterow incubatisns were carr~ed out for 10 min rather than 30 min to reduce total product formation to less than 20% s f the original substraw.

by duplicate preparations of fraction 0.05 B (2 1 -hydroxy-20a-hydroxysteroid 20a-dehydroge- nase), fraction 0.4 B (2 1 -desoxy-20a-hydroxy- steroid 20a-dehydrogernase), and the crude mix- ture of these enzymes (Jmction DS, - 20 Iq) from which they were derived. Raction 0.05 B com- pared with fmction 0.4 B shows the former to have a high specificity for the 21 -hydroxysteroids corticosterone and 1 1 -dehydrocortieosterone, whereas fraction 0.4 B exhibits specificity for the 21 -desoxysteroids 1 I p-hydroxyprogesterone and 1 1-ketoprogesterone. The nature of the oxygen function at C-f 1 influences the velocity of 20te reduction in the 21-hydroxy series, as illustrated by the substantially higher reaction rate given by 1 1-dehydrocorticosterone than by cortico- sterone. On the other hand, this difference does not occur in the 21-desoxy series, where both 1 1 p-hydroxyprogesterone and 1 1-ketoproges- terone show similar reaction rates.

Enzyme Inhibition: Fraction 0.05 B (2%-P-PyeiPoxy- 20a-hydroxysteroid 20a-Dehydrogenase)

Among several eortieoids tested with fraction 0.05 B, 1 1-dehydrocorticosterone showed a high reaction rate, therefore it was selected as a standard substrate for inhibition studies. Kinetic properties of this enzyme fraction with 11-dehy- drocorticosterone as substrate have been re-

I / S ~ p m e l e s / m l ) - I

FIG. 1. Effect of 500 pg of corticosterone (A) and 500 pg of llp-hyhxyprogesterone (0) on the effect of substrate concentration on reaction velocity of conversion of I 1-dehydrocorticosterone to 2Oa,21-dihydroxy-4-preg- nene-3,11 -dione (8). Standard incubation conditions with fiacfi~n 6.05 P (see text) except for veiriations in steroid concentrations. For cortico~~rone, Mi = 1.9 x 1W5 and for 1 lf3-hydraxypragestaozae, Ki = 3.2 x 10-5:

second series the effects of adding 0.5 mg 11 8- hydroxyprogesterone. In the mixtures contain- ing 1 1-dehydrocorticosterone and corticosterone both steroids were converted to 2001-hydroxy products, whereas in those containing 1 1-dehy- drocorticosterone and 1 1 p-Aydroxyprogesterone there was no conversion of 1 1 p-hydroxyproges- terone. This was evident from the chromato-

ported previously (1). Data in Fig. 1 show effects grams that showed, in the former series, both on the 1 1-dehydrocorticosterone substrate-con- 20u, 21 - dihydroxy - 4 - pregnene - 3, l l - dione and centration curve by the addition of 0.5 mg corti- 20a-diAydrocorticosterone, but in the latter, costerone to each incubation mixture, and in a only 20~,2l-dihydroxy-4-pregnene- 3,ll -dime ;

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1078 CANADIAN JOURNAL OF BIOCHEMISTRY. VOL. 46, 1968

1 1 ~,20a-dihydroxy-4-pregnen-3-one was absent. The nature of the curves in Fig. I indicate that both corticosterone and 11 - hydroxyprogester- one function as competitive inhibitors in this enzyme system, and while corticosterone also undergoes conversion as a substrate there is no change in the structure of 1 1 p-hydroxyproges- terone. With corticosterone as inhibitor, Ki = 1.9 x 10- ' , and with B 1 P-hydroxyprogesterone as inhibitor, Ki = 3.2 x 10- '. In separate exger- iments, variation of the inhibitor concentration with fixed concentrations of substrate gave the same constants (Fig. 2). IPS $gnrns~es~ml I-'

I I

0 250 500 Inhibitor, g ~ g

FIG. 2. Effect of 125, 250, a198 500 pg of corticoste- sone (A) md of 125, 250, and 500 pg of 1lf)-hydroxy- progesterone (8) on reaction velocity of the conversion of 500 pg of 11-dehydrocorticssterople to 20a321-dihy- drsxy-4-pregnene-3, I 1 -dione. Standard incubation con- ditions with fraction 8.05 P (see text) except for variations in steroid concentrations. Fsr corticosterone, Ki = 1-53 x lo;', and for 11 a-hydroxyprogesterone, K, = 3.2 x 10- .

Enzyme Inhibition: Fraction 0.4 P (21-Desoxy- 20a-/zydroxysteroid 20a-Dehydrogenase)

Although preliminary experiments showed several progestins to be active substrates in this enzyme system, 1 B B-hydroxyprogesterone gave a high rate of conversion and was, therefore, selected as a standard substrate for inhibition studies. Kinetic data for this enzyme system with 1 1 p-hydroxyprogesterone as substrate have been reported (1). Figure 3 shows the effect on the 1 1 P-hydroxyprogesterone substrate-concen- tration curve of adding 0.5 mg 1 l-dehydrocorti- costerone, and in another series the effect of adding 0.5 mg 1 1-ketoprogesterone. Chromato- grams prepared from incubation mixtures that contained I 1 p-hydroxyprogesterone and 1 1 - keto progesterone showed the 2Oa-hydroxy prod-

FIG. 3. Effect of 500 pg of 11-ketoprogeskrone (A) and 584) pg of Il-dekydrmorticosterone (O) on the effect of substrate concentration on reaction velocity s f eon- version of 8 1 B-hydroxyprogesterone to 1 f b,26a-dihy- droxy-4-pregnen-3-0ne (0). Standard incubation csndi- tisns withfraction 0.4 P (see text) except for variations in steroid conmtrations. For 1 1-ketoprogesterone, kll = 1.2 s lo-", and for 1 1-dehydrocortieoste~orae, KI = 3.7 x

ucts of both substrates. However, those pre- pared from mixtures of 11 p-hydroxyprogester- one and 1 1-dehydrocorticosterone exGibited only 1 1 8,20a- dihydroxy-4-pregnen-3-one ; 20a, 21-di- hydroxy-4-pregnene-0 laione was absent. It is clear from Fig. 3 that both 11-ketoprogestersne - - and 1 1-dehydrocorticosterone are competitive inhibitors in this enzyme system. Although there is no conversion of 1 1-dehydrocorticoster~ne, both 1 1-ketoprogesterone and the standard sub- strate 1 1 p-hydroxyprogesterone are reduced at C-20 to 2001-hydroxy products. With I l-dehydro- corticosterone as inhibitor, Ki = 3.7 x lo-", and with 1 1-ketoprsgesterone as inhibitor, Ki = 1.2 x Plots of data from experiments with fixed substrate and variable inhibitor concentra- tions gave the same constants (Fig. 4).

2001-Hydrsxy-4-paegnene-3,dd-dfone The structure 2Oa-hydroxy-4-pregneaae-3,11-

dione assigned to the product- of ihe reaction of fraction 0.4 P, NADPH,, and 1 l-ketoprogester- one was elucidated by the following-evidence. Neither ring A nor the keto function at C-11 were involved in the transformation of substrate to product, since the latter continued to exhibit an ultraviolet absorption band characteristic of A4-3,l 1-diketosteroids, with the wavelength of maximal absorption at 238 rnp (15 300). In view of the ease of esterifjing the unknown product to an acetate under mild acetylating

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KREI-iBIEE AND DARRACH: 2Oa-HYDROXYSTEROID DEHYDROGENASES

I I 1 1 I

0 5 08 I008 Inhibitor, ~g

FIG. 4. Effect of 250, 500, and 1000 pg of 11-ketoprogesterone (A) and of 250, 500, and 1000 pg of ll-dehydro- corticosterone (0) sn reaction velocity of the conversion s f 500 pg sf 11 $-hydrsxyprogestersne to 11 $,20a-dihydroxy- 4-pregen-3-one, Standard incubation conditions with flaction 0.4 B (see text) except for variations in steroid concen- trations. FOP I l-dehydr8co~ticoste~one, Ka = 3.7 x 1W4, and for 11-ketoprogesterone, Ki = 1.2 x

conditions, it was clear that the product of the enzymatic conversion had acquired a hydroxyl function at C-20. The above conclusions were confirmed by the infrared spectra in which the unknown exhibited an intense hydroxyl band at 2.95 y that disappeared on acetylation. Also the unknown exhibited a three-peaked band in the 5.8-6.2 p region characteristic of the A4-3,l 1- diketo structure; on acetylation this band ac- quired a fourth peak associated with the new carbonyl function of the acetate group. The critical question now concerned the configura- tion at C-20 which had become a new asym- metric center. By analogy with the 20a dihydro- genation of 1 1 P-hydroxyprogesterone in a similar enzyme system (I), the expected product was the 20a epimer. Evidence to support this con- clusion was provided by the different melting points and optical rotations of the unknown and its acetate when compared with those reported by Norymberski and Woods (8) for the free and acetylated 20j3-01. Of special significance in revealing the configuration of the unknown at C-20 was the fact that the specific rotation of the 20 P-acetoxy epimer (8) (reported : [a], + 2 18" (c, 0.81 in chloroform)) was more dextrorotatory than that of the unknown acetate ([a],25 + 147.8" (c, 0.912 in acetone)). Furthermore, the shift in molecular rotation on acetylation was strongly positive for the 20P-01 (8) (reported: AM, + 145") and weakly positive for the unknown ( A 6 + 5"). These optical rotation data con-

firmed a 20a-hydroxy configuration for the un- known in agreement with the rules established by Fieser (9) and Sarett (10) for other pairs of C-20 hydroxy epimers. Completion of the evidence for the formula assigned to the unknown was afforded by the fact that the acetoxy derivative was identi- cal with the product of chromic anhydride oxida- tion of the known 11 P-hydroxy-20a-acetoxy-4- pregnene-3,11 -dione.

Summary It is apparent from these experiments on the

20a-hydroxysteroid dehydrogenase activities of $-action 0.05 P and fraction 8.4 P that at least two such enzymes exist in the soluble fraction of homogenized male mouse liver. One shows specificity for 21-desoxysteroids (progestins) and the other for 21-hydroxysteroids (corticoids). In addition to this specificity, which depends on substitution at C-21, the nature of the oxygen function at C-11 (whether P-hydroxy or ketone) has a bearing on the velocity of C-2Q dihydro- genation in the 21-hydroxy-20a-hydroxysteroid 20a-dehydrogenase system when corticosterane and 1 1-dehydrocorticosterone are compared as substrates. On the other hand, the nature of the oxygen function at C-l 1 (whether P-hydroxy or ketone) has no effect in the 21-desoxy-20a-hy- droxysteroid 20a-dehydrogenase system when 1 1 P-hydroxyprogesterone and 1 l-ketoprogester- one are compared as substrates. Results of inhibi- tion experiments with the separated enzymes

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1080 CANADIAN JOURNAL OF BIOCHEMISTRY. VOL. 46, 1968

suggest that csrticoids and prsgestins can show mutual inhibition toward each other in the con- version of 20-keto hormones to their 20a-hydroxy metabolites.

The authors are grateful to the Medical Research Council of Canada and the National Cancer Institute sf Canada for financial assis- tance.

11. DARRACH, M., KRBHBPEL, R. E., and BEETH, E. A. Can. J. Biochem. 46, 715 (11968).

2. K R E ~ H E L , W. E., BURTON, A. F., and B A ~ C H , M. Can. J. Biochem. Physiol. 48, 1997 (1962).

3. SOUTHCO'BT, C. M., BANDY, H. E., NEWSOM, S . E., and DARRACH, M. Can. J. Bimhem. Physiol. 34, 913 (1956).

4. SO~HCO'FT, C. M., SPROULE, V. A., ~ ~ ~ I N T O S H , Ha, and BARRACH, M. Can. J. Biochern. Physisl. 36, 819 (1958).

5. DARRACR, M., and K ~ H B I E L , W, Can. J. Biochem. Physiol. 443, 1769 (1962).

6 NEHER, R., and WETFS~IW, A, Helv. Chim. Actas, 43, 1171 (1960).

7. Recornmadations (1964) of the international union of biochemistry on the nomenclature and elassifiea- tisn of enzymes. Elsevier Publishing CO., New Ysrk. 1965.

8. NORY~ERSKH, J. K., and W m ~ s , @. F. J. Chem. SOC. 3426 (1955).

9. FIESER, L. F., and FHESER, M. Steroids. Reinhold Publishing Gorp., New York. 1959. p. 614.

18. SARETT, L. H. 9. Am. Chem. Sw. 71, 1175 (1949).

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