purification of a 17@-hydroxysteroid dehydrogenase of ... · similar to that described by kunkel...

THE JOURNAL OF BIO~~OCYICAL CHEMISTRY Vol. 237, No. 2, February 1962

Printed in U.S.A.

Purification of a 17@-Hydroxysteroid Dehydrogenase

of Human Placenta and Studies on Its Transhydrogenase Function*

JOSEPH JAnAnAK,t JULIA A. ADAMS, H. G. WILLIAMS-ASHMAN, AND PAUL TALALAY:

From The Ben May Laboratory for Cancer Research and the Department of Biochemistry, University of Chicago, Chicago 37, Illinois

(Received for publication, July 24, 1961)

This paper describes methods for the extensive purification of a soluble 17@-hydroxysteroid dehydrogenasel of human placenta. This single enzyme appears to be responsible for the entire 17/3- estradiol-mediated transfer of hydrogen between pyridine nucleotides in soluble extracts of this tissue.

Experiments which were reported in 1958 (2, 3) showed that soluble enzyme preparations derived from human placenta pro- moted a reversible transhydrogenation between pyridine nucleotides in the presence of low concentrations of 17&hydroxy- st,eroids or 17-ketosteroids, notably 17/I-estradiol or estrone. These enzyme preparations also contained an active 17/3-hydroxysteroid dehydrogenase which interconverts 17@estradiol and estrone, and reacts with diphosphopyridine nucleotide (DPN) or triphosphopyridine nucleotide (TPN) (8, 9, 11). We suggested (2, 3) that the 17/3-estradiol-mediated transfer of hydro-

* This investigation was supported by grants from the American Cancer Society and the United States Public Health Service.

t Postdoctoral Fellow of the United States Public Health Serv- ice.

$ American Cancer Society Research Professor. 1 The interconversion of steroid hydroxyl and carbonyl func-

tions is catalyzed by a group of pyridine nucleotide-linked enzymes for which the name hydroxysteroid dehydrogenases has been widely adopted (1). It was subsequently recognized (24) that the same enzymes may catalyze the transfer of hydrogen between pyridine nucleotides or their analogues in the presence of catalytic levels of steroid substrates (transhydrogenase function) as well as stoichiometric reactions between steroids and pyridine nucleotides (dehydrogenase function). Evidence obtained from this and from previous studies (3,4) leaves no doubt that both functions are catalyzed by the same enzyme. It is therefore suggested that the enzyme under discussion be designated as a 17&hydroxysteroid dehydrogenase, and that reference be made to its dehydrogenase and transhydrogenase functions. It seems undesirable to use the term transhydrogenase alone inas- much as it is descriptive of only one aspect of the catalytic activity, and its function in this capacity under physiological conditions remains equivocal. In this way, possible confusion is avoided with unrelated transhydrogenases of animal tissue mito- chondria (5), of Pseudomonas jEuore.scens (6), and of vegetable sources (7).

The term i7&hydroxysteroid dehydrogenase is also preferable to estradiol-17,!%dehydrogenase (8, 9), since the enzyme reacts with other 17&hydroxysteroids, phenolic and nonphenolic (9). Since other enzymes are known to dehydrogenate steroids, e.g. by intro- ducing double bonds (lo), the term hydrozysteroid dehydrogensse specifies implicitly that the enzyme is an alcohol dehydrogenase.

gen between pyridine nucleotides is catalyzed by the 17/3-hydroxysteroid dehydrogenase in the following manner.

Estrone + TPNH + H+ e 17@-estradiol + TPNf

17@-Estradiol + DPN+ g estrone + DPNH + II+

TPNH + DPN+ = TPNf + DPNH

This formulation implies that, during the course of the hydrogen transfer between the pyridine nucleotides, 17&estradiol undergoes cyclic oxidation and reduction, and that transfer of hydrogen occurs by virtue of the interconversion of the steroid alcohol and ketone. Detailed evidence was presented in support of the identity of the hydroxysteroid dehydrogenase and the enzyme catalyzing the transhydrogenation (24). In addition, it was shown that other hydroxysteroid dehydrogenases, in conjunction with catalytic concentrations of their specific steroid substrates, catalyzed hydrogen transport between those pyridine nucleotides or their analogues which react with these enzymes (4, 12). This suggestion provided a simple and chemically well defined mechanism which could account for the hydrogen transfer reactions. However, numerous reports by Villee et a?. (13- 18) have claimed the separation of the estrogen-sensitive transhydrogenase from two hydroxysteroid dehydrogenases, specific for DPN and TPN, respectively. Villee has further stated that the transhydrogenase which is stimulated by 17/3-estradiol is devoid of hydroxysteroid dehydrogenase activity.

The present purification of the enzyme was undertaken to permit more detailed studies on the mechanism of its transhy- drogenating function and to resolve the conflicting statements which have appeared.

EXPERIMENTAL PROCEDURE

Materials and Methods

SolutionsThe media used in the isolation procedure had the following compositions: Medium A, 0.01 M potassium phosphate, 20% glycerol, 0.005 M EDTA, 0.007 M &mercaptoethanol, final pH 7.0; Medium B, 0.01 M potassium phosphate, 50% glycerol, 0.005 M EDTA, 0.007 M &mercaptoethanol, final pH 7.0; Me- dium C, 0.005 M potassium phosphate, 20% glycerol, 0.001 M

EDTA, 0.007 M /3-mercaptoethanol, final pH 7.0. Chemical Reagents-All reagents were prepared in glass-dis-

tilled water which was obtained by distillation of ordinary

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346 Placental 17p-Hydroxysteroid Dehydrogenase Vol. 237, I%o. 2

laboratory distilled water through a 50-cm Vigreux column. Ammonium sulfate, glycerol, disodium ethylenediaminetetra- acetate (EDTA), concentrated ammonium hydroxide, sodium pyrophosphate, and monobasic and dibasic potassium phosphates were all the best commercially available analytical reagents. Spectroscopic grade glycerol was used in all solutions from Steps 5 to 9 of the purification. Tris (hydroxymethyl) aminomethane was reagent grade supplied by the Sigma Chemical Company. 17/?-Estradiol (m.p., 175-177”, corrected) and estrone (m.p., 262-264”, corrected) were commercial preparations. Commer- cial /3-mercaptoethanol was distilled under reduced pressure (b.r., 48-50” at 7 mm Hg). Acetone and 95% ethanol were redis- tilled.

Preparations-DPN, TPN, DPNH, and acetylpyridine-*DPN were obtained from Pabst Laboratories. TPNH and disodium glucose 6-phosphate were supplied by the Sigma Chemical Company.

The following molar absorbancy indices have been used: for DPNH and TPNH, aM = 6220 at 340 rnp (19); for acetylpyridine-*DPNH, ay = 9100 at 363 rnp (20); and, for the difference at 385 rnp between ay for acetylpyridine-*DPNH and aM for DPNH, 5420. Appropriate enzymatic assays gave the following analyses for the nucleotides: DPN, 1.34 pmoles per mg; TPN, 1.08 pmoles per mg; and acetylpyridine-*DPN, 1.27 pmoles per mg. Direct absorbancy measurements of the reduced nucleotides gave the following values: DPNH, 1.10 pmoles per mg, and TPNH, 0.99 pmole per mg. Solutions of the nucleotides were prepared without neutralization and stored at -20”.

Purified yeast glucose 6-phosphate dehydrogenase was obtained from C. F. Boehringer and Sons, Mannheim, Germany. Of this preparation, 1 ml, or 5.2 mg of protein, reduced 564 pmoles of TPN and 0.05 pmole of DPN per minute, when tested in a system of 3.0-ml volume containing 5 pmoles of glucose 6-phosphate, 100 pmoles of Tris buffer at pH 7.4, and 5 pmoles of TPN or DPN. The enzyme was devoid of transhydrogenase activity. It was diluted lo-fold in 1% bovine serum albumin, and 0.01 ml was used for each transhydrogenase assay.

DiuZysis TubingSeamless regenerated cellulose dialysis tubing was obtained from the Visking Company, Chicago, Illinois. The commercial tubing was soaked in several changes of a solution containing 2.0 g per liter of EDTA, 1.37 g per liter of NaHC03, and 0.5 ml per liter of ,&mercaptoethanol. This procedure removed considerable quantities of yellow pigment and rendered the tubing colorless.

Protein Concentrations-These were determined by one of three methods. (a) Measurement of the absorbancy at 280 rnp, assuming that a solution containing 1 mg of protein per ml has an absorbancy of 1.0 in a cuvette of l-cm light path. (b) Meas- urement of the absorbancies at 280 and 260 rnk, and calculation of the protein concentration by the approximate formula, 1.50 X Azso - 0.75 X AZcO = protein concentration in milligrams per ml (21, 22). These optical measurements were carried out in silica cuvettes of l.O-cm light path. Optical measurements were made against blanks of stated composition. Small errors were unavoidable when fractions were analyzed from a column in which the composition of the eluting medium varied with respect to phosphate or glycerol concentration. (c) In case of turbid solutions (Steps 2 and 3 of purification), proteins were determined by the method of Lowry et al. (23)) with crystalline bovine serum albumin as standard.

Chromatography-DEAE-cellulose (24) was supplied by the Brown Company and had a rated capacity of 0.9 to 1.0 meq per g. Ecteola-SF (24) had a rated exchange capacity of 0.3 meq per g and was supplied by the Bio-Rad Corporation. Both cellulose ion exchangers were washed and equilibrated with Medium C. The exchangers were packed by gravity in glass columns provided with coarse sintered plates. The top of the exchanger was overlaid with 200-mesh glass beads to a depth of 5 to 10 mm, to avoid disturbance of the surface. The columns were connected to a mixing vessel of constant volume, provided with a magnetic stirring bar. This mixing vessel received the outflow of a reservoir containing the concentration limit buffer. This arrangement gives a convex gradient. The concentration (C) of the variable component at any time may be calculated from the formula, C = CI - (C, - C,Je-“/Vo, where Co is the initial concentration in the mixing reservoir, C’f is the concentration of the limit buffer, V is the volume of the solution which has been delivered from the mixing vessel at the indicated time, and VO is the volume of the mixing vessel (25). All chromatographic procedures were conducted with the column and eluting buffer at room temperature. The fractions eluted from the columns were cooled to 2-5” as soon as possible, and stored at this temperature. Phosphate concentrations were determined by the method of Gomori (26).

Xtarch Block Electrophoresis-This process was carried out in a home-made apparatus, in which the starch was contained in a grooved Lucite block and was enclosed in thin transparent plastic (Saran Wrap, Dow Chemical Company). Potato starch was obtained from the Central Scientific Company, Chicago. The material was washed 9 times by decantation with distilled water and then 4 times with sodium diethylbarbiturate buffer at pH 8.6 (p = 0.05). The fine material which did not settle readily was discarded. The material was handled in a manner similar to that described by Kunkel (27).

Column Electrophoresis in Density Gradient-The model 3340 C Column Electrophoresis Apparatus (manufactured by LKB- Produkter, Fabriksaktiebolag, Stockholm, Sweden) was used for eleetrophoresis in a density gradient. This apparatus consists of a jacketed vertical column and two electrode compartments. The column can be filled easily with a linear density gradient of glycerol, and provisions are made for the introduction of the enzyme at a suitable point in the density gradient and for the collection of the contents of the column in fractions at the ter- mination of the experiment. A full description of this apparatus is given by Svensson (28). Electrophoresis was carried out in a medium containing 0.01 M potassium phosphate buffer at pH 7.40 and a linear gradient of glycerol. The enzyme was usually introduced through the capillary tube at the top of the column, into a “density shelf,” the limits of which extended on either side of the glycerol concentration of the enzyme preparation. The column was cooled by circulating water through the jacket. Preliminary experiments showed that the gradient was stable after standing in the electrophoresis apparatus for 24 to 36 hours.

The enzyme migrated toward the anode (lower end) under these conditions, and the electrophoresis was continued for a sufficient period so that the enzyme which was usually introduced in 20% glycerol (in a density shelf from 15 to 25% glycerol) had migrated into the region of the column containing 40 to 50% glycerol.

Concentrations of Ammonium Sulfate and Organic Solvents-The

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February 1962 J. Jarabalc, J. A. Adams, H. G. Williams-Ashman, and P. Talalay 347

amount of ammonium sulfate to be added to a solution to attain a given degree of saturation was calculated by formula (29).

The mixing of solvents or solutions was assumed to result in strictly additive changes in volume. The compositions of solutions containing glycerol or other organic solvents are expressed as percentages in terms of relative volumes. When crystalline ammonium sulfate was added to solutions containing varying concentrations of glycerol, the quantities of ammonium sulfate added to attain a given degree of saturation were calculated and expressed as if the solutions were completely aqueous.

Measurement of Dehydrogenase and Transhydrogenase Activities

The dehydrogenase activities of the placental enzyme were measured by the following reactions.

170-E&radio1 + DPN+ = estrone + DPNH + H+ (1)

17&Estradiol + TPN+ ti estrone + TPNH + H+ (‘4

170.Estradiol + acetylpyridine-*DPN+ F=! (3)

The standard assay procedure for following the course of the purification of the enzyme was the reduction of acetylpyridine- *DPN by 17Sestradiol (Reaction 3), rather than the reduction of DPN (Reaction l), which was used in earlier purifications (3). The assay with the analogue was selected because of higher reaction rates (Table I) and the higher absorbancy index of the reduced analogue, and because the reaction remained linear with time over a much larger range of absorbancy than with the natural pyridine nucleotides. In contrast with that of DPN and TPN, the rate of reduction of acetylpyridine-*DPN is extremely sensitive to pH (4), and it is essential to control this factor carefully by adequate buffering.

The dehydrogenase function with the natural pyridine nucleotides was in many instances measured by the reduction of estrone by DPNH or TPNH (reverse of Reactions 1 or 2).

estrone + acetylpyridine-*DPNH + H+

Hydrogen transfer between pyridine nucleotides in the presence of catalytic concentrations of 17/3-estradiol was determined either by a coupled assay requiring the continuous enzymatic generation of low levels of TPNH, as follows,

TPN+ + glucose 6-phosphate ---f

6-phosphogluconolactone + TPNH + H+O

TPNH + H+ + estrone = TPN+ + 17&e&radio1

DPN+ + I7@-estradiol = estrone + DPNH + H+

Glucose 6-phosphate + DPN+ +

6-phosphogluconolactone + DPNH + H+ (4)

or by the use of acetylpyridine-*DPN as hydrogen acceptor from DPNH, as follows

Dehydrogenase Activities with DPN, TPN, and Acetylpyri- dine-*DPN-The reaction cuvettes contained, in a final volume of 3.0 ml, 440 pmoles of sodium pyrophosphate buffer at pH 10.2, 25 mg of bovine serum albumin (usually added as 0.5 ml of a 5% solution), 0.3 pmole of 17&estradiol in 0.04 ml of 95% ethanol, 1.9 pmoles of acetylpyridine-*DPN, or 1.35 pmoles of DPN or 1.1 pmoles of TPN, and appropriate quantities of enzyme which initiated the reaction. The final pH was between 9.3 and 9.4. Measurements were taken against a blank cuvette, from which the nucleotide was omitted. Control assays showed that the reaction was strictly dependent on both pyridine nucleotide and steroid. Measurements of acetylpyridine-*DPNH were made at 363 rnp, and those for DPNH and TPNH, at 340 rnp.

DPNH + H+ + estrone S 17p-estradiol + DPN+

acetylpyridine-*DPN+ + 17fl-estradiol =

acetylpyridine-*DPNH + H+ + estrone

Dehyclrogenase Activities with DPNH and TPNH-The reac-

tion cuvettes contained, in a final volume of 3.0 ml, 300 pmoles of Tris hydrochloride buffer at pH 7.4, 50 mg of crystalline bovine serum albumin (usually added as 1.0 ml of a 5% solution), 0.15 pmole of estrone in 0.02 ml of 95% ethanol, 0.22 pmole of DPNH or 0.20 pmole of TPNH, and appropriate quantities of enzyme which initiated the reaction. The final pH was 7.0. Additional blanks were frequently used to detect steroid-independent oxidations of the reduced nucleotides. Corrections for such reactions were made when necessary.

DPNH + acetylpyridine-*DPN+ =

DPN+ + acetylpyridine-*DPNH (5)

Although optical methods which are simple in principle are available for measurement of Reactions 1 to 5, numerous difficulties may be encountered in obtaining accurate and valid reaction rates, especially with crude enzyme preparations. Some of the reasons for these vicissitudes have been discussed earlier (3).

Spectrophotometric assays were carried out in silica or Pyrex cuvettes of l.O-cm light path in a Beckman DU spectrophotom- eter, in which the cell compartment was maintained at 25” f 0.5” by circulating water through thermospacers.

In several of the assay systems, the reaction kinetics were zero order only over relatively small changes in absorbancy of approximately 0.050 to 0.100. Consequently, it is essential to derive the velocities by graphic means, to take frequent readings, and to avoid excessive rates.2

Hydrogen Transfer from TPNH to DPN-The reaction cuvettes contained, in a final volume of 3.0 ml, 300 pmoles of Tris hydrochloride buffer at pH 7.4, 10 pmoles of sodium glucose 6-phosphate, purified yeast glucose 6-phosphate dehydrogenase (see “Materials and Methods”), 0.01 pmole of TPN, 1.35 pmoles of DPN, 0.015 pmole of 17/3-estradiol in 0.02 ml of 95% ethanol, and suitable quantities of the placental enzyme. The final pH was 7.2 to 7.3. Measurements were made at 340 rnp against blank cuvettes, which usually contained all the ingredients except for the nucleotides. The reduction of the TPN was complete within 1 to 2 minutes, and there was no detectable change in absorbancy thereafter until the placental enzyme was added to initiate the transhydrogenase reaction. In crude systems, a slow rate of reduction of DPN was observed in the absence of added 17/Sestradiol, and the rate of the estrogen-stimulated reaction was computed by the subtraction of an appropriate blank.

Hydrogen Transfer from DPNH to Acetylpyridine-*DPN-This

2 All reaction rates were calculated from the slopes of the initial reaction by addition of enzyme, and subsequent readings were linear portions of plots of absorbancy against time. The first taken at 15-second intervals for at least 4 minutes. If the rates reading was usually obtained 30 seconds after initiation of the were slow, less frequent readings were taken for longer periods.

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348 Placental 17p-Hydraxysteroid Dehydrogenase Vol. 237, No. 2

TABLE I Comparison of reaction rates of puri$ed i7p-hydroxysteroid dehydrogenase in various dehydrogenase

and transhydrogenase assay systems

All measurements were carried out under conditions specified in the section on assays. The enzyme preparation which had been carried through the entire purification had a specific dehydrogenase activity of 66,600 units per mg of protein. Suitable quantities of enzyme (0.58 to 58 pg of protein) were added to each assay system to obtain an initial rate of change in absorbancy of between 0.011 and 0.050 per minute. Each result is the mean of three determinations. The individual measurements did not deviate by more than &30jo from the mean figure. The absolute ratios of activities have been computed with the aid of the appropriate molar absorbancy coefficients (see “Materials and Methods”). The ratios of the activities (in units or in absolute terms) are expressed as percentages of the rate of reduction of acetylpyridine-*DPN.

Hydrogen donor or acceptor

Acetylp ri- dine-*D%N

Activity, in units per ml of enzyme. 199,000 Ratio of activities, in units.. 100 Activity, micromoles of pyridine nucleotide re-

21,700 10.9

19,300 9.70

22,000 11.1

11,000 1 6760 1 2450 5.53 3.39 1.23

duced or oxidized per ml of enzyme. 65.5 10.5 9.29 10.6 5.29 3.74 1.18 Absolute ratio of activities.. 100 16.0 14.2 16.2 8.07 5.71 1.80

DPN TPN DPNH

Dehydrogenase I Transhydrogenase

TPNH DPNH-acetylpyridine-‘DPN TPNH-DPN

method has been previously described (3) and was employed Comparison of Reaction Velocities in Assay Systems for with minor modifications. Three reaction vessels were required Dehydrogenase and Transhydrogenase Activities for the measurements. All of them contained, in a final volume of 3.0 ml, 300 pmoles of Tris hydrochloride buffer at pH 8.2,

The reaction velocities of a highly purified preparation of 17/3-

0.02 ml of 95% ethanol, and suitable quantities of the placental hydroxysteroid dehydrogenase in seven assay systems have been

enzyme which started the reaction. Cuvette 1 received no compared (Table I). Three measurements of each activity were

other components and served as the blank. Cuvettes 2 and 3 carried out on this enzyme preparation. The ratios of the activities apply only under the precise conditions described for each

each contained 2.54 pmoles of acetylpyridine-*DPN and 0.5 assay. pmole of DPNH. 17P-Estradiol (4 pg dissolved in 95% etha-

It should be noted that the activity ratios for the various

nol) was added to Cuvette 2. Cuvette 3 received 0.02 pmole of assays are extremely sensitive to changes in pH, to minor varia-

TPN. The final pH was 8.0 to 8.1. Measurements were tions in pyridine nucleotide concentrations, and even to tempera-

carried out at 385 mp. The rate of the steroid-mediated trans- ture. Temperature differences may exert remarkably large

hydrogenase activity was derived from the differences in the effects on the ratios of the activities. Preliminary measure-

initial rates of reduction observed in Cuvettes 2 and 3. As the ment@ indicate that when the temperature of the reaction was

enzyme was purified, the rate of reaction observed in Cuvette 3 raised from 25.7 to 36.8”, the rates of the dehydrogenation of

became a progressively smaller fraction of the total rate observed 17&estradiol were increased as follows: with DPN as acceptor,

in Cuvette 2 and could be neglected after Step 6 (Table IV). by a factor of 1.9; with TPN, 3.0; with pyridine aldehyde-*DPN,

There was a very slow nonenzymatic reduction of acetylpyridine- 1.8; and with acetylpyridine-*DPN, 1.0 (unchanged). Similarly,

*DPN by DPNH (30) under these conditions, with a change in the rate of the transhydrogenation from generated TPNH to

absorbancy of approximately 0.001 per minute, for which a DPN increased by a factor of 3.8 over the same temperature

correction is applied. range.

Units of Enzyme Activity Stabilization of Enzyme

The principal obstacle to the development of a satisfactory One unit of enzyme activity in all assay systems represents a procedure for the purification of the placental 17@-hydroxy-

change in absorbancy of 0.001 per minute at the stated wave lengths. steroid dehydrogenase was the extreme instability of the enzyme. Unless speci$ed otherwise, the dehydrogenase activities of all Langer and Engel (8)4 found that an ammonium sulfate fraction

preparations were determined by the assay which uses acetylpyri- of this enzyme in 0.05 M potassium phosphate at pH 6.85, con- dine-*DPN as acceptor (Reaction 3). taining 0.001 M EDTA and 0.001 M cysteine, lost 75% of its

It must be emphasized that the activity measurements expressed in such units are uncorrected for differences in the molar

3 Unpublished observations.

absorbancy indices of the reduced nucleotides. The activity 4 Dr. L. L. Engel has informed us that the magnitudes of the

measurements are given in most instances directly in terms of enzyme activities reported in reference (8) are incorrect because of an error in the conversion of the optical data into units. One

absorbancy changes, in order that their absolute magnitudes unit of enzyme activity was assumed to be equivalent to an ab-

may be appreciated. When enzyme activities have been con- sorbancy change of 0.0062 per minute in a 3.0.ml system and is,

verted to micromoles of pyridine nucleotide reduced or oxidized, in fact, equivalent to a change of 3 mpmoles per minute in the con-

the molar absorbancy indices given in “Materials and Methods” centration of reduced pyridine nucleotide, and not 1 pmole as

have been used. stated. Hence, all activities in units or in micromoles reported by Langer and Engel (8) should be divided by 333.

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activity upon storage for 1 day at 0”. If, however, the solutions contained 50% glycerol, or 50 pg per ml of 17&estradiol, full activity was retained after storage for 9 days at 0”. Glycerol and 17/3-estradiol were thus recognized to be highly effective stabilizers of the enzyme.

The purification procedure reported in 1958 was carried out in the presence of added 17&estradiol (3). The resultant enzyme preparations contained variable and unknown quantities of 17/?-estradiol, which was firmly bound to the enzyme and could not be completely removed. It was desirable to avoid the presence of 17P-estradiol as a stabilizing agent, especially if kinetic studies of the enzyme were to be carried out. The addition of glycerol proved to be a much more efficient and satisfactory mode of stabilization.

Preliminary experiments indicated that placental homogenates prepared in 50% glycerol possessed 2 to 3 times higher activities of the enzyme than those prepared in aqueous media, even when cysteine, EDTA, and nicotinamide were included in the solutions. Moreover, the activity of an ammonium sulfate fraction of a crude homogenate remained completely stable in 50% glycerol at 2-5” for at least 1 year, whereas 84% of this activity was lost in 4 months if 17&estradiol, 40 pg per ml, was present. The stabilizing effect of glycerol is dependent upon concentration. A homogenate prepared in 10 y0 glycerol lost 61 y0 of its activity upon storage at 2-5” for 3 months, whereas under similar conditions an enzyme prepared in 50$& glycerol retained full activity.

TABLE II Summary of fractionation of placental I7p-hydroxy-

steroid dehydrogenase

There were 51 placentas used in this purification. The frac- tionations of Steps 1 to 6 were carried out in portions, as explained in the text. The figures given for the volume, total activity, and specific activity in these steps are therefore composite values computed from the individual batches. Protein concentrations were determined as follows: Step 1, by the light absorption at 280 rnp; Steps 2 and3, by the method of Lowry et al. (23); Steps 4 to 8, from the light absorbancies at 260 and 280 rnp (21, 22). All dehydrogenase activities were measured by the rate of reduction of acetylpyridine-*DPN. In the chromatographies of Steps 6, 7, and 8, the enzyme yield is given for those fractions which were carried through to the next step. The total enzyme yields eluted in all fractions were slightly higher (see text).

step

-

_

Fraction Volume

The heat stability of an ammonium sulfate fraction of the placental enzyme in 50% glycerol was studied at three tempera- tures between 68” and 88”. In 10 minutes, the enzyme lost 40 $& of its activity at 75-78” and was completely inactivated at 85-88”. No activity was lost when the enzyme was heated at 67-68” for 30 minutes. In other experiments, only minor losses (0 to 20 %) in activity were observed upon heating for 180 minutes at 67- 68”, and some preparations have been heated for 360 minutes under similar conditions without loss of activity. The heat resistance of the enzyme is strongly dependent on the glycerol concentration. In 30% glycerol, approximately 60% of the activity was lost in 10 minutes at 68”, whereas in 20% glycerol, the activity was entirely destroyed under these conditions.

Centrifuged homogenate

First (NHI) 2S04 precipitate, 0 to 0.50 saturation

Supernatant from heat treatment

Second (NIL) 804 precipitate, 0.20 to 0.50 saturation

Dialysis and centrif ugation

First DEAE-cellulose column

Second DEAE-cellulose column

Ecteola-cellulose column

ml

34,900

3,406

4,300 43.1 600 67

1,160 31.9 710 50

1,840 30.4

670 24.8

107 20.4

140 18.5

-

It has been observed that certain concentrations of a number of glycols and glycol ethers, as well as sucrose and ethanol, but not acetone, have the ability to stabilize aqueous solutions of the placental 17@hydroxysteroid dehydrogenase.

* Volume occupied by the sypernatant fraction. t The initial protein concentrations were not determined on all

of the batches, and this value’is based on determinations for 1G placentas. The range of specific activities was 24.2 to 80.0 units per mg of protein.

Isolation Procedure

In the large scale isolation of the 17@-hydroxysteroid dehydrogenase, 51 fresh human placentas (16.44 kg, wet tissue weight) were used. All operations were carried out at 2-5”, unless specified otherwise. Since nearly 3 weeks were consumed by the collection of the tissue, each placenta (or at most two or three placentas) was processed individually and carried through Steps 1 and 2 without interruption. At the end of Step 2, the enzyme may be stored for prolonged periods without loss of activity. At subsequent stages of the purification, enzyme preparations derived from progressively larger numbers of placentas were pooled, until only a single batch was carried through Steps 7 and 8. The progress of the purification and the enzyme yields are summarized in Table II.

directly from the delivery rooms of the Chicago Lying-In Hos- pital. They were packed in cracked ice and transported to the laboratory, where homogenization was carried out usually within 30 minutes, and invariably within 45 minutes after the birth of the placenta. Portions (75 g each) of tissue free from connective tissue and fetal membrane were homogenized with 150 ml of Medium A. The homogenization was carried out for 15 seconds at full speed in the lOOO-ml cup of the Waring Blendor in a refrigerated cabinet maintained at 2-5”. The homogenate was centrifuged for 30 minutes at 10,000 x g, the supernatant solution was decanted, and the residue was discarded. Re-extraction of the residues increased the yield of enzyme by only lo%, and was not routinely carried out.

Step 1. Homogenization-The term placentas were obtained

Step d. First Ammonium Sulfate Precipitation-Without de- lay, solid ammonium sulfate was added to the supernatant fluids from Step 1 (originating from one to three placentas) to

- I Dehydrogenase

ZI

*

-7

Total activity

rlzils x units/mg 10-o protein

64.1 (44.5)’

53.3 224

11

2,100 47

34,706 39

62,700 32

14,000 29

I -

Specific activity Dver-all

%

(100)

83

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Placental lY&Hydroxysteroid Dehydrogenase Vol. 237, No. 2

- FRACTION

FIG. 1. First DEAE-cellulose chromatography (Step 6) of placental 17&hydroxysteroid dehydrogenase. The second ammonium sulfate precipitate designated as Batch C in the text was dialyzed against Medium C and centrifuged (Step 5). The supernatant solution had a volume of 729 ml and a total protein of 4460 mg. The enzyme activity totaled 9.4 X 106 units, and the specific activity was 2100 units per mg of protein. The enzyme was ap- olied at a rate of aporoximatelv 300 ml aer hour to a DEAE-cellu- iose column mea&&g 33 X 316 mm which had been previously equilibrated against Medium C.

Elution of the protein was carried out with a convex gradient of increasing phosphate concentration and rising pH. The gradient was established by means of a constant volume (1070 ml) mixing vessel initially containing Medium C, connected to a reservoir containing 0.4 M KtHPOa, 2Oyo glycerol, 0.001 M EDTA, and 0.007 Y &mercaptoethanol. The dead space between the top of the column packing and the mixing reservoir had a volume of 95 ml. The hold-up volume of the packed column itself was 200 ml. Frac- tions of 10.5-ml volume were collected at a rate of 360 ml per hour. The numbering of the fractions began with the initiation of the gradient. Not all of the unadsorbed protein in the initial enzyme solution had passed through the column when collection of Frac- tion 1 was begun. The increasing phosphate and pH gradient reached the bottom of the column coincident with the collection of Fraction 28. The measured phosphate concentration in Frac- tion 51 was 0.084 M, pH 7.79, and that of Fraction 72 was 0.158 M, pH 8.17. A total of 3220 mg of protein was not adsorbed by the column; 340 mg of this appeared in Fractions 1 to 10.

The graph illustrates the concentrations of protein (O--O, measured by light absorptions at 230 and 260 rnp) and of dehydrogenase activity (X--X) measured with acetylpyridine-*DPN in each fraction. The chromatography was carried out at room temperature. Fractions 52 to 71 contained 7.8 X 106 units (33%) of the total activity applied. The highest specific activity (54,200 units per mg of protein) was found in Fraction 59.

achieve 50% saturation. The pH was maintained near 7.0 during this procedure by the addition of 3.0 M ammonium hydroxide. The precipitate was permitted to accumulate for 2 hours and was then centrifuged for 30 minutes at 10,000 X g. The supernatant invariably contained less than 7% of the enzyme activity and was discarded. The precipitate was dissolved in a minimum of Medium B. The enzyme may be stored in this medium for at least 1 year at 2-5” without loss of activity.

Step S. Heat Treatment-The pooled products derived from four to five placentas (approximately 400 ml) were transferred to a a-liter Erlenmeyer flask provided with a mechanical stirrer and immersed in a bath maintained at 67-68”. Heating with

efficient stirring was continued for 3 hours, and the preparation was then cooled to 2-5”. The red solution became chocolate brown and developed an extremely viscous, sticky precipitate which was partially removed by centrifugation at 20,000 x g for 1 hour. The supernatant was not clear but could be easily decanted. The gummy precipitate was resuspended by stirring with a magnetized bar in one-half the original volume of Medium B. The suspension was centrifuged at 20,000 x g for 60 minutes, and the supernatant fractions were combined.

Step 4. Second Ammonium Sulfate Fractionation--For con- venience, each batch of enzyme which had undergone the heat treatment of Step 3 was fractionated individually. A saturated solution of ammonium sulfate at 2-5” containing 0.005 M EDTA and 0.007 M P-mercaptoethanol, adjusted to pH 7.0 with ammonium hydroxide, was added slowly with stirring to 20% saturation. The precipitate, which contained 1 to 10% (usually less than 5%) of the enzyme activity, was sedimented by centrifugation at 10,000 X g for 30 minutes and was discarded. The saturation with respect to ammonium sulfate was then raised in 10% increments, and the precipitates which had been permitted to accumulate for 3 hours or longer were collected separately. The precipitates were dissolved in Medium B. The major portion of the enzyme activity was found in the fractions precipitating between 20 and 50% saturation. In some cases, the quantities of enzyme precipitated between 40 and 50’% saturation were very small, and these fractions were not processed further. The total recovery of enzyme activity from all fractions was 33.2 X lo6 dehydrogenase units, which represents a 77% yield from the previous step. Only 31.9 x lo6 dehydrogenase units were carried through the subsequent steps (Table II).

Step 5. Dialysis--The enzyme fractions precipitating between 20 and 50 Y. saturation of ammonium sulfate were combined into three batches, as follows. Batch A (400 ml) was derived from placentas 1 to 19; Batch B (315 ml), from placentas 20 to 35; and Batch C (445 ml), from placentas 36 to 51. The fractions precipitating between 40 and 50% saturation of ammonium sulfate were not included if they contained relatively little activity. Batches A, B, and C were then carried through Steps 5 and 6 in an identical manner, and the following description is given for Batch C.

Portions of Batch C (30 to 40 ml) were placed in washed dialysis tubing (flat width, 3.3 cm), and dialyzed for 18 hours against two &liter changes of Medium C. A copious precipitate formed. The contents of the bags were combined, and the bags were washed out with small volumes of Medium C. The preparation was then centrifuged for 30 minutes at 10,000 x g, and the precipitate was discarded. Table II presents the combined results of the dialysis and centrifugation for Batches A, B, and C.

Step 6. First DEAE-Cellulose Chromatography-After dialysis, Batches A, B, and C were individually subjected to chromatography on DEAE-cellulose. A description which applies to Batch C is given in Fig. 1. Batches A and B were treated in an analogous fashion. The fractions collected were analyzed for protein and for dehydrogenase activity (Fig. 1).

The solutions containing enzymatic activity were pale yellow, in contrast with the protein peak eluted just ahead of the enzyme (Fractions 28 to 45, Fig. l), which was intensely yellow. No dehydrogenase activity was found in the column washings or in Fractions 1 to 40.

Chromatography on DESE-cellulose resulted in a 16.5-fold

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February 1962 J. Jarabak, J. A. Adams, H. G. Williams-Ashman, and P. Talalay 351

5 2.5 - 4

E

2 ‘0

2.0-

;i

F - f 1.5 3

z

:: OC

0 - FRACTION

FIG. 2. Second chromatography of placental 17p-hydroxysteroid dehydrogenase on DEAE-cellulose (Step 7). The most highly purified fractions obtained from the first DEAE-cellulose chromatography (Step 6) of Batches A, B, and C were dialyzed for 18 hours against Medium C and applied to a DEAE-cellulose column (31 X 156 mm) which had been equilibrated against the same medium. The preparation applied to the column contained 24.8 X 106 units of dehydrogenase activity and had a specific activity of 34,766 units per mg of protein. The total quantity of protein was 716 mg. The enzyme was applied at a rate of approximately 306 ml per hour. Elution was carried out under conditions identical with those described for the first DEAE-cellulose chromatography (Fig. 1). Fractions of 10.5-ml volume were collected at a rate of 360 ml per hour. The dehydrogenase activity of each fraction was determined with acetylpyridine-*DPN as acceptor (X--X). Protein concentrations (e--O) were determined by measurements of light absorptions at 260 and 280 rnp.

purification and a total recovery of 25.3 X lo6 dehydrogenase units of the activity (83 %) in the three batches.

Step 7. Second DEAE-Cellulose Chromatography-The fractions containing the major enzyme activities in each of the three ehromatographies of Step 6 were combined and rechromato- graphed. Details are given with Fig. 2, which also shows the distribution of protein and dehydrogenase activity eluted from this column. The total yield of enzyme in Fractions 45 to 67 (Fig. 2) was 22.78 X lo6 units (92%). The largest quantities of enzyme were found in Fractions 49 and 50 and had specific dehydrogenase activities of 70,000 and 73,000 units per mg of protein, respectively. The highest specific activity (79,800 units per mg of protein) was found in Fraction 52.

Fractions 47 (specific activity of 37,200 units per mg of protein) to 60 (specific activity, 33,100 units per mg of protein) were combined. The preparation was assayed and contained a total of 20.45 X lo6 units and had a specific activity of 62,700 units per mg of protein. Since it was not convenient to proceed with Step 8 at once, the enzyme was dialyzed against a solution of pH 7.0 containing 0.005 M potassium phosphate, 50% glycerol, 0.001 M EDTA, and 0.007 M ,&mercaptoethanol and stored in this medium at 2-5’.

Step 8. Chromatography on Ecteola-Cellulose-At a later time, the preparation was diluted with 1.5 volumes of a solution of pH 7.0, composed of 0.005 M potassium phosphate, 0.001 M

EDTA, and 0.007 M P-mercaptoethanol, in order to reduce the glycerol concentration to 20%. The resultant solution (268 ml) was applied to an Ecteola-cellulose column. The elution

- FRACTION

FIG. 3. Chromatography of placental 17p-hydroxysteroid dehydrogenase on Ecteola-cellulose (Step 8). The enzyme preparation derived from the second DEAE-cellulose chromatography (Step 7) was diluted to the composition of Medium C, and applied at the rate of 60 ml per hour to an Ecteola-cellulose column (31 X 155 mm; 30 g of ion exchanger) which had been equilibrated with Medium C. The enzyme solution (268 ml) contained 20.4 X 100 units (Fractions 47 to 60 of Step 7) and had a specific activity of 62,700 units per mg of protein. Application of the enzyme was followed by a 60-ml wash of Medium C. The column was developed with a convex gradient of increasing phosphate concentration and rising pH in a manner which was identical with that described for Steps 6 and 7 (Figs. 1 and 2). Each fraction had a volume of 6.5 ml, and the rate of elution was 75 ml per hour. Collection of fractions shown on the graph was begun when the gradient device was connected to the column. The dead space between the top of the column packing and the mixing reservoir had a volume of approximately 190 ml. The graph shows the distribution of eluted protein (O--O), which was measured by light absorptions at 280 and 260 rnp, and the dehydrogenase activity measured with acetylpyridine-*DPN (X--X).

of the enzyme was carried out according to the same schedule as used for Steps 6 and 7. The distribution of enzyme activity and of protein in the various fractions is shown in Fig. 3. Fractions 44 to 62 contained the major portions of the dehydrogenase activity. The specific activities in these fractions varied between 88,000 and 119,000 units per mg of protein. Fraction 45 contained the highest concentration of enzyme and had a specific activity of 101,000 units per mg of protein. Chromatography on Ecteola-cellulose achieved almost a a-fold further purification, which was not obtainable by two chromatographies on DEAE- cellulose. The protein eluted in advance of the dehydrogenase (Fig. 3) was examined for the possible presence of transhydrogenase activity. No 17/3-estradiol-dependent hydrogen transfer between DPNH and acetylpyridine-*DPN was detectable in Fractions 31 to 43.

Concentration of Enzyme on DEA E-Cellulose-The fractions eluted from the Ecteola-cellulose column were somewhat dilute (1.16 mg of protein per ml). The solution comprising Fractions 44 to 62 from Step 8 was divided into two portions and applied directly, without intervening dialysis, to two small DEAE-

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352 Placental I?+@-Hydrozysteroid Dehydrogenase Vol. 237, No. 2

-O20 I I 1 -.--a

-15 -10 -5 0 t5 +I0 t15 +20 POSITION CM

FIG. 4. Starch block electrophoresis of a placental 17&hydroxysteroid dehydrogenase preparation obtained from the second ammonium sulfate precipitation (Step 4). The starch block measured 420 X 30 X 3 mm. The starch was thoroughly equilibrated with sodium diethylbarbiturate buffer at pH 8.6 6.1 = 0.05). The block was equilibrated for 30 minutes at 2-5” before the introduction of 0.2 ml of the enzyme (5.3 mg of protein) into a groove at the center of the block (Posifion 0, located 21 cm from either electrode). The enzyme contained a total of 6600 units of acetylpyridine-*DPN, 560 units of DPN, and 440 units of TPN-dehydrogenase activities. The total transhydrogenase activity was 80 units, measured by hydrogen transfer from TPNH to DPN. After application of the enzyme, the block was sealed in Saran Wrap and allowed to equilibrate for a further 30 minutes, and a potential of 500 volts was applied for 11 hours. The current varied between 6.5 and 8.6 ma. At the end of this period, the block was sectioned into l-cm segments perpendicular to the applied potential. Each segment was transferred to a tube and eluted by mixing with 2.0 ml of a solution of pH 7.0 composed of 0.005 M potassium phosphate, 50% glycerol, 0.001 M EDTA, and 0.007 M &mercaptoethanol. The assays were carried out on aliquots of the centrifuged supernatant.

The graph shows the distribution of dehydrogenase activities with acetylpyridine-*DPN as acceptor (X-X, in units X lo+) and with TPN as acceptor (Ic - -m, in units X 10-l). The dehydrogenase activity was also measured with DPN (not shown), and was found to follow a distribution identical with the other enzyme activities. The 17p-estradiol-dependent hydrogen transfer from TPNH to DPN is also presented (A....A, in units). An approximate measure of the distribution of protein is given by the absorbancy measurements at 284 me (O--O) which were carried out in cuvettes of l-cm light path, against a blank composed of the eluting medium.

cellulose columns (10 X 215 and 10 x 250 mm, respectively) which had been equilibrated against Medium C. The columns were washed with the same medium, and the enzyme was eluted by the application of a solution of pH 7.0, containing 0.5 M

potassium phosphate, 50% glycerol, 0.001 M EDTA, and 0.007 M

P-mercaptoethanol. The final yield of enzyme was 14 X lo6 dehydrogenase units in a volume of 35.3 ml.

Electrophoretic and Chromatographic Properties at Various States of PuriJkation

Electrophoresis on Starch-A portion of the enzyme from Step 4 of the purification was subjected to electrophoresis on a starch block, and the migration of the dehydrogenase and transhydrogenase activities was examined. A partial separation of dehydrogenase and transhydrogenase activities has been described (16) with preparations of comparable purity. The experimental conditions (Fig. 4) were patterned closely after

those described by Hagerman and Villee (16), although these authors provide rather little experimental detail. Four enzyme activities were measured: dehydrogenase activities with DPN, TPN, and acetylpyridine-*DPN, as well as the 17@-estradiol- mediated hydrogen transfer from TPNH to DPN. These assays were carried out on the enzyme before its application and on the eluates of the segments cut from the starch block. In 11 hours, the peaks of all the activities had migrated 13 cm toward the anode. The total recovery of the enzyme activities varied from 28 to 35% in the four assay procedures, and the distribution of the activities was closely parallel in each segment (Fig. 4). There was considerable loss of enzyme activity during the electrophoresis, since no stabilizer, such as glycerol, was added. However, the loss of all of the enzyme activities was proportional and there was no indication of their separation by this procedure.

Column Electrophoresis of Enzyme Obtained from First DEAE- Cellulose Chromatography-A partially purified enzyme preparation obtained from Step 6 was subjected to electrophoresis in a vertical density gradient of glycerol in the LKB column. After the electrophoresis, the contents of the column were collected in fractions. Each was analyzed for protein and for dehydrogenase activity with acetylpyridine-*DPN, and in some cases TPN, as acceptors. The transhydrogenase function of each fraction was measured by the 17P-estradiol-dependent transfer of hydrogen from DPNH to acetylpyridine-*DPN. Full experimental details and the distribution of enzyme activities are presented in Fig. 5. The enzyme traveled downward toward the anode into the increasing concentration of glycerol. The dehydrogenase activities measured with acetylpyridine-*DPN and with TPN migrated at the same rate and followed the same distribution as the 17/3- estradiol-mediated transhydrogenase activity. A substantial fraction of the protein did not travel as rapidly as the bulk of the enzyme and was consequently eluted after the enzyme peak from the electrophoresis column.

Electrophoresis and Chromatography of PuriJiecl Enzyme-The purified enzyme obtained from the fractionation procedure was finally examined by electrophoresis in a density gradient of glycerol, followed by chromatography on a column of Ecteola- cellulose. The protein concentrations and dehydrogenase activities with DPN, TPN, and acetylpyridine-*DPN were determined on the individual fractions obtained from each procedure. The 17/3-estradiol-mediated hydrogen transfer from TPNH to DPN was also measured.

Electrophoresis in a density gradient of glycerol was carried out in the LKB column (Fig. 6). Of the 210,000 units of dehydrogenase activity (specific activity, 69,000 units per mg of protein) which were applied to the column, 176,000 units (84%) were recovered. Almost the entire activity of enzyme (160,000 units) was found in three fractions (27 to 29), which contained enzyme with specific dehydrogenase activities of 115,000,128,000, and 114,000 units per mg of protein, respectively. Only these fract,ions contained sufficient protein for a reasonably accurate determination by the method of light absorbancies at 280 and 260 rnp. Dehydrogenase and transhydrogenase activities migrated in an entirely parallel manner, and there was no evidence for their separation. The recoveries of the other enzyme activities were as follows: DPNH-dehydrogenase, 91 To, TPNH-dehydrogenase, 850/,, and transhydrogenase, 91%. Since the yield of all the enzyme activities was high, and essentially similar, it appears unlikely that a significant fraction of any enzyme ac-

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- FRACTION

FIG. 5. Electrophoresis of a placental 17p-hydroxysteroid dehydrogenase preparation carried through the first DEAE-cellulose chromatography (Step 6) in a vertical density gradient of glycerol in an LKB column. An aliquot of the enzyme obtained from DEAE-cellulose chromatography was dialyzed against 0.01 M potassium phosphate, pH 7.4, 20$Zo glycerol, for 21 hours. The dialyzed solution had a volume of 6.0 ml and contained a total of 105,000 dehydrogenase units and 3.84 mg of protein. The LKB column was filled from below with 0.01 M potassium phosphate at pH 7.4 containing varying concentrations of glycerol. First, 50 ml of 15% glycerol were introduced, followed by 6.0 ml of the enzyme. This was followed by 230 ml of a linear gradient beginning with 25% glycerol and approaching 50’% glycerol. The linear gradient was produced by a simple device consisting of two vertical plastic cylinders (A and B) of identical cross section (diameter, 4 cm), maintained at the same horizontal level and interconnected at the bottom by a capillary tube (31). Cylinder A, which was stirred vigorously, contained initially 133 ml of 25% glycerol and was connected by flexible Tygon tubing, which passed through an ice bath for cooling purposes, to the filling tube at the bottom of the electrophoresis column. Cylinder B was filled with 124 ml of 50’% glycerol. The cathode (top) vessel contained 0.01 M potassium phosphate, pH 7.4, whereas the anode (bottom) vessel was filled partially with 5Ooje glycerol in 0.01 M potassium phosphate buffer, pH 7.4, and partially with 0.01 M potassium phosphate buffer, pH 7.4, alone.

The thin band of enzyme solution was initially located 345 mm above the bottom plate of the electrophoresis tube. Its position is shown by the arrow near Fraction 118. The electrophoresis vessel was cooled by circulating water at 3”. A constant current of 10 ma was applied, with the voltage varying from 450 to 510 volts, for 22 hours. The contents of the column were then collected in 2-ml fractions through the outlet at the bottom. The graph demonstrates the distribution of the protein (0-O) in the various fractions as determined by the optical density at 280 rnH read against 25% glycerol, 0.01 M potassium phosphate, pH 7.4. The optical density of the 50% glycerol-O.01 M phosphate buffer mixture was 0.020, and this accounts for the elevation of the protein base line at the left of the graph. Dehydrogenase activities were determined with acetylpyridine-*DPN (X--X, in units X 10m2), as well as in some cases with TPN (W- - I, in units X 10-r). The graph also shows the distribution of the 17p- estradiol-dependent transfer of hydrogen between DPNH and acetylpyridine-*DPN (A . . . . A, units X 10-r). Approximately 90,000 units of dehydrogenase activity (acetylpyridine-*DPN) were recovered in the various fractions (approximately 85%). The enzyme peak had migrated 90 mm toward the anode.

tivity was separated widely from the principal enzyme activities,

thereby going undetected.

Fig. 6 also shows the ratios of the activities of the enzyme activities in Fractions 25 to 31. For each fraction, the various dehydrogenase and transhydrogenase activities are expressed

- FRACTION

FIG. 6. Electrophoresis of purified placental 17&hydroxysteroid dehydrogenase in a density gradient of glycerol by means of the LKB column electrophoresis apparatus.

The entire apparatus, including the electrode vessels, contained 0.01 M potassium phosphate buffer at pH 7.4. The upper part of the column contained 15% glycerol in the phosphate buffer. The lower part of the column was filled with a linear gradient extend- ing from 25yo to 50% glycerol in phosphate buffer (cf. Fig. 5). The purified enzyme was dialyzed against a solution containing 20% glycerol in 0.01 M potassium phosphate at pH 7.4. A 6-ml aliquot of the enzyme (210,000 dehydrogenase units and 3.04 mg protein) was introduced from above into the “density shelf” between 15 and 25% glycerol. The enzyme formed a sharp band of 2 mm width which was located 295 mm above the lower end of the electrophoresis tube. The jacket was cooled with circulating water at 8”. After electrophoresis for 33+ hours at a constant current of 10 ma (400 to 425 volts), the contents of the column were eluted in fractions of 4.5 ml.

In the lower part of the figure are presented the dehydrogenase activities with acetylpyridine-*DPN as acceptor (X--X, in units X lo-*), with DPNH as hydrogen donor (O--O, in units X 10-l), and with TPNH as hydrogen donor (O- - -0, in units X 10-r). The 17p-estradiol-dependent transhydrogenase activity was measured from TPNH to DPN (A+ . . .A, in units). Protein concentrations (O-----O) were determined from the light absorptions at 280 and 260 9.

The upper section of the figure gives the dehydrogenase and transhydrogenase activities of each fraction as percentages of the dehydrogenase activity measured with acetylpyridine-*DPN as hydrogen acceptor. The ratios are calculated with all activities expressed in units. DPNH (O--O), TPNH (O----O), and transhydrogenase(A....A).

as percentages of the dehydrogenase activity for acetylpyridine- *DPN. The mean relative activities are as follows: dehydrogenase DPNH, 9.7% (range, 7.7 to 12.2); dehydrogenase TPNH, 3.57% (range, 2.75 to 4.70); and transhydrogenase function from TPNH (generated) to DPN, 1.20% (range, 0.92 to 2.15).

The three fractions which contained the principal activity in the column electrophoresis and appeared to behave in a substan- tially homogeneous manner were next subjected to chromatography on Ecteola-cellulose, with a convex gradient of increasing phosphate concentration at pH 7.0. The details of this experi-

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354 Placental I?‘@-Hydroxysteroid Dehydrogenase Vol. 237, No. 2

6 IO 14 16 22

- FRACTION

FIG. 7. Chromatography of purified placental 17@-hydroxysteroid dehydrogenase on Ecteola-cellulose. Fractions 27,28, and 29 (Fig. 6) were combined. They contained, in a volume of 13.3 ml, 1.34 mg of protein and 133,066 acetylpyridine-*DPN dehydrogenase units. The enzyme was diluted with an equal volume of water and applied without dialysis to a column of Ecteola-cellulose (85 X 4 mm) which had been equilibrated against Medium C. The enzyme was eluted with a convex gradient, with a constant volume (270 ml) mixing vessel containing the equilibrating buffer, which was connected to a reservoir containing 0.2 M potassium phosphate, 20% glycerol, 0.961 M EDTA, and 0.007 M &mercaptoethanol at pH 7.0. Fractions of 5.0-ml volume were collected at a rate of 9 ml per hour. The chromatography was carried out at room temperature. The absorbancy of each fraction was determined at 289 rnp against a blank of Medium C. Individual frao- tions were assayed for dehydrogenase activity with acetylpyridine-*DPN (X--X, units X 30e2), with DPNH (O--O, in units X lo-r), and with TPNH (n- - -0, in units X 10-r). The transhydrogenase activity was determined from generated TPNH to DPN (A-- A). The phosphate concentrations were as follows: Fraction 8, 0.025 M; Fraction 10, 0.026 M; Fraction 12, 0.035 M; Fraction 20,0.038 M.

The upper se&on of the figure gives the dehydrogenase and transhydrogenase activities of each fraction as percentages of the dehydrogenase activity measured with acetylpyridine-*DPN as hydrogen acceptor. The ratios are calculated with all activities expressed in units. The assay systems are designated as follows: DPNH (C--C), TPNH (O---O), and transhydrogenase (A---A).

ment and the results of the enzyme assays are shown in Fig. 7. It should be pointed out that only 1.34 mg of protein were subjected to chromatography, and the protein concentrations of the eluted fractions were too low to be determined with any degree of accuracy. The total recovery of the dehydrogenase activity with acetylpyridine-*DPN was 113,000 units out of a total of 133,000 units which were applied to the column (84 ye). The specific activities of Fractions 10, 11, and 12 were approximately 105,500, 104,000, and 100,000 units of dehydrogenase per mg of protein, respectively. The total yield of dehydrogenase activities was 88% for measurements with DPNH and 82% for

measurements made with TPNH. The recovery of the transhydrogenase activity was 80 %. The graph illustrates that there is good agreement between the distributions of the enzyme activities as measured by four different assay systems.

Comparison of Dehydrogenase and Transhydrogenase Activities at Various Rages of Purijication

Efforts were made to separate the dehydrogenase activities (with different nucleotides) of the purified enzyme from each other and from the transhydrogenase activity. No indication of even a partial separation of these activities was obtained when the purified enzyme was subjected to column electrophoresis in a density gradient of glycerol (Fig. 6), and when the most active fractions obtained from this electrophoresis were subsequently chromatographed on Ecteola-cellulose (Fig. 7). There was close agreement in the recovery of each enzyme activity measured in both the electrophoresis and the chromatography. Dehy- drogenase and transhydrogenase activities of even the relatively crude enzyme (Step 4) could not be resolved by starch block electrophoresis (Fig. 4), contrary to the claims of Hagerman and Villee (16).

These experiments did not exclude the possibility that the original homogenate contained separate enzyme(s) concerned with pyridine nucleotide-linked dehydrogenations of 17P-estradiol or with the 17&estradiol-mediated transfer of hydrogen between pyridine nucleotides. It could be argued that such enzymes, if present, might have been separated and discarded during the early stages of the purification. However, numerous measurements of the dehydrogenase and transhydrogenase activities on enzyme preparations at each stage of the purification, from Step 1 to Step 8, did not support this supposition. A few of these measurements of dehydrogenase and transhydrogenase activity ratios are presented in Table III. It is immediately apparent that variations in the activity ratios by as much as a factor of 2, and rarely a factor of 3, have been observed. It is equally apparent that these variations in the ratios of enzyme activities are found with preparations which vary widely in their specific activities (by approximately 2500-fold). There are no systematic variations in the activity ratios, and, in fact, the majority of the measurements agree closely.

Hydrogen Transfer from DPNH to Acetylpyridine-*DPN- This system provides a useful and accurate measure of the 17@ estradiol-dependent transhydrogenase activity. It permits the measurement of the transhydrogenase function in the absence of auxiliary enzymes and presents certain other advantages, which have been detailed previously (3). Crude soluble extracts of human placenta, as well as many other tissues, contain a variety of enzymes, probably flavoproteins, which catalyze steroid-independent oxidations of DPNH and TPNH by pyridine nucleotide analogues of higher potential, such as acetylpyridine-*DPN (32). It was found that the steroid-independent oxidation of DPNH by acetylpyridine-*DPN is unaffected by the addition of even large quantities of TPN, whereas the 17p- estradiol-mediated component of this reaction is completely obliterated by low concentrations of TPN. Hence, it was possible to devise an accurate assay of the 17/3-estradiol-dependent component of the reaction, even when the preparations contained bound steroids, by using two reaction vessels, both of which contained DPNH, acetylpyridine-*DPN, and the placental enzyme preparation; in addition, the first vessel contained TPN, and the second, 17/3-estradiol. This assay system was the

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step

TABLE III

Comparison of dehydrogenase and transhydrogenase activities of placental iY@-hydroxysteroid dehydrogenase at different stages of purity

-

Fraction

- -

Centrifuged homogenate

First (NH,)kSOa precipitate, 0 to 0.5 saturation

Supernatant from heating

Second (NHI) zSO~ precipitate : 0 to 0.2 saturation 0.2 to 0.3 saturation 0.3 to 0.4 saturation 0.4 to 0.5 saturation

Second (NHI)&SO~ precipitate, 0 to 0.5 saturation

First DEAE-cellulose column (Fig. 1) : Batch C, Fraction 51 Batch C, Fraction 72

First DEAE-cellulose column, combined eluates from Batches A, B, and C

Second DEAE-cellulose column (Fig. 2) : Fraction 45 Fractions 47 to 48 Fractions 49 to 54 Fractions 55 to 60 Fraction 61

Ecteola-cellulose column (Fig. 3) : Fraction 43 Fractions 44 to 63

LKB column electrophoresis (Fig. 6), Fractions 27 to 29

second Ecteola-cellulose column (Fig. 7), Fraction 10

Purified enzyme (Table I)

kverages

Dehydrogenase actwity with

acet;pPr;dme-

Uf&S/?d

1,800

17,000

6,600

7,200 18,000 19,200 13,600 21,600

8,000 8,200

36,000

8,600 59,000

100,000 38,000 26,000

70,000 56,000

10,000

5,800

199,000

Relative enzyme activities*

Dehydrogenase

Acqtyl- P$ge-

100

100

100

100 100 100 100 100

100 100

100

100 100 100 100 100

100 100

100

100

100

100

DPNH TPNH TPNH-DPN

9.4

10.3

3.9

5.9

6.1

0.30

0.74

9.55 1.29

8.35 5.0 0.69 10.0 5.55 1.11

7.29 4.16 1.25 8.10 5.66 1.69 9.26 3.47 1.04

10.0 8.53

11.2

3.38 0.88 3.54 0.98

2.57 1.00

9.33 3.37 0.93 10.2 4.41 1.02 9.00 4.80 1.00 9.47 4.74 1.11

10.0 5.77 1.00

9.35 8.03

10.0

8.63

11.1

9.39

5.72 4.64

4.00

3.88

5.53

4.58

1.11 1.09

1.10

1.03

1.23

1.03

- Transhydro-

genase

* The relative activities are expressed in terms of units, and related to the rate of reduction of acetylpyridine-*DPN = 100 (see Ta- ble I). It should be noted that the dehydrogenase activities reported in this table were measured by the reduction of acetylpyridine- *DPN in the presence of 17p-estradiol and by the oxidation of DPNH or TPNH in the presence of estrone.

principal method used for measuring the transhydrogenase activity in the previous (3) partial purification of the placental enzyme. It was shown in these earlier experiments (3) that the proportion of the estrogen-independent component of this reaction decreased as the enzyme became more highly purified. Similar measurements have now been carried out on enzyme preparations at various stages of the present purification (Table IV).

Less than one-half of the observed activity is steroid-dependent in the crude homogenate and the first ammonium sulfate frac-

tions. The proportion of the steroid-mediated component rises,

reaching 100% after the first chromatography on DEAE- cellulose. Careful measurements on the latter fraction indicate that, in the presence of TPN, the rate of reaction was certainly less than 0.5% of the rate observed in the estrogen-containing cuvette devoid of TPN. However, if neither l’l&estradiol nor TPN was added to the reaction system, a rate of approximately 2.5% of the estrogen-stimulated rate was observed. Since this slow reaction was completely suppressed by the addition of TPN, it appears likely that it represents hydrogen transfer mediated by small quantities of bound 17/I-estradiol in the enzyme preparation.

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356 Placental 17/3-Hydroxysteroid Dehydrogenase Vol. 237, No. 2

TABLE IV Relative rates of l’Y&estradiol-dependent and independent reactions

between DPNH and acetylpyridine-*DPN catalyzed by fractions of placental enzyme of varying purity

Steroid-dependent proportion of total activity

Purification step Fraction

% 2 First (NH,)B04 fraction, 0 to 0.50 46

saturation 3 Supernatant from heat treatment 73 5 Dialysis and centrifugation 93 6 First DEAE-cellulose chromatog- 100

raphy -

DISCUSSION

A soluble 17P-hydroxysteroid dehydrogenase of human placenta has been isolated by an eight-step procedure which resulted in an over-all purification of approximately 2500-fold, with a yield of 29%. In no single step was the yield lower than 74 ‘j& The most active preparations (114,000 dehydrogenase units per mg of protein) catalyzed the reduction of 37.5 /Imoles of acetylpyridine-*DPN, 6.08 pmoles of DPN, or 5.32 pmoles of TPN per minute per mg of protein at 25” in the presence of 17/3-estradiol. The most active preparations of the placental dehydrogenase previously available had the following specific activities. That of Hagerman and Villee (16) reduced 0.064 Imole of DPN per minute per mg of protein; that of Langer and Engel (8)l reduced 0.096 pmole of DPN per minute per mg of protein; and that of Talalay, Hurlock, and Williams-Ashman (3) reduced 0.119 Fmole of DPN per minute per mg of protein at 25”. Consequently, the earlier preparations of this enzyme possessed only 1 to 2oj, of the activity of the enzyme obtained in high yield by the purification described in this paper.

When dehydrogenase and transhydrogenase activities were measured by a variety of reactions, only minor variations in the ratios of these activity measurements were observed during the course of the entire 2500-fold purification (Table III). Deviations from constant ratios were most marked in the least purified enzyme preparations. This may be related to the technical difficulties inherent in obtaining accurate and repre- sentative measurements in the various assay procedures (3). We should like to point out that large variations in the activity ratios of a single enzyme preparation in various assay procedures may result, inter alia, from minor changes in pH, in temperature, and in the concentrations of steroid and pyridine nucleotides. In the present experiments, the yield of each of the enzyme activities remained reasonably constant throughout the purification, and no separation of dehydrogenase and transhydrogenase activities could be achieved by various procedures, including electrophoresis and chromatography.

The information obtained from this purification supplements the extensive evidence reported earlier from our laboratory (24) that (a) dehydrogenase and transhydrogenase functions are reflections of the catalytic activity of a single 17&hydroxysteroid dehydrogenase; (b) this enzyme reacts with TPN and DPN as well as with analogues of these nucleotides; (c) it is responsible for most, if not all, of the 17/?-estradiol-mediated transfer of hydrogen which takes place in soluble preparations of this tissue; and (d) transfer of hydrogen involves reversible oxidation-reduction of the steroid.

In the last 3 years, numerous claims have been made by Villee et al. (13-18) which purport to show the separation and differentiation of dehydrogenase and transhydrogenase activities. It has been claimed that “purified” placental preparations can be separated by starch block electrophoresis, by continuous flow curtain electrophoresis, and by DEAE-cellulose chromatography into components which contain only dehydrogenase or only transhydrogenase activity. By paper curtain electrophoresis, Hagerman and Villee (16) reported the separation of three fractions which contained, respectively, a DPN-specific 17/Sestradiol dehydrogenase, a TPN-specific 17/3-estradiol dehydrogenase, and a steroid-stimulated transhydrogenase which is devoid of dehydrogenase activity. The only paper (16) which contains any experimental support for these findings unfortunately gives no information on the quantities of enzyme applied or recovered, or on the absolute magnitudes of the activities measured. These claims for the separation of three distinct enzymes are at complete variance with our experiments, in which enzyme preparations have been obtained which are 50 to 100 times more refined than those described by other authors. In our hands, all preparations showing transhydrogenase activity also catalyze the oxidation of 17/%estradiol, and vice versa. For this reason, the experiments of Kellogg and Glenner (33), which appear to have demonstrated by histochemical methods the existence of separate DPN- and TPN-linked hydroxysteroid dehydrogenases for 17@-estradiol in placenta, require further examination (cf. also Kellogg (34)).

It has been stated that dehydrogenase and transhydrogenase functions of the placental enzyme can be differentiated on the basis of thermal lability and the action of certain inhibitors (13-B). Careful experiments on thermal inactivation and measurements of losses of activity under a variety of conditions of storage have provided no evidence for such separation of activities (3, 4). Differences in the degree of inhibition of dehydrogenase and transhydrogenase activities by certain inhibitors (thyroxine, 2’,5’-ADP, 2’-AMP) are to be expected and do not argue for the existence of separate enzymes, as pointed out previously (4). Such differential inhibitions are the inevita- ble consequence of the measurement of dehydrogenase and transhydrogenase activities under conditions which differ with respect to the types and concentrations of nucleotides and in the levels of steroids. Large differences in the binding constants for the nucleotides and their competition for a common binding site(s) result in competitive interactions and differences in reactivity to a variety of inhibitors (4).

SUMMARY

The purification of a soluble 17@-hydroxysteroid dehydrogenase from human placenta has been accomplished in the presence of varying concentrations of glycerol. The eight-step procedure resulted in an approximately 2500-fold purification with an over-all yield of 29%. The recovery of enzyme in the individual steps was 74 to 90%. One milligram of the purified enzyme catalyzed the reduction of 37.5 pmoles of the acetylpyridine analogue of diphosphopyridine nucleotide, 6.08 pmoles of diphosphopyridine nucleotide, or 5.32 pmoles of triphosphopyridine nucleotide per minute at 25” in the presence of 17Sestradiol under specified conditions.

Measurements of the dehydrogenase and transhydrogenase activities demonstrated that the yields and ratios of these activities are essentially constant over the entire course of purifica-

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tion of the enzyme. At various stages of purity, the enzyme has been subjected to electrophoresis on starch, to electrophoresis in a density gradient of glycerol, and to chromatography on modified cellulose anion exchangers. No separation of any dehydrogenase from the transhydrogenase activities of the enzyme could be achieved by these procedures.

It is concluded that a soluble 17&hydroxysteroid dehydrogenase in human placenta reacts with both diphosphopyridine nucleotide and triphosphopyridine nucleotide, and that this enzyme is responsible for most, if not all, of the 17/3-estradiol- mediated transfer of hydrogen between pyridine nucleotides which is demonstrable in soluble extracts of this tissue.

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Joseph Jarabak, Julia A. Adams, H. G. Williams-Ashman and Paul TalalayStudies on Its Transhydrogenase Function

-Hydroxysteroid Dehydrogenase of Human Placenta andβPurification of a 17

1962, 237:345-357.J. Biol. Chem.

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