ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 101, 278-285 (1963)

Purification of 3o~-Hydroxysteroid Dehydrogenase Obtained from the Soluble Fraction of Rat Liver ~

SAMUEL S. K O I D E

From the Division of Clinical Investigation, Sloan-Kettering Institute for Cancer Research, New York, New York

Received December 5, 1962

The 3~-hydroxysteroid dehydrogenase obtained from tim soluble fraction of rat liver was partially purified by centrifugation, ammonium sulfate precipitation, C-/-aluniina adsorption, and hydroxylapatite-DEAE-cellulose column chromatog- raphy. The purified enzyme preparation was subjected to repeat hydroxylapatite- DEAE-cellulose column chromatography and starch-block electrophoresis in order to separate the transhydrogenase from the dehydrogenase activities. This separation was not attained during any of these steps of purification. Attempts at further puri- fication were not successful due to the instability of the purified enzyme preparation.

INTRODUCTION

Talalay and co-workers (1) have shown tha t 17B-estradiol acts as a coenzyme by undergoing an alternating oxidation-reduc- tion in the mediation of hydrogen transfer between the pyridine nucleotides. Thus the 17fl-hydroxysteroid dehydrogenase obtained f rom the soluble fraction of human placenta was responsible for the transhydrogenation. Hagerman and Villee (2), however, re- ported tha t the pyridine nucleotide trans- hydrogenase sensitive to 17~-estradiol was distinct and separate from the D P N 2 and TPN-l inked 17fl-hydroxysteroid dehydro- genases. The two proposed mechanisms apply equally to the hydrogen transfer s t imulated by 3a-hydroxysteriods. Hurlock and Talalay

I This work was supported by Grant C-3809 fronl the National Cancer Institute, U. S. Public Health Service, and by a contract from the U. S. Atomic Energy Commission, AT (30-1)-910.

~Abbreviations used: oxidized and reduced di-and triphosphopyridine nucleotides, DPN, DPNH, TPN, TPNtt; ethylenediamine tetra- acetate, EDTA; tris(hydroxymethyl)aminometh- ane, Tris; diethylaminoethylcellulose, DEAE cel- lulose; adenosine mono-, di-, and triphosphates, AMP, ADP, ATP; flavine adenine mono- and di- nueleotides, FMN and FAD.

(3) concluded tha t the t ranshydrogenat ion st imulated by 3a-hydroxysteroids was medi- ated by the a-hydroxysteroid dehydrogen- ase obtained from rat liver. Baron, Gore, and Williams (4), however, reported tha t an androgen-activated t ranshydrogenase de- void of the dehydrogenase act ivi ty was present in ra t and human prostate glands.

We have a t t empted to resolve this prob- lem by further purification of the 3~-hy- droxysteroid dehydrogenase from the soluble fraction of rat liver. No separation of the dehydrogenase and transhydrogenase activi- ties of the enzyme could be achieved at the present stage of purification. For identifica- tion in this paper the enzyme activities were considered as D P N - and TPN-dehydro- genase and transhydrogenase.

METHOD

MATERIALS

Glass-distilled deionized water was used to pre- pare all solutions. Ammonium sulfate, analytical grade, was recrystallized once from 0.001 M EDTA solution and twice from glass distilled water. Tris, EDTA, monobasic and dibasic sodium phosphates, cysteine, nicotinamide, and sodium bicarbonate were commercially available analytical grades. Androsterone (m.p. 185 ~ and testosterone (m.p.

278

P U R I F I C A T I O N OF 3a -HYDROXYSTEROID D E H Y D R O G E N A S E 279

150-151 ~ were recrystallized from appropriate solvents. Glucose 6-phosphate, DPN, TPN, DPNH, TPNH, glucose-6-phosphate dehydrogen- ase, type V, and C~/-alumina were obtained from Sigma Chemical Company. Cellulose dialysis tub- ing was obtained from the Visking Company, Chicago, Ill. The tubing was soaked in several changes of a solution containing 0.001 M EDTA, 0.001 M cysteine, and 0.01 M phosphate buffer, pH 7.6. Sodium dithionite was purchased from Mathe- son Coleman and Bell Company. Tri ton X-100, Lot 3798, was a gift from RoAm & Haas Co. Phosphate buffers and Tris buffers were prepared according to Gomori (5). The protein content was measured by the biuret method (6) with crystalline bovine serum albumin as standard. Approximate protein content of the fractions collected from chroma- tography was est imated by measurements of ab- sorbancy at 278 mm

CHROMATOGRAPHY

DEAE-cellulose (medium mesh, 0.9 meq./g. capacity) was purchased from Sigma Chemical Company and treated with 1 N NaOH, 1 N HC1, and 1 N NaOH as described by Keller and Block (7). The cellulose was washed ten times with glass- distilled water and three times with 0.01 M phos- phate buffer, pH 7.6, before being used. Calcium phosphate gel (hydroxylapatite) was prepared ac- cording to Tiselius, Hjert6n, and Levin (8). A mix- ture of 20 g. DEAE-cellulose (dry wt.) and 350 ml. hydroxylapati te solution (89 mg./ml.) was pre- pared and washed three times with 0.01 M phos- phate buffer, pH 7.6. The mixture was poured into a 3 X 50 cm. column and allowed to settle by gravi ty to give a column length of 25 cm. I t was washed with 1 1. of 0.01 M phosphate buffer, pH 7.6. The top of the column was overlaid with filter paper and glass wool to avoid disturbance of the surface. A second column measuring 2.2 X 40 cm. was prepared in a similar manner.

ASSAY FOR DEHYDROGENASE AND TRANSHYDROGENASE ACTIVITIES

TPN and DPN dehydrogenase activities were assayed in a total volume of 3 ml. containing 200 t~moles Tris buffer, pH 7.6, 1 t~mole TPN or DPN, 200 t~moles (NH'I4)2SO4, and appropriate amounts of enzyme. (Pietruszko, Gore, and Baron (10) re- ported that various inorganic sulfates, including (NH,)2SO4, st imulated the act ivi ty of 3a-hydroxy- steroid-dependent transhydrogenating system of rat liver.) The reaction was initiated by the ad- dition of 35 ~g. androsterone dissolved in 10 ~1. dioxane to the reaction cuvette. The control cuvette contained all ingredients except the steroid. The transhydrogenase system contained

0.6 Kornberg unit of glucose 6-phosphate dehy- drogenase, 10 ~moles glucose 6-phosphate, 5 ~moles MgC12, 1 ~mole DPN, 0.02 #mole TPN and ap- propriate amounts of enzyme, 200 ~moles (NH4)~- SO4, 200 ~moles Tris buffer, pH 7.6 in a total vol- ume of 3 ml. The reaction was initiated by the addition of 10 ~g. androsterone dissolved in 10 ~l. dioxane to the reaction cuvette. The control cuvet te contained all ingredients except the steroid. The changes in absorbancy were measured with a Cary recording spectrophotometer employ- ing a 1-cm. light path at 340 mu. One unit of all three enzyme activities represents a change in ab- sorbancy of 0.001/mAn.

STARcH-BLocK ELECTROPHORESIS

Potato starch was obtained from Fisher Scien- tific Company and washed ten times by decanta- tion with distilled water and three times with 0.01 M Tris, pH 8.6. The procedure was carried out as described by Kunkel (11) in an electrophoresis unit manufactured by the Laboratory Glass and Instru- ments Corporation, New York.

ISOLATION P R O C E D U R E

STEP 1. PREPARATION OF HOMOGENATE

Adult male rats were killed with a blow on the head; the livers were excised immediately and placed in an ice-cooled container. (Livers obtained from rats anesthetized with sodium pentobarbital have since been found to give satisfactory enzyme preparations.) Subsequent steps were carried out in a cold room at 2-4~ Liver tissues (50 g.) were homogenized in a Waring blendor at 60 v. for 1 mAn. with 150 ml. of 0.25 M sucrose, 0.001 M EDTA, 0.001 M cysteine, 0.01 M nicotinamide, and 0.03 M NaHCO3. The homogenate was centrifuged at 13,000 X g for 15 mAn. in the Servall Superspeed centrifuge, model RC-2. The supernatant solution was centrifuged at 59,000 X g for 1 hr. in a Spinco preparative ultracentrifuge, model L. The above two eentrifugation steps were later modified by centrifuging once at 13,000 X g for 1 hr. in the Servall Superspeed centrifuge.

STEP 2. AMMONIUM SULFATE FRACTIONATION

The volume of the supernatant solution from Step 1 was measured. Ammonium sulfate was added to the solution in small increments to give 0-0.3, 0.3-0.5, and 0.5-0.7 saturation. The mixture was stirred constantly with a magnetic stirrer. The pH was maintained at 7.6 with the addition of dilute ammonium hydroxide solution. After the addition of ammonium sulfate to the appropriate saturation point, the mixture was stirred for 1 hr. and centrifuged at 13,000 X g for 30 man. in the

280 KOIDE

Servall Superspeed centrifuge. The precipitates obtained at 0.3 and 0.5 ammonium sulfate satura- tion were discarded. The precipitate obtained from the 0.5-0.7 ammonium sulfate saturation fraction was dissolved in 0.01 M phosphate buffer, pH 7.6 and dialyzed against a continuous flow of 10 1. of 0.001 M EDTA adjusted to pH 7.6 with 0.2 M Tris for 2 hr. The solution was centrifuged at 27,000 X g for 15 rain. (SS-34 rotor, Servall Superspeed centri- fuge). This supernatant preparation can be stored frozen and retains its activity for at least 6 months.

STEP 3. CT-ALuMINA EXTRACTION

The enzyme preparation from Step 2 was diluted with 0.01 M phosphate buffer pH 7.6 to give a protein concentration of 20 mg./ml. C~-Almnina solution (10 mg./ml.) was added to the enzyme preparation to give a ratio of a milligram of C'~- alumina to a milligram of protein. The lnixture was stirred for 15 rain. and centrifuged at 27,000 X g for 10 rain. The protein adsorbed by the C-y- alumina was recovered by resuspending the gel in the original volume of 0.1 M phosphate buffer, pH 7.6 and stirring for 15 min. The mixture was centrifuged at 27,000 X g for 10 min. The extraction procedure was repeated with 0.1 M phosphate buffer, 1 M (NtI4)2SO4, pit 7.6. A small amount of enzyme can be recovered by a third extraction with 0.1 M phosphate buffer, pH 7.6, 1 M (NH4)2SO4. The major portion of enzyme activity was re- covered in the initial two extractions. The two fractions were pooled and dialyzed overnight against two changes of saturated ammonium sulfate solution, pH 7.6. The mixture was centri- fuged at 27,000 X g for 10 min. The precipitate was dissolved in 0.01 M phosphate buffer, pH 7.6.

STEP 4. HYDROXYLAPATITE-DEAE-CELLU- LOSE COLUMN CHROMATOGRAPHY

The enzyme preparation from Step 3 was dialyzed for 3 hr. against three changes of 0.01 M phosphate buffer, pH 7.6. The preparation was centrifuged at 27,000 X g for 10 rain. The small amount of precipitate which resulted was dis- carded. The supernatant solution was added to a hydroxylapatite-DEAE-cellulose column. The total protein content of supernatant solution ranged from 1.0 to 1A g. Elution was accomplished by first placing 0.01 M phosphate buffer above the column. The successive elating solutions were dripped in at the same rate as the formation of the eluate so that at all times there was 100 ml. solu- tion above the column. The column was elated successively with 0.01 M phosphate buffer, pH 7.6; 0.1 M phosphate buffer, pH 7.6; 0.1 M phosphate buffer, 0.2 M (NH4):SO4, pH 7.6; and 0.1 M phos- phate buffer, 0.5 M (NH4):SO4, pH 7.6. One experi-

meet is illustrated in Fig. 1. Fractions of 20 ml. were collected at a rate of 60 ml./hr, and tested for DPN-dehydrogenase activity. The tubes contain- ing the enzyme activity were pooled and dialyzed overnight against two changes of saturated am- monium sulfate solution. The mixture was centri- fuged at 27,000 X g for 10 rain. The precipitate was dissolved in 0.01 M phosphate buffer, pH 7.6 and tested for DPN- and ~lPN-dehydrogenase and transhydrogenase activities (Table I).

STEP 5. SECOND HYDROXYLAPATITE-DEAE- CELLULOSE COLUMN CHROMATOGRAPHY

The enzyme preparation from Step 4 was di- alyzed for 4 hr. against three changes of 0.1 M phosphate buffer, pH 7.6 and centrifuged at 27,000 X g for 10 rain. The supernatant was added to the hydroxylapatite-DEAE-cellulose colmnn measuring 2.2 X 40 cm. Elution was initiated by placing 0.1 M phosphate buffer, pH 7.6 above the column. The successive eluting solutions were dripped in at the same rate as the formation of the eluate so that at all times there was 100 ml. solu- tion above the column. The column was eluted successively with 0.1 M phosphate buffer, pH 7.6; 0.1 M phosphate, 0.05 M (NH4)2SO~, pH 7.6; 0.1 M phosphate buffer, 0.1 M (NH4)~SO4, pH 7.6; and 0.i M phosphate buffer, 0.2 M (NH4)2SO4. One of the experiments is presented in Fig. 2. Fractions of 6.8 ml. were collected at a rate of 40 ml./hr, and tested for DPN- and TPN-dehydrogenase and transhydrogenase activities.

RESULTS

The enzyme preparation obtained from ammonium sulfate precipitation at 0.5-0.7 saturation (Step 2) when heated at 57~ for I0 rain. lost all its dehydrogenase and transhydrogenase activities. Preliminary studies were carr ied ou t to stabil ize this enzyme f rac t ion to hea t t r ea tmen t . T h e following compounds were tes ted: D P N , D P N H , T P N , T P N H , A M P , A D P , A T P , F A D , F M N , nicot inamide, E D T A , andros- terone, e t iocholanolone, tes tos terone, cys- teine, sodium di thioni te , ascorbic acid, 50 % glycerol solution, and 1% Tr i t on X-100 solution. None of these compounds were effect ive in p reven t ing loss of enzyme ac- t i v i t y of the crude enzyme p repa ra t ion by hea t ing a t 57~ for 10 min. Moreover , all th ree ac t iv i t ies were found to be labile to ace tone and e thanol p rec ip i ta t ion and to lyophi l iza t ion bu t f a M y stable to fu r the r a m m o n i u m sulfate precipi ta t ion. B e y o n d the first h y d r o x y l a p a t i t e - D E A E - c e l l u l o s e

PURIFICATION OF 3a-HYDROXYSTEROID DEHYDROGENASE 281

A t

]20 I---

I

._o =

-~ 8o

2 6 0

.g

, 20

O : - - - - ' : : 0 i 0 I0 40 90

- Fraction

- - !.~f

c '1 D

i I t I l I 20 30 50 60 70 80

1.2 1.0

0.8 --! 0.6 0.4 0.2

FIG. 1. Hydroxylapatite-DEAE-cellulose column chromatography of a 3a-hydroxy- steroid dehydrogenase preparation obtained from C~,-alumina adsorption (Step 3). In this experiment 27 ml. of enzyme preparation containing 1.4 g. protein with a total DPN- linked dehydrogenase activity of 34,100 units was placed on the cohmm after dialysis against 0.01 M phosphate buffer, pH 7.5 for 4 hr.

Elution of the protein was carried out as described in the text under Step 4. The column was eluted successively with 300 ml. of 0.1 M phosphate buffer, pH 7.6; 800 ml. of 0.1 M phosphate buffer, pH 7.6; 500 ml. of 0.1 M phosphate buffer with 0.2 M (NH~)2SO~, pH 7.6; and 300 ml. of 0.1 M phosphate buffer with 0.5 M (NH4)~SO4, pH 7.6. The solutions were added at fractions 1, 11, 51, and 70 as indicated by arrows A, B, C, and D, respectively.

The graph illustrates the distribution of the DPN-linked dehydrogenase activity in 3-ml. aliquots ( - -0 - - ) . The assay system contained 1 t, mole of DPN, 3 ml. fraction, and 35 ~g. androsterone. Every other fraction was assayed. An approximate measure of protein is given by the absorbancy measurements at 278 m~ ( - - 0 - - ) which were carried out in cuvettes of 1-cm. light path, against a blank of eluting medium. Fractions 9-11 and 26-31 had absorb- ancy greater than 1.5.

TABLE I

SUMMA/~Y OF FRACTIONATION OF 3o-~-~-~u DEHYDROGENASE AND TRANSHYDROGENASE

FROM RAT LIVER

Total Activity, units

S tep Fractions Volume Dehydrogenase Transhydro- genase

DPN TPN

ml.

1 Ultracentrifuged homogcnate 690 51,700 44,800 16,550 2 (NI-L)2SO4 precipitation 0.5-0.7 saturation 84 45,400 38,200 13,100 3 C7-Alumina adsorption 19 37,200 30,100 9,880 4 Hydroxylapatite-DEAE-cellulose chromatography 9 8,600 4,280 1,250

282 KOIDE

~40

=

:~ 20

5 l0 *

- Fraction

FIG. 2. Second hydroxylapatite-DEAE-cellulose eolumn chromatography of 3a-hy- droxysteloid dehydrogenase obtained from Step 4 (See text and Fig. 1). In this experiment 8 ml. of enzyme preparation containing 215 mg. protein with a total DPN- and TPN-linked dehydrogenase and transhydrogenase activities of 3680, 2560 and 800 units, respectively, were utilized.

Elution of the protein was carried out as described in the text under Step 5. The column was eluted with 300ml. of 0.1 M phosphate buffer, pH 7.6; 200 ml. of 0.1 M phosphate buffer with 0.05 M (NH4)~SO4, pH 7.6; 200 ml. of 0.1 M phosphate buffer with 0.1 M (NH4)2SO4, pH 7.6; and 500 ml. of 0.1 M phosphate buffer with 0.2 M (NH4)2SO4. The solutions were added at fractions 1, 35, 58, and 85 as indicated by arrows A, B, C, and D, respectively.

The graph shows the distribution of the DPN-linked ( - - 0 - - ) and TPN-linked ( 0 - - 0 ) dehydrogenase and transhydrogenase ( - - 0 - - ) activities. The assay system was carried out as described in the text except that 3 ml. of the fraction was used with the omission of Tris buffer and (NH4)2SO4. Every third fraction was assayed for the respective activities. An approximate measure of protein is given by the absorbancy measurements at 278 m~ ( ~ O - - ) which were carried out in cuvettes of 1-cm. light path, against a blank of eluting medium.

TABLE II SPECIFIC ACTIVITIES OF 3(~-HYDROXYSTEROID ~)EHYDROGENASE AND TRANSHYDROGENASE OF RAT LIVER

Units/mg. Protein

Steps Fractions Dehydrogenase Transhydro- genase

DPN TPN

Ultracentrifuged homogenate 1.2 0.7 0.2 (NH4)2SO4 precipitation 0.5-0.7 saturation 13.8 11.7 3.7 CT-Alumina adsorption 30.6 21.1 5.2 Hydroxylapatite-DEAE-cellulose column chromatography 98 53.2 14.9

co lumn c h r o m a t o g r a p h y (Step 4), however , the enzyme p r e p a r a t i o n was found to be exceedingly uns tab le .

A s u m m a r y of a r ep resen ta t ive experi- ment , showing the r ecovery of enzyme ac- t iv i t i es a t va r ious s teps of pur i f ica t ion, is p resen ted in Tab le I. The final percen tages of the act iv i t ies , recovered a f te r hyd roxy l - a p a t i t e - D E A E - c e l l u l o s e co lumn chroma-

t o g r a p h y (Step 4), were 17, 10, and 8 % for D P N - and T P N - d e h y d r o g e n a s e and t r ans - hydrogenase , respec t ive ly . Specific ac t iv i t i es ob t a ined f rom a r ep re sen t a t i ve p r e p a r a t i o n are p resen ted in Tab le I I . The specific ac- t iv i t i e s of D P N - ~nd T P N - d e h y d r o g e n u s e and t r a n s h y d r o g e n a s e fol lowing hyd roxy l - a p a t i t e - D E A E - c e l l u l o s e co lumn ch romatog- r a p h y ranged f rom 64 to 189, 29 to 117,

PURIFICATION OF 3a-HYDROXYSTEI~OII) DEHYDROGENASE 283

and 7 to 33 units/rag, protein, respectively, in six separate experiments. The average specific activities obtained from the six experiments were 99, 56, and 16 units/rag. protein, respectively.

One of the experiments on the fractiona- tion of the enzyme preparat ion by hydroxyl- apat i te-DEAE-cel lulose column chromatog- raphy (Step 4) is illustrated in Fig. 1. A broad zone with two peaks of dehydrogcnase act ivi ty was consistently observed. Further- more, the fraction with the max imum ac- t ivi ty usually did not correspond to the greatest absorbancy at 278 rag. The fractions between 40 and 75 were pooled and precipi- ta ted with ammonium sulfate solution (Step 4). This enzyme preparat ion was re- chlvmatographed (Step 5). One of the experiments of the second hydroxylapat i te - DEAE-cellulose column chromatography is illustrated in Fig. 2. Two peaks of act ivi ty were observed. Dehydrogenase and trans- hydrogenase activities were present in both peaks. Fraction 58 had the greatest act ivi ty; whereas, fractions 48-53 contained the maximum amounts of protein. The recovery of the enzyme by ammonium sulfate precipi- tat ion was less than 20 % for this step.

Enzyme preparations obtained from am- monimn sulfate precipitation (Step 2) were subjected to starch-block electrophoresis. The active fraction was found to migrate toward the cathode a t p H 8.6. For this reason the samples were subsequently placed 5 cm. from the anode. Figure 3 illustrates an experiment on the distribution of an enzyme preparat ion obtained from C~- alumina adsorption (Step 3). The dehydro- genase and transhydrogenase activities were present in the same segment. Starch-block electrophoresis was found to be less feasible for the separation of protein impurities than hydroiy lapa t i te~ DEAE-cellulose column chromatography (Step 4). The enzyme preparat ion obtained from Step 4, however, was subjected to starch-block electrophoresis as described in Fig. 3. Although the enzyme protein distribution as measured by ab- sorbaney at 278 m~ was identical with tha t described in Fig. 3, no dehydrogenase or transhydrogenase activities were detected in any of the segments.

ee~

u ~

!

1 0 - -

8 - -

6 - -

]f ,l

i l . l +5 0 -5 -10 -15

Position in crn

1.6

1.4

1.2

t 3.6

0.4

0.2

FIG. 3. Starch-block electrophoresis of a rat liver 3a-hydroxysteroid dehydrogenasc prepara- tion obtained from CT-alumina adsorption (Step 3). The starch block measured 15 X 60 X 200 ram. It was equilibrated with 0.01 M Tris buffer, pH 8.6 at 2-5~ for 1 hr. One milliliter of the enzyme preparation (40 rag. protein) was placed in a groove 5 cm. from the anode (position 0). The enzyme contained a total of 1220, 940, and 260 units of DPN- and TPN-dehydrogenase and transhydro- genase activities, respectively.

Potential of 150 v. was applied for 11 hr. with a current variation of 12-14 ma. The block was sectioned into 1-cm. segments and eluted twice with 10 ml. of 0.1 M Tris buffer with 1 M (NH4)~SO~, pH 8.2. The supernatant fractions were collected by centrifugation at 25,000 X g for 15 rain. The assay was carried out on 3-ml. aliquots of the centrifuged supernatant solution.

The graph illustrates the distribution of the DPN- and TPN-dehydrogenase ( - -0-- ) and trans- hydrogenase (- - 0 - -) activities. All the activities were found in segment - 9 cm. Approximate pro- tein measurement was made at 278 mt~ in cuvettes of 1-cm. light path against a blank of eluting medium ( - - 0 - - ) .

284 KOIDE

DISCUSSION

The 3a-hydroxysteroid dehydrogenase from the soluble fraction of rat liver was partially purified by Tomkins (9) and by Hurlock and Talalay (3). They purified the enzyme by centrifugation, ammonium sul- fate precipitation, calcium phosphate gel adsorption, and alcohol or acetone precipita- tion. In our hands, the activity of the enzyme preparation obtained from the last two steps of purification involving calcium phosphate gel adsorption and alcohol or acetone precipitation was erratic and the recovery was poor. The present outlined pro- cedure of C~,-alumina gel adsorption and hydroxylapatite-DEAE-cellulose column chromatography gave superior yield and reproducibility (Steps 3 and 4). However, we were unable to purify further the enzyme preparation obtained from hydroxylapatite- DEAE-cellulose column chromatography (Step 4) by starch-block electrophoresis or by repeat chromatography due to its lability.

Two peaks of dehydrogenase activity were observed during hydroxylapatite-DEAE- cellulose column chromatography (Figs. 1 and 2). The second peak is probably caused by the change in concentrations of the eluting buffers and is not evidence of multi- plicity of enzymes. The TPN-dehydrogenase activity is usually 0.60.8 of the DPN- dehydrogenase activity. We have noted, however, that in approximately one out of five preparations, the TPN-dehydrogenase activity was equal to or exceeded the DPN- dehydrogenase activity. We have not pursued this finding and are unable to explain it at this time.

The activity of the enzyme preparations reported by Tomkins (9) and that reported in this study are not comparable since they were assayed under different conditions. Tomkins (9) utilized DPNH and dihydro- cortisone; whereas DPN or TPN and androsterone were used in this study. He defined a unit of activity as a decrease in optical density of 0.01/min. at 340 m~ measured during the interval of 15-30 see. The relative rate of oxidation of DPNH with androstane-3,17-dione was reported as 20 % of dihydroeortisone (9). The enzyme

preparation obtained by Tomkins (9) had a specific activity of 375 units/mg, protein. This was approximately a 75-fold purifica- tion from the initial extract. Hurlock and Talalay (3) reported a 20-fold purification of their preparation from the initial extract, which was obtained by a similar procedure.

In this study, we were primarily interested in the dehydrogenation and transhydrogena- tion mediated by androsterone. The activi- ties of the enzyme preparation were deter- mined by measuring the rate of reduction of the pyridine nucleotides with androsterone. A unit of activity was defined as an increase in absorbancy of 0.001/rain. at 340 m~ measured during the initial minute. The enzyme preparation obtained in this study possessed a DPN-dehydrogenase activity of about 99 units/mg, protein. This was approximately a 90-fold purification from the initial extract.

By the method outlined, the 3a-hydroxy- steroid dehydrogenase and transhydrogenase activities were not separated either by hydroxylapatite DEAE-cellulose column chromatography or by starch-block elec- trophoresis. These findings support the proposition of Hurlock and Talalay (3) that the hydrogen transfer stimulated by 3a- hydroxysteroids is probably mediated by a dual pyridine nucleotide-linked dehydro- genase. They further postulated that the 3a-hydroxysteroid acts as a coenzyme in the hydrogen transfer by undergoing an alternating oxidation-reduction at position 3. To resolve definitely the question of whether the hydrogen transfer is mediated by the 3a-hydroxysteroid dehydrogenase or a distinct and separate transhydrogenase, a homogeneous protein must be obtained.

ACKNOWLEDGMENTS

The author is grateful to Dr. Rulon W. Rawson and Dr. Martin Sonenberg for their interest and encouragement of this project, and also to Mrs. Margaret Abad for technical assistance.

R E F E R E N C E S

1. JARABAK, J. , ADAMS, J. A., WILLIAMS-ASH- MAN, H. G., AND TALALAY, P., J . Biol. Chem. 237,345 (1962).

PURIFICATION OF 3a-HYDROXYSTEROID DEHYDROGENASE 285

2. HAGERMAN, D. D., AND VILLEE, C. A., J . Biot_ Chem. 234, 2031 (1959).

3. I{URLOCK, B., AND TAI,ALAY, P., J. Biol. Chm.e 288, 886 (1958).

4. BARON, D. N., GORE, M. B. R., AND WILLIAMS, D. C., Biochem. J. 74, 20P (1960).

5. GoMoxI, G., in "Methods in Enzymology" (S. P. Co]owick and N. O. Kaplan, eds.), Vol. 1, p. 144. Academic Press, New York, 1955.

6. VON HIPPEL, P. H., ANn WAUGH, I). F., J. Am. Chem. Soc. 77, 4311 (1955).

7. KELLER, S., AND BLOCK, ~[:~. J., in "Laboratory Manu~l of AnalytieaI Methods of Protein Chemistry" (P. Alexander and R. J. Block, eds.), Vol. 1, p. 76. Pergamon Press, New York, 1960.

8. TISELIUS, A., HJERTI~N, S., AND LEVIN, (~., Arch. Biochem. Biophys. 65, 132 (1956).

9. TOMKINS, G. M., J. Biol. Chem. 218, 437 (1956).

10. PIETRUSZKO, R., GORE, M. B. R., AND BARON, D. N., Biochem. J. 84, 77P (1962).

l l . KUNKEL, H. G., Methods Biochem. Anal. 1, 14l (1954).