ANALYTICAL

BIOCHEMISTRY

95, 286-292 (1979)

Preparation of Highly Purified 3~ and 3P-Hydroxysteroid Dehydrogenases from Pseudomonas sp.l MIKIO Department

of Pharmacology School

SHIKITA~

AND PAUL TALALAY~

and Experimental Therapeutics, Baltimore, Maryland

The Johns 21205

of Medicine,

Hopkins

University

Received December 15, 1978 A method is described for preparing highly purified 3a- and 3P-hydroxysteroid dehydrogenases (EC 1.1.1.50 and EC 1.1.1.145, respectively), essentially uncontaminated with one another, from extracts of a steroid-induced Pseudomonas species. These enzymes are suitable for the microanalysis of 3a-hydroxy-, 3@hydroxy-, and 3-ketosteroids.

The first purified hydroxysteroid dehydrogenases were named and isolated just 25 years ago (l-3). They are a family of widely distributed NAD- and NADP- linked alcohol dehydrogenases that promote the interconversion of hydroxyl and carbonyl functions on the steroid skeleton and side chain and exhibit a high degree of steric and positional specificity (4). Considerable effort has been expended on the purification and the elucidation of the mechanism of action of these enzymes and much is known about their specificities and the equilibria of the reactions which they promote (4). Interesting and as yet not fully explored applications of these isolated enzymes are their use in the microanalysis of steroids by groupspecific reactions (5-8), in carrying out highly specific oxido-reductions (3), and in the resolution of enantiomeric steroids (9). Although relatively crude enzymes are adequate for some of these purposes, highly refined preparations are essential for certain analytical and preparative purposes, especially where mixtures of steroids are involved. 1 Dedicated to the memory of Dr. Alvin Nason. * Present address: National Institute of Radiological Sciences, 9-1, 4-chome, Anagawa, Chiba-shi, Japan. 3 To whom correspondence regarding this manuscript should be addressed. 0003-2697/79/070286-07$02.00/O Copyright All rights

0 1979 by Academic Press. Inc. of reproduction in any form reserved.

We have recently revived our interest (5-8) in the microanalytical possibilities of the applications of these enzymes, and have extended the sensitivity of measurement by the application of the enzymatic pyridine nucleotide cycling methods of Lowry and colleagues (10) to the measurement of steroids at the picomolar level and below.4 The contribution of Harkonen, Adlercreutz, and Groman (11) in demonstrating the feasibility of this approach in measuring corticosteroids and 17P-estradiol was an important stimulus for this work. For the measurement of steroids in small fragments of endocrine tissue directly, we required highly purified 3a- and 3P-hydroxysteroid dehydrogenases (EC 1.1.1.50 and EC 1.1.1.145, respectively). Several methods for obtaining purified 3a-hydroxysteroid dehydrogenase preparations of high specific activities

from

Pseudomonas

testosteroni

(12- 16) have been described. These preparations have been widely used for the quantitation of bile acids and salts by the specific oxidation of the 3cr-hydroxyl group of these compounds and the measurement of the NADH formed (17-19). The situation is more complex with respect to the 3/3-hydroxysteroid dehydrogenase of P. 4 P. Talalay, manuscript in preparation. 286

3~ AND 3p-HYDROXYSTEROID

testosteroni. This microorganism was originally believed to contain a single enzyme with specificity for both the 3p- and 17phydroxyl groups of steroids and was designated first as /3-hydroxysteroid dehydrogenase (2,3,20), and subsequently as (3 and 17)@hydroxysteroid dehydrogenase (4). Homogeneous preparations of (3 and 17)/?hydroxysteroid dehydrogenase have now been obtained by Schultz, Groman, and Engel (2 1,22) from Pseudomonas testosteroni and the properties of this enzyme have been examined in some detail. However, P. testosteroni also contains at least one and possibly several distinct 17@-hydroxysteroid dehydrogenases, devoid of 3P-hydroxysteroid dehydrogenase activity (23,24). Because of the presence of a complex mixture of these enzymes in P. testosteroni, we turned our attention to the report of Teller and Bongiovanni (25), who found in their preparations of P. testosteroni a contaminating Pseudomonas sp. which showed only 3/3-hydroxysteroid dehydrogenase activity. These authors suggested that their organism was a “mutant” of P. testosteroni, but since many Pseudomonas species have steroid-oxidizing capacity (14), the organism may have been a contaminating species. We shall refer to the Teller-Bongiovanni organism as Pseudomonas sp. TB. It contains 3cw-and 3/3-hydroxysteroid dehydrogenase activities and little, if any, activity for the 17P-hydroxyl groups of steroids. The present paper describes the isolation of highly purified 3a- and 3&hydroxysteroid dehydrogenases from Pseudomonas sp. TB grown on steroid-containing media. EXPERIMENTAL

PROCEDURES

Materials. All reagents were of the best commercial quality available. Reagentgrade methanol was redistilled. Glycerol was of spectroscopic quality. Distilled water was passed through a mixed-bed ion exchanger and then redistilled in an all-glass system. Ammonium sulfate had low heavy metal ion content (special enzyme grade,

DEHYDROGENASES

287

Schwarz/Mann, Orangeburg, N. Y.). The lithium salt of P-NAD was obtained from Boehringer-Mannheim Biochemicals, Indianapolis, Indiana. DEAE-cellulose (Type DE 52; swollen microgranular form) was obtained from Reeve Angel, Clifton, New Jersey. Agarose-hexane-NAD (Type 1) was obtained from P-L Laboratories, Milwaukee, Wisconsin. The preparation of this material is based on the procedure of Mosbach et al. (26), which involves reaction of cyanogen bromide-activated agarose with 6-aminocaproic acid and the coupling of the resultant product with NAD+ in the presence of dicyclohexylcarbodiimide. CHESS [(2cyclohexylamino)ethanesulfonic acid] was obtained from Calbiochem, La Jolla, California. Solutions of CHES were adjusted to pH 9.0 with NaOH. Crystalline bovine plasma albumin (Armour Pharmaceutical Company, Chicago, Ill.) solutions were neutralized. 19-Nortestosterone-agarose was prepared as described (23). Lyophilized steroid-induced preparations of the “mutant” of P. testosteroni (STDHM) were obtained from Worthington Biochemical Corporation, Freehold, New Jersey. Enzyme asscrys. Spectrophotometric measurements were made at 340 nm in cuvettes of I.O-cm light path maintained at 25°C. The reaction systems contained in a final volume of 3.0 ml: 100 mM CHES, pH 9.0; 0.33 mM NAD; 0.015% bovine plasma albumin: 34.5 PM androsterone (for 3ahydroxysteroid dehydrogenase) or dehydroepiandrosterone (for 3/3-hydroxysteroid dehydrogenase) in 30 ~1 of methanol, and the appropriate amounts of enzyme (usually I20 ~1) to achieve initial absorbance changes of 0.005-O.O6O/min. If necessary, the enzyme preparations were diluted in 1% bovine plasma albumin. The enzyme activities are expressed as micromoles of NAD+ reduced per minute under these conditions, by the use of the value 6,270 M-’ cm-’ for the extinction coefficient of NADH. Enzyme ’ Abbreviation used: CHES, (2-cyclohexylamino) ethanesulfonic acid.

288

SHIKITA

AND TALALAY

specific activities were expressed per milligram of protein as determined by the method of Lowry et al. (27), with bovine plasma albumin as standard. Polyacrylamide gel electrophoresis. Analytical polyacrylamide gel electrophoresis (28,29) was performed in 8-cm double-stack gels containing a 3% acrylamide stacking gel on a 5% acrylamide running gel. The gels were equilibrated and run in Trisglycine buffer, pH 8.5, containing 5 mM 2mercaptoethanol. Electrophoresis was carried out at 4°C at 3 mA/tube until the tracking dye (bromphenol blue) was about 0.5 cm from the end of the tube. The enzyme activities were detected as described by Schultz et al. (21). The protein bands were located with 0.01% Coomassie brilliant blue. TABLE PURIFICATION

OF&AND

3pHYDROXYSTEROID

Purification procedure Streptomycin supernatant Ammonium sulfate precipitate (40-60%)

All operations were carried out at 0-4”C, unless otherwise specified. Step 1. Extraction,

removal of nucleic

1

3/3-HSD

1180

502

758

425

3a-HSD”

PSEUDOMONAS

Specific activity (~mol/min/mg protein) 3a-HSD

3p-HSD

2794

0.422

0.180

398

1.90

1.07

3a-HSD

3/3-HSD

100

100

64.3

22.9

56.7

489 355 421

7.2 3.9 3.2

67.9 91.0 132

41.4 30.1 35.7

dehydrogenase.

84.7

dehydrogenase

29.2

3/3-Hydroxysteroid

sp.TB

Yield (%)

669

DEAE-cellulose chromatography effluent (200-270 mM phosphate) Sephadex G- 150 chromatography Affinity chromatography Final (after concentration) ‘* HSD, hydroxysteroid

Purijication of3w and 3/3-Hydroxysteroid Dehydrogenases

Total protein 0x4

3wHydroxysteroid DEAE-cellulose chromatography effluent (125- 175 mM phosphate) SephadexG-150chromatography Affinity chromatography Final (after concentration)

The initial extracts of lyophilized powder of Pseudomonas sp. TB contained about two to three times as much 3a- as 3/?hydroxysteroid dehydrogenase activity. Separation and purification of these two specific dehydrogenases were accomplished by DEAE-cellulose ion exchange, gel filtration, and affinity chromatography. The purification procedure is summarized in Table 1, which presents the mean values from two purifications.

DEHYDROGENASEACTIVITIESFROM

Total activity (~mol/min) Step

RESULTS AND DISCUSSION

dehydrogenase

319

46.3

206 206 160

10.5 1.8 1.8

6.89 19.6 114 88.9

63.6 41.0 41.0 31.9

3w AND

3p-HYDROXYSTEROID

The lyophilized bacterial powder (14 g) was suspended in 140 ml of 50 mM potassium phosphate buffer containing I mM EDTA, 20% glycerol, and 0.05% 2-mercaptoethanol, final pH 7.0. Smooth suspension was obtained by treatment (3 x 15 s) with a Polytron (Model PTlO-35, Brinkmann Instruments) homogenizer. The suspension (ca. 150 ml) was then treated for 30 min in a sonic oscillator (MSE Model 60W; 0.9-cmdiameter probe) while being circulated by means of a peristaltic pump between the sonication vessel (3 x 10 cm; 40 ml volume) and a 2-m-long coil of silicone rubber tubing (6-mm internal diameter) which was immersed in a mixture of ice and water. The rate of circulation was adjusted so that the temperature in the sonication vessel never rose above 8°C. After sonication, a yellow, turbid supernatant fluid was obtained by centrifugation at 20,OOOg for 20 min, and the residue was washed by suspension in the same buffer and centrifuged. The combined supernatant fluids were treated with one-third of their volume of a 10% streptomycin sulfate solution, and the resultant precipitate of nucleic acids was removed by centrifugation. The hydroxysteroid dehydrogenases were precipitated from the solution by the addition of ammonium sulfate to between 40 and 60% saturation, while the solution was maintained at pH 7 by the addition of NH,OH. acids,

and precipitation

Step 2. DEAE-cellulose

of enzymes.

chromatography.

The ammonium sulfate precipitate was dissolved in a minimum volume of 10 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA and 0.05% 2-mercaptoethanol, and dialyzed against the same buffer overnight. The solution was then applied to a column (2 x 20 cm) of DEAEcellulose which had been equilibrated with the same buffer. The hydroxysteroid dehydrogenases were eluted from the column with a linear concentration gradient of potassium phosphate (from 10 to 300 mM; 400 ml total) at pH 7.0 in the presence of 1 mM

289

DEHYDROGENASES

EDTA and 0.05% 2-mercaptoethanol. The majority of the 3c-u-hydroxysteroid dehydrogenase activity was eluted at phosphate concentrations between 125 and 175 mM, and 3/3-hydroxysteroid dehydrogenase was eluted between 200 and 270 mM. The fractions containing each activity were pooled and the enzymes were precipitated with ammonium sulfate at 70% saturation (pH 7). Each precipitate was dissolved in 3.0 ml of 100 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA and 0.05% 2-mercaptoethanol. Step 3. Gel filtration

on Scphadex

G-150.

The hydroxysteroid dehydrogenases were chromatographed separately on a column of Sephadex G-150 (1.5 x 80 cm) which had been equilibrated with 100 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA and 0.05% 2-mercaptoethanol. The 3a-hydroxysteroid dehydrogenase activity was eluted between 1.5 and 1.8 void volumes, whereas the 3P-hydroxysteroid dehydrogenase activity was eluted between 1.3 and 1.7 void volumes. Step 4. Afjnity chromatography. The solution of 3a-hydroxysteroid dehydrogenase (14 ml) was dialyzed against 20 vol of 1 mM EDTA-0.05% 2-mercaptoethanol for a few hours and then brought to 20 ml with water. The solution was recirculated overnight through a column (0.9 x 12 cm) of NAD-hexane-agarose at a flow rate of 6 ml/h. The column was washed with 25 ml of 10 mM potassium phosphate, pH 7.0, containing 1 mM EDTA and 0.05% 2-mercaptoethanol. The 3a-hydroxysteroid dehydrogenase was eluted from the column with 50 ml of a linear concentration gradient of potassium phosphate buffer (between 10 and 300 mM), pH 7.0, also containing EDTA and 2-mercaptoethanol. The eluted enzyme was concentrated to about 1 ml by ultrafiltration under reduced pressure and finally by dialysis against an excess of a solution containing 50% glycerol and 2 mM potassium phosphate, pH 7.0. The pooled fractions (14 ml) of the 3p-

290

SHIKITA

AND TALALAY

hydroxysteroid dehydrogenase obtained from the gel filtration (Step 3) were treated with solid lithium salt of NAD+ to a final concentration of 0.5 mM. The solution was applied to a column (1.5 x 15 cm) of 19nor-testosterone-agarose and equilibrated by recirculation of the solution through the column overnight (flow rate, 6 ml/h). The column was then washed with 60 ml of 50 mM potassium phosphate buffer, pH 7.0, containing 1 M ammonium sulfate, 20% glycerol, 1 mM EDTA, and 0.05% 2-mercaptoethanol. After all the NAD+ had been washed out of the column, the 3/?-hydroxysteroid dehydrogenase was eluted with 100 mM potassium phosphate, pH 7.0, containing 1 mM EDTA and 0.05% 2-mercaptoethanol (flow rate, 12 ml/h). The active fractions (56 ml) were combined and concentrated to about 1 ml by a combination of ultrafiltration and dialysis against a solution containing 50% glycerol and 2 mM potassium phosphate, pH 7.0. The final preparations were stored at TABLE SPECIFIC

ACTIVITIES OF PURIFIED FROM Pseudomonas

-20°C and retained about 30-50% of their activities after 18 months. Properties of Purified ~CX-and 3PHydroxysteroid Dehydrogenases Specific activities. The final purified preparations obtained by the procedures described in this paper had 3a-hydroxysteroid dehydrogenase specific activities of 88- 168, and 3P-hydroxysteroid dehydrogenase specific activities of 120- 170 pmol of steroid oxidized/min/mg of protein. Various other procedures for preparing 3cr-hydroxysteroid dehydrogenase from P. testosteroni and Pseudomonas sp. TB gave preparations of comparable specific activities, when allowance is made for the somewhat different conditions of assay (Table 2). Only Roe and Kaplan (14) appear to have purified the 3phydroxysteroid dehydrogenase activity of Pseudomonas sp. TB and report a specific activity of 73 ~moYmin/mg protein using somewhat different assay conditions and a 2

&HYDROXYSTEROID DEHYDROGENASE testosteroni AND Pseudomonas sp.

Assay conditions

Year

Buffer’ (mM)

NAD b=f)

(13)

1964 1965

33.3 66.7

0.167 0.167

(14)b

I%9

33.3

0.47

115

(15)”

1976

66.7

0.167

35

(16)”

1977

66.7

0.25

50

0.33

34.5

Reference

(12)

Present workb

a Pseudomonas b Pseudomonas

100

Androsterone (KM) 17.3 34.5

Organic solvent (vol %) 0 Dioxane (0.67%) CH,OH (3.3%) CH,OH (0.33%) C,H,OH (3.3%) CH,OH (1%)

PREPARATIONS

TB Specific enzyme activity (~mol/min/mg protein) Initial

Final

0.24 1.67

1% 341

0.372

160

0.99

300

0.79

300

0.9- 1.3

88- 168

testosteroni.

sp. TB. c Sodium pyrophosphate buffer in all systems, except present work which used CHES buffer. The pH was 9.0, except in the report of Battais et al. (16), who used pH 9.5.

3o- AND 3p-HYDROXYSTEROID

I!1c+

0 +

FIG. 1. Drawing of polyacrylamide gels after electrophoresis of purified 3P-hydroxysteroid dehydrogenase (A and B) and 3a-hydroxysteroid dehydrogenase (C and D). Gels A and C were stained with Coomassie brilliant blue, and gels B and D were stained for 3P-hydroxysteroid dehydrogenase (dehydroepiandrosterone) and 3a-hydroxysteroid dehydrogenase (androsterone) activities, respectively. Experimental details are given in the text.

DEHYDROGENASES

291

of protein and enzyme activity were observed. The two species which migrated most rapidly contained the highest enzyme activities. In addition, a faint band with R, = 0.46 stained with the dye but was devoid of enzyme activity. These findings are entirely consistent with the report of Skalhegg (30) that 3a-hydroxysteroid dehydrogenase activity of P. testosteroni resides in two major and one minor catalytic species, and that the purified enzyme contained two types of subunits of approximately equal molecular weight (ca. 25,000) but carrying different charges. The enzymatically inactive minor component with R, = 0.46 may be a polymeric species (31).

different steroid substrate (epiandrosterone) ACKNOWLEDGEMENTS from those in our work. The highly puriThese studies were supported by National Institutes of Health Grants AM 07422 and AM 19300. We thank fied enzymes are of comparable and high specific activities. Dr. Trevor M. Penning for carrying out the polyFor the microestimation of steroids, an acrylamide gel electrophoreses. even more important property of these prepNote udded in proof: Since this manuscript was subarations is the lack of contamination of one mitted, it has come to our attention that T. Uwajima, K. enzyme by the other. Measurements on the Ta kayama, and 0. Terada (Agric. Biol. Chem. 42, most highly purified preparations have 1577-1583. 1978) have reported the purification and shown that in the conventional assay sys- crystallization of a 3a-hydroxysteroid dehydrogenase from Pseudomonu.s pufida tern, the contaminations of 3o-hydroxysteroid activity in 3P-hydroxysteroid prepREFERENCES arations, and vice versa, were 0.48% and 0.039%, respectively. 1. Talalay, P., and Dobson, M. M. (1953) J. Biol. Polyacrylamide gel electrophoresis. ElecChem. 205, 823-837. trophoresis of the purified preparations was 2. Marcus, P. I., and Talalay, P. ( 1956) .I. Biol. Chem. 218, 661-674. carried out in polyacrylamide gels, which 3. Talalay. P., and Marcus, P. I. (1956)J. Biol. Chem. were stained for protein with Coomassie 218, 675-691. blue and tested for hydroxysteroid de4. Talalay. P. (1963) in The Enzymes (Boyer, P. D., hydrogenase activities with androsterone Lardy. H., and Myrblck, K., eds.). 2nd ed.. and dehydroepiandrosterone as substrates. Vol. 7, pp. 177-202, Academic Press. New York. The results (Fig. 1) show that the 3/3hydroxysteroid dehydrogenase activity is 5. Hurlock, B., and Talalay, P. (1956) Proc. Sot. Exp. Biol. Med. 93, 560-564. associated with a single band of protein 6. Hurlock, B.. and Talalay, P. (1957) J. Biol. Chem. which migrates with an Rf = 0.61 relative 227, 37-52. to the tracking dye, and that there are no 7. Hurlock, B., and Talalay, P. (1958) Etldocrino/o,q 62, 201-215. other detectable components. Upon electro8. Talalay. P. (1960) Methods Biochem. Anal. 8, phoresis of the purified 3a-hydroxysteroid 119- 143. dehydrogenase preparations, two major 9. Talalay, P., and Levy, H. R. (1959) in Steric bands (R, = 0.66-0.68 and 0.59-0.60) and Course of Microbiological Reactions, Ciba one less prominent band (R, = 0.53-0.55) Foundation Study Group No. 2 (Wolstenholme.

292

10.

11. 12. 13. 14. 15.

16.

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SHIKITA

AND TALALAY

G. E. W., and O’Connor, C. M., eds.), pp. 53-78, Churchill, London. Lowry, 0. H., Passoneau, J. V., Schulz, D. W., and Rock, M. K. (1961) J. Biol. Chem. 236, 2746-2755. Hsrkanen, M., Adlercreutz, H., and Groman, E. V. (1974) J. Steroid Biochem. 5, 717-725. Delin, S., Squire, P. G., and Porath, J. (1964) Biochim. Biophys. Acta 89, 398-408. Boyer, J., Baron, D. N., and Talalay, P. (1965) Biochemistry 4, 1825- 1833. Roe, C. R., and Kaplan, N. 0. (1968) Biochemistry 8, 5093-5103. Augrust, L. E., Norum, K. R., and Skalhegg, B. A. (1976) Biochim. Biophys. Acta 438, 1322. Bat&is, E., Terouanne, B., Nicolas, J. C., Descamps, B., and Crastes de Paulet, A. (1977) Biochimie 59, 909-917. Iwata, T., and Yamasaki, K. (1%4) J. Biochem. (Tokyo) 56, 424-431. Palmer, R. H. (1969) Methods Enzymol. 15, 280-288. Bruusgaard, A. (1970) C/in. Chim. Acta 28, 495504.

20. Delin, S., and Porath, J. (1963) Biochim. Biophys. Acta 67, 197-200. 21. Schultz, R. M., Groman, E. V., and Engel, L. L. (1977) J. Biol. Chem. 252, 3775-3783. 22. Schultz, R. M., Groman, E. V., and Engel, L. L. (1977) J. Bioi. Chem. 252, 3784-3790. 23. Benson, A. M., Suruda, A. J., Barrack, E. R., and Talalay, P. (1974) Methods Enzymol. 34, 557-566. 24. Groman, E. V., and Engel, L. L. (1977) Biochim. Biophys. Acta 485, 249-254. 25. Teller, J. D., and Bongiovanni, A. M. (1%3) Nature (London) 197, 11 I2- 1113. 26. Mosbach, K., Guilford, H., Ohlsson, R., and Scott, M. (1972) Biochem. J. 127, 625-631. 27. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265275. 28. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427. 29. Omstein, L. (1964) Ann. N. Y. Acad. Sci. 121, 321-349. 30. Skglhegg, B. A. (1975)Int. J. Peptide Protein Res. 7, 335-339. 31. Skglhegg, B. A. (1974) Eur. J. Biochem. 46, 117125.