PURIFICATION AND PROPERTIES OF A &HYDROXYSTEROID DEHYDROGENASE*

BY PAUL TALALAYt AND MARIE MOLLOMO DOBSON

(From the Ben May Laboratory for Cancer Research, the University of Chicago, Chicago,

Illinois, and the Medical Research Council Unit for Chemical Microbiology, School of Biochemistry, University of Cambridge, Cambridge, England)

(Received for publication, June 5, 1953)

Certain species of Pseudomonas may utilize testosterone and related steroids as sole sources of carbon (l-3). “Resting” cell suspensions of these bacteria carry out vigorous oxidative degradation of such steroids by means of adaptive enzymes. A study of the mechanism of this process has led to the characterization of one enzymatic step: the reversible intercon- version of 3/3- and 17/3-hydroxysteroids and their respective ketosteroids by a DPNI-linked dehydrogenase (1). The purification and spectrophoto- metric assay of this enzyme are reported here, together with information on kinetics, substrate specificity, and competitive inhibition by other ster- oids. Consideration is also given to a sensitive enzymatic assay of selected groups of steroids.

The metabolism of androgenic steroids by mammalian enzymes has been recently reviewed (4, 5). Samuels et al. (6) have demonstrated that slices and crude homogenates of liver and kidney catalyze the aerobic conversion of testosterone to 17-ketosteroids and the disappearance of the ultraviolet absorption due to the or,/?-unsaturated ketone group. The formation of 17-ketosteroids is stimulated by the addition of DPN. A concentration of the steer liver enzyme converting testosterone to 4-androstene-3,17- dione has been described (7), and the requirement for DPN demonstrated. Conversion of A5-3&hydroxysteroids to A4-3-ketosteroids has been claimed to occur in crude homogenates of certain endocrine tissues fortified with DPN, but not in liver or other organs (8).

Interest in microbiological transformations of steroids was stimulated by Mamoli and Vercellone (9) who demonstrated the conversion in approxi- mately 80 per cent yield of 4-androstene-3,17-dione to testosterone by fer-

* These investigations were supported by grants from the American Cancer So- ciety, recommended by the Committee on Growth of the National Research Council, and by the Jane Coffin Childs Memorial Fund for Medical Research.

t Senior Assistant Surgeon, United States Public Health Service; assigned from the National Cancer Institute, Bethesda, Maryland, to the University of Chicago.

1 The following abbreviations are used in this paper: DPN = diphosphopyridine nucleotide; DPNH = reduced diphosphopyridine nucleotide; TPN = triphosphopyr- idine nucleotide.

823

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824 P-HYDROXYSTEROID DEI-IYDROGENASE

menting yeast (or possibly by bacteria contaminating the yeast). Mamoli (10) and subsequently Wettstein (11) studied the reduction of estrone to 17/3-estradiol by fermenting yeast. More recently, the reverse reaction was described by Heusghem and Welsch (12) who demonstrated that rest- ing cell suspensions of certain species of Strepfomyces carried out the quan- titative conversion of 17P-estradiol to estrone. Microbiological oxidation of 3-hydroxysteroids has likewise been observed in the conversion of dehy- droepiandrosterone (13) or 5-androstene-3,17-diol (14) to 4-androstene-3, - I7-dione by yeast under aerobic conditions. The reduction of S-ketoster- oids proceeded less rapidly and completely t,han that of 17-ketosteroids, especially with increasing length of the side chain at position 17.

Methods and Materials

Microorganism-A species of Pseudomonas was used in these investiga- tions (1).

DPN--Approximately 60 per cent pure DPN was obtained commer- cially, and further purification was accomplished by adsorption on a Dowex 1 formate column and elution with formic acid according to an unpublished procedure of A. Kornberg and B. L. Horecker. The purity of the final preparations was measured enzymat.ically with alcohol dehydrogenase by the method of Racker (15) and was in excess of 80 per cent on a weight basis, without special precautions to dry the material. The molecular extinction coefficient for pure DPNH was assumed to be 6220 (16). The purified DPN was stored frozen, and solutions were prepared by dissolving the requisite amount in water and neutralizing with NaHC03.

Testosterone-U. S. P. grade testosterone was incorporated into the growth medium without additional purification. For spectrophotometric measurements, further purification was carried out by two crystallizations from ethyl acetate, followed by a sublimation in VCLCUO (0.001 mm. of Hg at 118’). The final product had a melting point of 154.5-155” (corrected) and a specific rotation of [cy]i2 + 111.7” (1.95 gm. per 100 ml. of ethanol solution).

Solutions of testosterone were prepared either in methanol or in water. Saturated aqueous solutions were prepared by adding an excess of finely ground test.osterone to water and bringing the solution to 100” for 1 minute. After cooling to room temperature, the turbid suspension was centrifuged for 10 minutes at 2000 X g to give a clear supernatant solution containing 25 to 30 y of testosterone per ml. The precise testosterone concentration of such solutions was determined spectrophot.ometrically. The molecular extinction coefhcient of purified testosterone in methanol was found to be E = 15,600 at the absorption peak X,,,. = 240 to 241 rnp. In water the peak shifted considerably toward longer wave-lengths, A,,,,,. = 250 to 251

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P. TALALAY .4ND M. M. DOBSON 825

rnp and E = 16,200. These values checked closely with those determined by Dannenberg (17).

Other Steroids-The identity of all steroids was checked by melting point and specific rotation, and, when necessary, additional purification was carried out by crystallization and sublimation. The physical constants of samples used were in good agreement with those recorded in the literature.

Solvents-Common solvents were redistilled reagent grade materials. Formamide was distilled at 98-101” and 14 mm. of Hg. Commercial hexane was treated with concentrated sulfuric acid, followed by alkaline potassium permanganate, and then distilled through a fractionating column.

Growth, Harvesting, and Enzyme Extraction-The microorganisms were grown in 5 to 10 liter batches in well aerated liquid cultures containing 5 gm. per liter of yeast extract (Difco) and 0.5 gm. per liter of testosterone on a mineral medium.2 The testosterone was finely ground with a small portion of the medium and the eutire mixture autoclaved for 10 minutes at 15 pounds per sq. in. of steam pressure. The cultures were grown on a platform shaker at 30” and harvest,ed after 10 to 15 hours, at the time when the testosterone-oxidizing enzyme activity was at a maximmn2 The en- tire culture was then filtered t,hrough Whatman No. 40 filter paper on a large Biichner funnel in order to retain unused testosterone and precipi- tated salts.

The cells were harvested from the filtrate by passage through a Sharples continuous supercentrifuge. They were washed twice with 0.03 M phos- phate buffer, pH 7.2, by centrifugation. The resultant cell paste was then mixed with 2 to 3 times its wet weight of levigated grinding alumina (Nor- ton Abrasive Company) in a cold mortar and the chilled mass gently ground for approximately 2 to 3 minutes according to the method of McIlwain (18). The mixture was extracted with two portions of 0.03 M

phosphate buffer, pH 7.2, and the extracts pooled (total volume approxi- mately 3 to 5 times the wet weight of the cells). These extracts were used for the purification procedure described below. Centrifugation at 10,000 X g for 15 minutes at 0” resulted in a sharp separation of the clear straw-yellow supernatant fluid from particulate matter, cell envelopes, and alumina.

Spectrophotometric Assay-The enzyme activity was measured by the rate of reduction of DPN at 340 rnp in a Beckman spectrophotometer, with cuvettes of 1 cm. light path. The lamp housing was water-cooled, and enzymatic reactions were run in a compartment in which the temperature was regulated by means of Beckman “thermospacers” through which water at 24-25” was circulated. The temperature of the reaction cell could in this manner be maintained within f0.5” or less.

2 Talslay, P., and Dobson, M. M., to be published.

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826 /?-HYDR~XY~TER~ID DEHYDR~GENA~E

The reaction mixture consisted of 0.6 ml. of 0.166 M sodium pyrophos- phate buffer, pH 9.0, 1.0 ml. of saturated aqueous solution of testosterone containing approximately 25 to 30 y (about 0.1 PM) of testosterone (or 0.1 ml. of a methanolic solution of testosterone containing 25 y), 0.1 ml. of solution of DPN (about 0.48 PM), and 0.05 to 0.10 ml. of appropriate dilu- tions of the enzyme. The volume was made up to 3.0 ml. with water. Unless otherwise indicated, the reaction was initiated by the addition of enzyme. Measurements of the optical density were made, at 1 minute intervals, against a blank cell contaming all components except testosterone. The enzyme activity was measured in terms of the rate of optical density

2 4 6 8 10 12 14 16 18 -MICROGRAMS OF PROTEIN PER 3ML.

FIG. 1. Proportionality of initial reaction rate to enzyme concentration. Condi- tions described under “Spectrophotometric assay.”

change with time at 340 rng. 1 unit of enzyme activity was defined as a change of logI lo/l = 0.001 per minute in a 1.00 cm. cell at 25” f 0.5” and pH 9.0. Rates were calculated from zero order reaction curves. For density changes of 0.020 per minute or less, the rate of reaction was propor- tional to the amount of enzyme (Fig. 1).

Protein Determinations-The protein content of crude extracts was meas- ured by the turbidimetric method of Stadtman et al. (19), by means of a calibration curve made with crystalline bovine serum albumin. The light absorptions at 260 and 280 rnp were determined at each step of purifi- cation in 0.03 M phosphate buffer, pH 7.2. With progressive purification of the enzyme, the ratio 280:260 increased, and, when it exceeded 0.95, calculations of the protein concentration were made by use of the Kalckar formula (20).

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Paper Chromatography of Steroids-Ascending or descending paper chro- matography was used for identification of steroids and reaction products. Whatman No. 43 paper was impregnated with formamide by applying a mixture of equal volumes of formamide and methanol, blotting, and drying at room temperature. The moving phase consisted of a mixture of equal volumes of hexane and benzene. The positions of (Y , p-unsaturated ketonic compounds were detected by visual inspection of the fluorescence with ultraviolet light and a fluorescent screen; other steroids were detected by treating the paper with a saturated solution of antimony trichloride in chloroform (21). With this system testosterone (RF = 0.39), 4-andro- stene-3,17-dione (RR = 0.70), and dehydroepiandrosterone (RR = 0.59) could be readily separated from each other and a number of related steroids.

Results

Enzymatic Activities of Cell-Free Extracts-Extracts of testosterone- adapted cells contained a number of enzymes. By spectrophotometric methods, it was possible to demonstrate the reduction of DPN by ethanol and methanol and the presence of certain enzymes of the tricarboxylic acid cycle such as fumarase, isocitric dehydrogenase, and malic dehydro- genase. In the presence of testosterone or related hydroxysteroids, these cell-free extracts catalyzed a rapid reduction of DPN, whereas in the ab- sence of substrate there was a relatively slow endogenous reduction of the pyridine nucleotide. TPN could not act as an acceptor for the oxidation of hydroxysteroids by the enzyme.

Cell-free extracts of bacteria grown in the absence of steroids contained virtually no hydroxysteroid dehydrogenase. The adaptive enzyme was induced by the addition of testosterone, dehydroepiandrosterone, or 4- androstene-3,17-dione to the growth medium. These compounds were oxidized at the same rate by washed whole cell suspensions, independently of which steroid was used in the growth medium. Crude extracts of cells grown on each of the three steroids contained the hydroxysteroid dehy- drogenase.

Enzyme PuriJication-All operations were carried out as close to 0” as possible. The pooled extracts in 0.03 M phosphate buffer, pH 7.2, were clarified by recentrifugation in the cold for 10 minutes at 10,000 X g and then fractionated according to the following scheme: (1) Ammonium sul- fate precipitation at 33 to 50 per cent saturation. (2) The precipitate was redissolved and protamine sulfate added to remove nucleic acids. (3) The supernatant fluid from the protamine sulfate precipitation was then sub- jected to two subsequent ammonium sulfate fractionations.

In a typical preparation, 7.2 liters of filtered culture medium (0.71 mg. of dry weight per ml.) were harvested by continuous centrifugation. The

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828 P-HYDROXYSTEROID Dm~D~ommsE

cell paste was washed twice with 0.03 M phosphate buffer, pH 7.2. The total wet weight of the cells was 22 gm. 55 gm. of alumina were added, and the mixture was ground in a chilled mortar and extracted with 150 ml. of 0.03 M phosphate buffer, pH 7.2, in two portions (Fraction A, Table I). Saturated, cold ammonium sulfate (neutralized with ammonium hy- droxide) was then added slowly to 33 per cent saturation. The precipitate was removed by centrifugation and the saturation of the supernatant fluid increased to 50 per cent by the further addition of saturated ammonium sulfate solution. After standing overnight, the precipitate was centrifuged and dissolved in 50 ml. of water (Fraction B). To this mere added 10 ml. of a solution of protamine sulfate (Lilly) containing 20 mg. per ml., pH

TABLE I

Purification of Enzyme

Fraction Volume

I- ( nrl.

A. Initial extract B. 33-50yo (NH,)zSOa ppt. C. Supernatant from prota-

mine sulfate ppt.

149 50 63

+wly

8.75 7.05 1.91

?vg. units fier ml.

1304 1500 353 4670 120 1960

units units ger mg

224,000 188 235,000 663 123,500 1021

0.52 0.54 1.30

2.58 38.7 8000 120,000 3100 D. 30-40yo (NH,)zSOa ppt. 15 1.56

Enzyme assays and protein determinations were carried out as described under

Protein :oncen- tration

Total activity

Specific activit)

---, -~

“Methods and materials.” Aqueous testosterone solutions were used for Fractions A and B, but following the protamine step, the absence of DPN reduction by meth- anol permitted the more convenient addition of testosterone in methanol.

Ratio, 280:260

6.4. The copious precipitate was again permitted to accumulate and re- moved by centrifugation. To the supernatant solution (Fraction C), am- monium sulfate was added slowly to 30 per cent saturation and the precipi- tate discarded. The saturation of the supernatant fluid was then increased to 40 per cent and the precipitate collected on the centrifuge and dissolved in 15 ml. of mater (Fraction D). A third, careful ammonium sulfate pre- cipitation increased the purity of the product only slightly. Attempts to purify the enzyme further by adsorption on calcium phosphate gel and fractional elution with phosphate buffers of increasing ionic strengths pro- duced at most a a-fold increase in purity. The most purified preparations exhibited specific activities of about 4800 units per mg. of protein, repre- senting 25- to 50-fold purifications with preservation of 40 to 50 per cent of the initial enzyme activity.

Effect of pH on Reaction Equilibrium-The enzyme activity and reaction equilibrium were markedly sensitive to changes in pH, as already pointed

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out for other reactions catalyzed by DPN-requiring enzymes (15). The influence of hydrogen ion concentration on the equilibrium of the following reaction was studied.

Testosterone + DPN G 4-androstene-3,17-dione + DPNH + H+

Measurements were carried out in orthophosphate and pyrophosphate buf- fers between pH 6.1 and 9.2 at 25.5” with a large excess of enzyme in order to attain equilibrium rapidly. The spectrophotometer cuvettes contained 100 PM of buffer, 0.483 ,UM of DPN, 0.173 PM of testosterone in 0.1 ml. of CH$OH, and 0.24 mg. of enzyme in a total volume of 3.0 ml. Equilibrium constants were designated in accordance with the suggestion of Racker (15) as follows :

k = [4-androstene-3,17-dione][DPN,,a.] [testosterone][DPN,,.]

k n

= [4-androstene-3,17-dione][DPNH][H+] [testosterone][DPN]

A logarithmic plot of k against the pH was linear (Fig. 2), and calculation of lcH gave an average value of 3.60 X 1OW. Increasing the pH of the solution favored the complete oxidation of steroid as well as higher enzyme activity. For purposes of assay of DPN or testosterone, the equilibrium was practically completely in favor of the forward reaction at pH 9.0.

Enzyme Stability; Protection by DPN-Purified enzyme solutions slowly lost activity at 4”, but were completely stable for at least 6 months at - 10”. Dilute solutions of the purified enzyme (containing less than 1 mg. of protein per ml.) lost activity at 25”, and this instability was aggravated at high pH. Thus, preincubation of the diluted enzyme at 25” for 5 min- utes at pH 9.0 resulted in loss of 80 per cent of the activity. DPN ex- erted a powerful stabilizing action on the enzyme.

In Fig. 3, the residual activity of a known quantity of enzyme, following preincubation for 5 minutes at 25” and pH 7.2 and 9.0 in the absence of DPN, is compared with the activity of the same amount of enzyme when this was used to initiate the reaction in the presence of DPN. Evidently, considerable inactivation occurred during the preincubation at pH 9.0 in the absence of DPN, whereas this was negligible at pH 7.2. It is also seen that the reaction rate was much slower at pH 7.2 t,han at pH 9.0. Efficient protection required not merely trace amounts of DPN, since solutions con- taining 0.01 PM per ml. of DPN did not protect the enzyme and protection was observed only when concentrations reached 0.1 PM per ml. or higher.

Sulfhydryl reducing agents such as cysteine and glutathione did not efficiently stabilize the enzyme. Each of these compounds in final concen- trations of 0.005 M or 0.01 M did not prevent rapid enzyme inactivation at

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830 P-HYDROXYSTEROID DEHYDROGENASE

pH 9.0 and 25”, conditions under which DPN exercised a profound pro- tective effect. Bovine serum albumin, 1 to 5 mg. per ml., and Versene (ethylenediaminetetraacetic acid), 50 parts per million, provided only slight protection; i.e. 10 to 25 per cent of the efficiency of DPN.

p-Chloromercuribenzoate in a concentration of lop5 M inhibited the ac- tivity 65 per cent if the enzyme was briefly preincubated with this reagent.

2-

.2- 6.0 7.0 8.0 9.0

-PH

FIG. 2. Influence of pH on the equilibrium of conversion of testosterone to 4- androstene-3,17-dione, showing proportionality between log,0 k and pH.

If DPN was added to the enzyme prior to the preincubation with p-chloro- mercuribenzoate, the inhibitory effect was virtually abolished.

Stabilization of the enzyme could also be demonstrated by the addition of certain substrates. Preincubation at 25” and pH 9.0 in the presence or absence of testosterone or dehydroepiandrosterone resulted in the same loss of enzyme activity, whereas 17p-estradiol, which itself is a substrate, was a strong protector.

Assay of DPN or Steroids-With limiting amounts of DPN and excess of other reactants, the nucleotide could be conveniently assayed enzymati- tally, provided the pH was sufficiently alkaline so that the equilibrium point was in favor of complete reduction of the coenzyme. Small amounts of

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testosterone could be assayed under similar conditions when DPN was pres- ent in excess. Fig. 4 shows a plot of the equilibrium density of the reduced DPN against the amount of testosterone added to the reaction mixture. It can be clearly seen that there is an equimolar relation between the amount of testosterone oxidized and the amount of DPN reduced. In a 3 ml. volume 1 to 2 y of testosterone (about 0.005 PM) could be measured with

0 1 2 3 4 5 6 -MINUTES

FIG. 3. Inactivation of enzyme in the absence of DPN. The reaction cuvettes contained 100 pM of pyrophosphate buffer, pH 9.0, or orthophosphate buffer, pH 7.2, 25 y of testosterone, 0.48 MM of DPN, and 50 y of enzyme in a volume of 3.0 ml. In the two upper curves the enzyme was used to initiate the reaction, whereas in the lower curves the enzyme was preincubated in the absence of DPN for 5 minutes at 25” and the reaction initiated by adding DPN.

accuracy, and the method is subject to refinement in sensitivity with micro cells containing smaller volumes.

Identification of Product-The following incubation mixture was used for a large scale experiment: 10 ml. of aqueous solut,ion of steroid (containing 1.0 FM), 1.0 ml. of 0.5 M phosphate buffer, pH 8.4,0.5 ml. of enzyme extract, 0.5 ml. of DPN (1.4 /*M), and 3.0 ml. of HzO. The reaction was run at 25” for 20 minutes, and the mixture then extracted three times with 5 ml. of ethyl acetate. The combined extracts were dried over anhydrous so- dium sulfate, filtered, reduced to dryness in DCGCUO, dissolved in 0.5 ml. of CH30H, and 0.1 ml. was used for paper chromatography. Under these

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832 /~-HYDR~XY~TEROID DEHyrmoGmAsE

conditions, the principal oxidation product of testosterone, dehydroepi- androsterone, and 5-androstene-3@, 17fl-diol was 4-androstene-3,17-dione. Dismutations also occurred under these conditions; thus, dehydroepian- drosterone was converted not only to 4-androstene-3,17-dione but also to testosterone.

-j.o4 TESTOSTERONE

OV .OEOt

-MICROGRAMS TESTOSTERONE IN 3ML

FIG. 4. Enzymatic assay of testosterone. The experimental points represent the equilibrium optical density of DPNH plotted against the amount of testosterone added to the spectrophotometer cuvette (3 ml. volume; 1 cm. light path). The line was drawn for an equimolar relation between micromoles of testosterone and micro- moles of DPNH. Experimental conditions as described under “Spectrophotometric assay.”

Substrate SpeciJicity-The specificity of the reduction of DPN by the purified enzyme in the presence of a variety of substrates has been partly defined. All substrates were added in methanol which, in concentrations up to 10 per cent in the presence of DPN, neither inactivated the enzyme nor reduced DPN.

The following simple aliphatic and cyclic alcohols did not participate in the reaction : methanol, ethanol, propan-2-01, cyclopentanol, and cyclo- hexanol. In Table II the rates of enzymatic oxidation of 3p- and 17/3- hydroxysteroids as measured by the formation of DPNH are given relative to that of testosterone. In the case of compounds having two reactive hydroxyl groups, the over-all rate of reduction of DPN was measured.

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P. TALALAY AND M. M. DOBSON 833

Other steroids with potentially oxidizable hydroxyl groups did not react. Thus cholesterol and the adrenal cortical hormones desoxycorticosterone, cortisone, and 17-hydroxycortisone (Kendall’s Compound F) all resisted oxidation. cu-Hydroxysteroids such as epitestosterone, androsterone, and 17a-estradiol were likewise inert. The phenolic hydroxyl groups of es- trogens were not. attacked; estrone and 17cr-estradiol were resistant to oxidation, and the amount of reduced DPN at equilibrium with 17gestra- diol indicated the oxidation of only a single hydroxyl group. Both hy- droxyl groups of 5-androstene-3@, 17p-diol underwent oxidation, whereas equilibrium data indicated that only one hydroxyl group in l’icu-methyl-5- androstene-3B, 17p-diol was oxidizable.

TABLE II

Relative Rates of Enzymatic Oxidation of 3& and lY&Hydrox@eroids

Steroids Relative rates of oxidation

Testosterone............................................ 5-Androstene-3fl,17p-diol. Androstan-178~ol-3-one (dihydrotestosterone) Androstan-3p-ol-17-one (epiandrosterone). 5-Androsten-3&ol-17-one (dehydroepiandrosterone) 5.Pregnen-3P-01.20-one. 17@-Estradiol . 17ol-Methyl-5-androstene-3p,l7~-diol.

_____-

100 79.0 46.5 35.4 23.6 17.5 17.2 11.0

The rates were calculated from zero order reaction curves. The rate of formation of DPNH was measured spectrophotometrically at 340 rnp and 25” in a 3 ml. system containing 100 PLY of pyrophosphate buffer, pH 9.0, 25 y of steroid dissolved in 0.1 ml. of CHSOH, 0.48 pM of DPN, and 0.1 ml. of enzyme.

Reduction OJ Ketosteroids with DPiVH-DPNH prepared by the method of Gutcho and Stewart (22) was reoxidized in the presence of certain 17- ketosteroids and purified preparations of the dehydrogenase at pH 6.3. The reduction of the 3-keto group of testosterone occurred relatively slowly under such conditions. The 17-keto group of androsterone was readily attacked, although the 3~OH group of this compound could not be oxidized by the enzyme and DPN. Apparently a reactive group at position 3 was unnecessary for binding of the substrate to the enzyme surface, since androst,an-17-one, which lacks such a group, was reduced quite readily. Progress curves of the enzymatic oxidation of reduced DPN by various steroids are shown in Fig. 5.

inhibition of Enzyme Reaction-Preliminary investigations of the inhibi- tion of the oxidation of testosterone by structurally related compounds have been carried out. The oxidation of testosterone was powerfully and competitively inhibited by 17a-estradiol. 50 per cent inhibition of the

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834 ,B-HYDROXYSTI~R~ID DmyDRoGENAsE

reaction at pH 9.0 was obtained, with a ratio of substrate to inhibitor of 2.5 to 3.0. 17a-Estradiol also inhibited the oxidation of 17p-estradiol. Stilbene derivatives such as diethylstilbestrol or meso-hexestrol were likewise powerful inhibitors of testosterone oxidation. cu-Hydroxysteroids other than 17a-estradiol did not interfere with the oxidation of their p

1 2 3 4 5 - MINUTES

FIG. 5. Time curve for reduction of 17-ketosteroids by DPNH. The rate of oxi- dation of DPNH was measured at 25” in a 3 ml. system containing 100 PM of phos- phate buffer, pH 6.3,50 y of steroid dissolved in 0.1 ml. of CHaOH, 0.68 NM of DPNH, and 58 y of enzyme (specific activity 3100 units per mg.). Optical densities were measured at 1 minute intervals against a blank cell containing all the components except steroid. W, 4-androstene-3,17-dione; 0, androsterone; A, androstan-17- one; l , epiandrosterone.

epimers. Thus in equimolar quantities, androsterone (androstan-3ar-ol-17- one) did not inhibit the oxidation of epiandrosterone (androstan-3/?-ol-17- one), nor did epitestosterone (4-androsten-i7Lu-ol-3-one) inhibit the oxida- tion of testosterone (4-androsten-17@-ol-3-one).

DISCUSSION

Adaptive enzymes of microorganisms provide a powerful tool for the study of selected enzymatic reactions. The dehydrogenase isolated from

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P. TALALAY AND M. M. DOBSON 835

testosterone-adapted Pseudomonas cells oxidizes 3p- and 17@-hydroxyster- oids with DPN as acceptor. These reactions can be demonstrated to occur stoichiometrically, for example, according to the following equations.

(1) Testosterone + DPN * 4-androstene-3,17-dione + DPNH + H+

(2) Dehydroepiandrosterone + DPN G 4-androstene-3,17-dione + DPNH + H+

(3) 5-Androstene-38,17&diol+ PDPN e 4-androstene-3,17-dione + BDPNH + 2H+

cr-Hydroxysteroids do not participate in the reaction. All attempts to dissociate the 3- and 17-OH oxidizing activities have been unsuccessful. The relative rates of oxidation of testosterone and dehydroepiandrosterone are constant, independently of whether the enzyme is induced by growing the cells in the presence of testosterone, dehydroepiandrosterone, or 4- androstene-3,17-dione. The possibility exists that in mammalian tissues the 3- and 17-OH oxidizing activities, which have been considered enzymat- ically distinct (6, S), are also associated with a single enzyme. The 25- to 50- fold purified enzyme does not oxidize simple aliphatic or cyclic secondary alcohols and appears to be steroid-specific. The oxidation proceeds more slowly at position 3 than at position 17, and with increasing side chain length at position 17 the rate of oxidation of 3p-hydroxyl groups decreases. Although ac-hydroxyl groups are not oxidized, and do not appear to antag- onize the oxidation of P epimers (with the exception of 17a-estradiol), the CC configuration does not prevent binding of the molecule to the enzyme surface, since the 17 keto group of androsterone is readily reduced by DPNH. In fact, no reactive site at position 3 appears necessary, since androstan-17-one participates in this reaction.

DPN is a powerful stabilizer of the enzyme under conditions producing inactivation, such as elevated temperature, alkaline pH, or sulfhydryl bind- ing reagents. The protective effect of DPN on sulfhydryl enzymes has been known since the work of Rapkine (23), and these observations suggest that free sulfhydryl groups are necessary for activity.

Significant protection of the enzyme is produced by certain steroid sub- strates such as 17p-estradiol, whereas testosterone and dehydroepiandros- terone are relatively ineffectual. 17/3-Estradiol has a much greater affinity for the enzyme than has testosterone, as may be inferred from the reaction rates of mixtures of these steroids, although Michaelis constants are not available, since they are extremely low and their measurement presents certain technical problems. This may account for the protective effect of 17Sestradiol for the enzyme, and the tight binding of phenolic groups of the estrogens may likewise be responsible for the competitive inhibition of testosterone oxidation by 17a-estradiol, whereas other cr-hydroxysteroids do not inhibit oxidation of @-hydroxysteroids.

The enzyme /3-hydroxysteroid dehydrogenase produces interconversions

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836 P-HYDROXYSTEROID DEHYDROGENASE

between such steroids, as is illustrated in Fig. 6. The major pathway of the oxidation of 5-androstene-SP , 17/?-diol to 4-androstene-3,17-dione re- mains to be determined. Similar types of reactions probably occur in analogous compounds of the androstane series.

The possibility of using this enzymatic reaction for the assay of selected groups of steroids is being studied. The assay of 3@- and 17P-hydroxy- steroids, as exemplified by measurement of testosterone in amounts of 1 to 2 y per 3 ml. system and 1 cm. light path, appears quite feasible. The sensitivity could be increased IO-fold by using micro cells. Since the reduc-

OH

m FIG. 6. Interconversions of steroids catalyzed by fl-hydroxysteroid dehydrogen-

ase. I, testosterone; II, 4-androstene-3,17-dione; III, dehydroepiandrosterone; IV, 5-androstene-3fi, 17fi-diol.

tion of the 3-keto group at pH 6.3 proceeds so very much more slowly than that of the 17-keto group, the enzyme could probably be used for the assay of 17-ketosteroids. Preliminary experiments with biological mate- rials are being conducted.

SUMMARY

A DPN-linked dehydrogenase capable of reversibly interconverting cer- tain 3& and 17p-hydroxysteroids and the respective ketosteroids has been extracted from Pseudomonas cells grown on testosterone. This enzyme has been purified twenty-five to 50 times and conditions for the spectrophoto- metric assay of activity have been described. It appears to require free sulfhydryl groups for activity and is rapidly inactivated at high pH. DPN

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I’. TALALAY AND M. M. DOBSON 837

exerts a strong protective effect, as do certain substrates such as 17@- estradiol, but not testosterone or dehydroepiandrosterone. Under suitable conditions, the enzyme performs stoichiometric,conversion of steroids, such as the oxidation of testosterone to 4-androstene-3,17-dione. At alkaline pH the equilibrium is greatly in favor of such oxidation, whereas the reduc- tion of ketosteroids by reduced DPN readily occurs at lower pH. 3~ and 17a-hydroxysteroids do not participate in the reaction and cannot inhibit the oxidation of their /3 epimers, with the exception of 17a-estradiol which powerfully and competitively inhibits the oxidation of testosterone. The enzyme may be employed for the sensitive spectrophotometric assay of DPN, 3/?- and 17P-hydroxysteroids, or ketosteroids in microgram quanti- ties.

The authors are indebted to the Chemical Specialties Company, New York, for generous gifts of testosterone and other steroids. Samples of steroids were also kindly provided by the Schering Corporation, Bloomfield, New Jersey, and Charles E. Frosst and Company, Montreal. Thanks are due to Mr. Richard Leek for technical assistance.

BIBLIOGRAPHY

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Paul Talalay and Marie Mollomo DobsonDEHYDROGENASE

-HYDROXYSTEROIDβA PURIFICATION AND PROPERTIES OF

1953, 205:823-837.J. Biol. Chem. 

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