BlOcHEMICA ET BIOPRYSICA ACTA

BBA 65247

189

FURTHER STUDY ON THE PURIFICATION OF 3a-HYDROXYSTEROID

DEHYDROGENASE OF RAT LIVER

SAMUEL S. KOIDE

Division of Clin-ical Investigation, Sloan-Kettering Institute for Cancer Research, Sloan-KetteringDivision, Graduate School of Medical Sciences, and Department of Medicine, Cornell UniversityMedical College, New York. N.Y. (U.S.A.)

(Received March I 6th, I96S)

SUMMARY

The 3u-hydroxysteroid dehydrogenase (3a-hydroxysteroid:NAD(P) oxido­reductase, EC I.1.I.50) obtained from the soluble fraction of rat liver was purified bycentrifugation, ammonium sulfate precipitation, gel filtration and DEAE-cellulosechromatography. The NAD- and NADP-dehydrogenating and transhydrogenatingactivities ofthe purified preparation ranged from 281-464,832-1087 and 8-12 m,umo­les of pyridine nucleotides reduced per min per mg of enzyme protein, respectively.The dehydrogenating and transhydrogenating activities were not separated duringthe purification procedure. An increase in the dehydrogenating activities, however,was proportionately greater than that of the transhydrogenating activity.

INTRODUCTION

A partial purification of the 3a-hydroxysteroid dehydrogenase (3a-hydroxy­steroid:NAD(P) oxidoreductase, EC I.r.I.50) obtained from the soluble fraction ofrat liver was reported 1. The marked instability of the final enzyme preparationhindered further purification. Subsequently, glycerol and sulfites were found toprotect the enzyme from heat denaturations. In the present study glycerol was usedin the media. The steps of purification included gel filtration and DEAE-cellulosecolumn chromatography.

MATERIALS AND METHODS

Sucrose, enzyme grade, was purchased from Mann Research Lab., N.Y,U.S.A. Sephadex G-100 was obtained from Pharrnacia Fine Chemicals, Uppsala,Sweden. Other materials were reported in a previous paper!. Solution A contained30% glycerol, 0.01 M phosphate buffer, pH 8.0; solution B contained 30% glycerol,0.1 M phosphate buffer, pH 8.0; solution C contained 30% glycerol, 0.5 M phos­phate buffer, pH 8.0. Cellulose dialysis tubing was soaked overnight in a solution

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containing 10 mM Na 2S20 S' I mM EDTA, pH 7.0 and washed with glass distilledwater before use.

The protein content was measured by the method of LOWRY et at.3• Approximateprotein content of fractions collected by chromatography was estimated by measure­ments of absorbance at 280 mfl which were carried out in cuvettes of r-em lightpath, against a blank of eluting medium.

Assay for dehydrogenating and trans hydrogenating activitiesNAD-dehydrogenating activity was assayed in a total volume of 3 ml containing

200 pmoles of phosphate buffer (pH 8.0), I flmole NAD and appropriate amounts ofenzyme. The NADP-dehydrogenating activity was assayed in a system containing200 pmoles of glycine-NaOH buffer (pH 9.5), 0.5 flmole NADP and appropriateamounts of enzyme in a total volume of 3 ml. The reaction was initiated by the addi­tion of 35 j-tg of androsterone dissolved in IO fll methanol to the reaction cuvettes.The control cuvette contained all ingredients except the steroid. The transhydro­genating system contained 0.6 Kornberg unit of glucose-6-phosphate dehydrogenase(Ee 1.1.1.49), 10 j-tIDoles disodium glucose-6-phosphate, 5 flIDoles MgCl2 • r zzmoleNAD, o.oz emole NADP, 200 flmoles phosphate buffer (pH 8.0), and appropriateamounts of enzyme in a total volume of 3 ml. The reaction was initiated by theaddition of 8 flg androsterone dissolved in 10 fll methanol. The changes in absorbancewere measured with a Cary spectrophotometer equipped with a o.r-absorbance slidewire attachment, employing a I em light path at 340 uip; 22-23°. One unit of allthree activities represents the reduction of a mrzmole of pyridine nucleotides per minunder the prescribed assay conditions. The molar absorbance index for the reducedpyridine nucleotides at 340 mfl was taken as 6220 (ref. 4).

Get filtration and chromatographySephadex G-IOO was washed ten times with glass-distilled deionized water and

three times with soln. A. The fine sediment was decanted off. A column of 4.2 cm X

142 em was prepared by gravity. The column was further washed with 3 1of soln. A.DEAE-cellulose (medium mesh, 0.9 mequivjg capacity) was treated as described byKELLER AND BLOCK5. 20 g (dry wt.) of treated DEAE-cellulose was washed ten timeswith glass-distilled water and three times with soln. A. The DEAE-cellulose waspacked to give a column of 2.6 em X 26 em under a pressure of 150-200 mm Hg. Itwas washed with 3 1 of soln. A.

ISOLATION PROCEDURE

Step I. Preparation of homogenate. Fasted adult male rats were anesthesizedwith sodium pentobarbital. The liver was perfused with 0.9% saline and excised.All subsequent steps were performed at 0-4°. 50 g of tissue were homogenized in aWaring blendor at 60 V for I min with 150 ml of O.I M potassium phosphate buffer,pH 8.0. The homogenate was centrifuged at 59 000 X g in a Spinco preparativeultracentrifuge for I h.

Step 2. Ammonium sulfate fractionation. This step was carried out as pre­viously reported- except that the precipitate obtained from the 0.5-0.7 ammonium

Biochim, Biopbys. A eta. IID (1965) 189-19+

3a-HYDROXYSTEROID DEHYDROGENASE 191

sulfate saturation was dissolved in soln. A and dialyzed against soln. A until thedialysate gave a negative test for ammonia with Nesslers solution.

Step 3. Sephadex gel filtration. 10 ml of the ammonium sulfate preparationwere added to the Sephadex column and eluted with 21 of soln. A. Fractions of13-15 ml were collected at a rate of about 12 mljh and tested for NAD-dehydro­genating activity (Fig. I). The tubes containing the enzyme activity were pooled anddialyzed overnight against several changes of soln. A.

Step 4. DEAE-cellulose chromatography. The enzyme preparation from Step 3

35 .

..i. 30

1.4

1.2

1.01'3.E

o.sal(IJ

+'a0.6 <1J

uCa.0

0.4 6U1.0<{

0.2

I 0100 110

Fig.!. Sephadex G-lOO gel filtration of 3 a-hydroxysteroid dehydrogenase preparation fromammonium sulfate precipitation (Step 2). In this experiment 10 ml of enzyme preparation con­taining 300 mg of protein with a total NAD-dehydrogenating activity of 3660 units were placedon the column. The recovery of the activity for this experiment was 15 %. Elution of the proteinwas carried out with soln. A. Void volume was 520 ml.

was added to the DEAE-cellulose column and eluted with a liter of soln. A followedby 500 ml of soln. B. Fractions of 10-IZ ml were collected at a rate of about 60 mljhand tested for NAD-dehydrogenating activity (Fig. 2). The tubes containing theenzymic activity were pooled and dialyzed overnight against several changes ofsoln. A.

Because of the low transhydrogenating activity of the dialyzed fractions fromthe gel filtration and chromatography column (Steps 3 and 4). it was found necessaryto concentrate the fractions. Aliquots of the fractions were placed in Visking tubings.The tubings were covered with granulated or powdered sucrose overnight. Theresulting volumes were less than 20% of the original volumes.

RESULTS AND DISCUSSION

In a previous study. we demonstrated that the 3 a-hydroxysteroid dehydro­genase from the soluble fraction of rat liver was partially protected by glycerol andsulfites from heat denaturation". For this reason glycerol was added to the medium.

Biochim: Biophys, Acta. lIO (1965) 189-194

192

A B

t t I0.8

0.6 10co

0.4ss""0"0.2 uc0

JJ

0 '-0

10 20 30 40 50 60 70 80 90 100 110 120 130 140 IIIJJ

Fractions «

S. S. KOIDE

Fig. 2. DEAE-cellulose column chromatography of preparation obtained from Sephadex G-IOO

gel filtration (Step 3). In this experiment 90 ml of enzyme preparation containing 31 mg proteinwith a total NAD-dehydrogenating activity of 570 units were placed on the column. The recoveryof the activity for this experiment was 24 %. The column was eluted with IDOO ml of soln, A and300 ml of soln, B at fractions 10 and 107 as indicated by arrows A and B, respectively.

Purified enzyme preparation was stable in the glycerol medium for at least 6 weekswhen stored at 0-40 or kept frozen.

The recovery of the NAD-dehydrogenating activity of a representative ex­periment is presented in Table 1. In five separate runs the final recovery after DEAE­cellulose column chromatography (Step 4) ranged from 5 to 10%. About 15-30%of the added activity was recovered on gel filtration and on DEAE-cellulose chro­matography.

The unit of enzyme activity as defined in the previous paper' differs from that

TABLE I

RECOVERY OF NAD-DEHYDROGENATING ACTIVITY OF 3 U-HYDROXYSTEIWID DEHYDROGENASE OF

RAT LIVER AT VARIOUS STEPS OF PURIFICATION

Step Fractions Total NAD- Recoverydehydrogenating (%)activities(units)

I Ultracentrifuged homogenate 40go 100

2 (NH{J gS04 fractionation 0.5-0.7 saturation 3660 go3 Sephadex G-IDO gel filtration 897 224 DEAE-eellulose column chromatography 257 6·3

of the present study. In the previous report, one unit was defined as a change inabsorbance of o.oorjmin which is equivalent to 0-483 mzzmoles of reduced pyridinenucleotides formed per min or 0.483 unit in the present study.

The specific activities of the enzyme preparations of a representative experi­ment at various steps of purification are shown in Table II. The specific activitieswere greatest with the fractions obtained from DEAE-cellulose column chromato­graphy (Step 4). The NAD~ and NADP-dehydrogenating and transhydrogenatingactivities ranged from 281-464, 832-1087 and 8-12 units per rng of protein, respec-

Biochim. Biophys. Acta, lIO (lg6S) 189-194

3a-HYDROXYSTEROID DEHYDROGENASE

TABLE II

193

SPECIFIC ACTIVITIES OF DEHYDROGENATING AND TRANSHYDROGENATING ACTIVITIES OF 3U­HYDROXYSTEROID DEHYDROGENASE OF RAT LIVER

Steps Fractions Unitslmg' of protein

Dehydrogenating Transbydro-genating

NAD NADP

I Ultracentrifugecl homogenate 3·4 7·3 0·42 (NH.hS04 fractionation 0.5-0.7 saturation 12 41 0.63 Sephadex G-roo gel filtration 184 334 7.84 DEAR-cellulose column chromatography 375 949 9.2

tively. The NAD-dehydrogenating activity of the preparation obtained from theDEAE-cellulose column chromatography (Step 4) was five to eight times moreactive than that of the previous reported preparations". The transhydrogenatingactivity, however, was in the same range. The NADP-dehydrogenating activity wasnot comparable since it was assayed at different pH's and with different buffers.

Representative experiments on the fractionation of the enzyme preparationby Sephadex G-roo gel filtration and by chromatography on DEAE-cellulose columnare illustrated in Figs. I and 2. respectively. An additional protein peak appearedlater on Sephadex filtration (not illustrated) which did not possess any of the enzymicactivities tested. On the DEAE-cellulose column, an additional protein peak can beeluted with soln. C. This fraction did not possess any ofthe enzymic activities tested.

The dehydrogenating and transhydrogenating activities of the 3a-hydroxy­steroid dehydrogenase were not distinctly separated during the purification procedure.The increase in the dehydrogenating activities, however, was proportionatelygreater than that of the transhydrogenating activity (Table II). This dissociation ofthe dehydrogenating and transhydrogenating activities was observed previouslywhen the enzyme was treated with (f-mercaptoethanol and testosterone". In thecourse of this study, we have observed that crystalline albumin stimulated theNADP-dehydrogenating activity. The NAD-dehydrogenating and transhydro­genating activities were unchanged or slightly inhibited by the addition of albumin.This effect of albumin on the activities of the purified preparation is presently understudy.

ACKNOWLEDGEMENT

The author is grateful to Dr. R. RAWSON for his interest in this project andto Miss M, T. TORRES for technical assistance. This investigation was supported bya U.S. Public Health Service Research Career Program Award No. r-K3-AM-SSIJ-OIfrom the National Institute of Arthritis and Metabolic Diseases, and Grant C-3809from the National Cancer Institute, U.S. Public Health Service.

REFERENCES

I S, S. KOIDE, Arch. Biochem., 101 (1963) 278.2 S. S. KOIDE A~D M. T. TORRES, Biochim, Biophys. A eta. 89 (I964) ISO.

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I94 S. S. KOIDE

3 O. H. LOWRY, N. J. ROSEBROUGII, A. L. FAltR AND R. J. RANDALL, J. Bioi. Chem., 193 (1951)265·

4 B. L. HORECKER AND A. KORNBERG, J. Bioi. Chem., 175 (194 8) 385.5 S. KELLER AND R. J. BLOCK, in P. ALEXANDER. AND R. J. BLOCK, Laboratory Monua; of

Analytical Methods of Protein Chemistry, VoL I, Pergamon Press, New York, 1960, p. 76.6 S. S. KOIDE, Steroids, 3 (1964) 85.

Bioobim, Biopbys, Acta, no (1965) 189-194