BIOCHIMICA ET BIOPHYSICA ACTA

BBA 65206

Ir5

DISTRIBUTION OF 3-,B-HYDROXYSTEROID DEHYDROGENASE AND

LJ5-3-OXOSTEROID ISOMERASE IN HOMOGENATE FRACTIONS OF HUMAN

TERM PLACENTA

SAMUEL S. KOIDE AND MARIA T. TORRES

Division of Clinical Investigation, Sloan-Kettering Institute, Sloan-Kettering Division, GraduateSchool of Medical Sciences, and The Department of Medicine, Memorial Hospital and CornellUniversity Medical College, New York, N.Y. (U.S.A.)

(Received January t rth, 1965)

SUMMARY

The activities of the 3-,B-hydroxysteroid dehydrogenase (3-,B-hydroxysteroid:NAD(P) oxidoreductase, EC LI.L5I) and the LJ5-3-oxosteroid isomerases (3-oxoste­roid LJ4-LJ5-isomerases EC 5.3.3.1), in the soluble, mitochondrial and microsomalfractions of human term placenta were determined. Both enzymic activities werefound in all subcellular fractions. The greatest activities were in the particulatefractions. The optimum pH for the 3-,B-hydroxysteroid dehydrogenase was in thevicinity of pH 10-10.5. The results of this study suggest that the 3-,B-hydroxysteroiddehydrogenase and the isomerase might be separate enzymes and that there mightbe distinct isomerases for different substrates in the particulate fractions.

INTRODUCTION

The conversion of LJ5-3-,B-hydroxysteroids to LJ4-3-oxosteroids in the humanterm placenta has been attributed to the action of LJ5-3-,B-hydroxysteroid dehy­drogenase-, The 3-,B-oxidation and isomerization might be mediated in two distinctsteps. The LJ5-3-oxosteroid isomerases (3-oxosteroid LJ4-LJ5-isomerases, EC 5.3.3.1)have been demonstrated in the bovine adrenal gland and many rat organs 2- 4 • Itwould appear from these reports that the conversion of dehydroepiandrosterone to4-androstene-3,I7-dione by the placental LJ5-3-,B-hydroxysteroid dehydrogenasemight be mediated by a 3-,B-hydroxysteroid dehydrogenase (3-,B-hydroxysteroid:NAD(P) oxidoreductase, EC 1.1.1.51) and a separate LJ5-3-oxosteroid isomerase. Thepretent study was undertaken to observe the distribution of the 3-,B-hydroxysteroiddehydrogenase and J5-3-oxosteroid isomerase activities in subcellular fractions ofhuman term placenta.

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MATERIALS AND METHODS

S. S. KOIDE, M. T. TORRES

The 3-,B-hydroxysteroids were obtained from Steraloids, Inc., New York andPreparations Lab., New York, recrystallized from acetone, benzene or ethyl acetate,purified on alumina or silica-gel column and sublimed when possible. The meltingpoints and infrared spectra corresponded with published data. The purity of thesteroids was checked by paper chromatography. A single spot was obtained withtwo elution systems. s-Androstene-3,I7-dione and 5-pregnene-3,20-dione were pre­pared according to the method described by DJERASSI et al.". and purified on analumina column according to the procedure described by NES et al». They wereused without further purification. 5(ro)-Estrene-I7-,B-ol-3-one and 5(ro)-estrene-3­~-01-I7-one were gifts of C. Chen, Chicago, Ill. NAD and NADP were purchasedfrom Sigma Chemical Co., St. Louis, Mo. Protein determinations were carried outaccording to the method of LOWRY et at.? All solvents were purified by simple distil­lation.

Assay procedurefor 3-,B-hydroxystero-id dehydrogenaseThe dehydrogenase activities were assayed in a system containing I,umole

NAD or 0.5,umole NADP, zoo cmoles glycine-NaOH buffer (pH 10.0) and appro­priate amounts of enzyme in a total volume of 3 ml. The reaction was initiated bythe addition to the reaction cuvette of 0.1 ,umole of the appropriate steroids dissolvedin IO,ul of methanol. The control cuvette contained all ingredients except the steroid.The activity was measured by observing the changes in absorbancy at 340 m,u at25°. The molar absorbancy index for the reduced pyridine nucleotides at 340 m,uwas taken as 6220 (ref. 8).

Assay procedurefor L15-3-oxosteroid isomerase activityThe isomerase activity was assayed by two different procedures. The spectro­

photometric method was used to assay the conversion of s-androstene-3,I7-dione to4-androstene-3,I7-dione. Although EWALD et al.2,4 were able to adapt this methodto assay the conversion of s-pregnene-3,20-dione to progesterone, in our hands, theresults were inconsistent probably because of the low isomerase activity and poorsolubility of the steroid. The procedure described by KRUSKEMPER et az.a was usedto assay the conversion of s-pregnene-3,20-dione.

Procedure I. The isomerase activity Jas assayed in a system containingzoo rzmoles phosphate buffer (pH 7.0) and appropriate amounts of enzyme in a totalvolume of 3 ml. The reaction was initiated by the addition of 0.15 ,urnole s-androstene­3,I7~dione dissolved in 10 ,ul of methanol to the reaction cuvette. The control cuvettecontained all ingredients except the steroid. The activity was measured by observingthe changes in absorbancy at 248 m,u at 25°. Non-enzymic controls were run with thesame system, using I mg of crystalline bovine albumin in place of the enzyme. Theactivities were corrected by subtracting the non-enzymic rates. The molar absorbancyindex for 4-androstene-3,I7-dione at 248 m,u was taken as 16300 (ref. 9). Under theseconditions a change in absorbancy of 0.001 per min is equivalent to the isomerizationof 0.184 m,umole of s-androstene-3,I7-dione per min. The dehydrogenase and iso­merase activities were measured with a Cary spectrophotometer equipped with a 0.1

absorbancy slide wire attachment, employing a r-em light path.

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HYDROXYSTEROID DEHYDROGENASE AND ISOMERASE II7

Procedure 2. I ml of enzyme preparation was pipetted into ro-ml glass­stoppered flasks containing I ml of 0.1 M Tris-Hf'l buffer (pH 7.0). All determina­tions were run in duplicate. The flasks were pre-incubated for 10 min at 37°. At theend of this period, o.a zzmole of 5-pregnene-3,20-dione dissolved in 0.03 ml of metha­nol was added to the flasks. The flasks were incubated in a Dubnoff shaker for 10 minat 37° under air. The reaction was terminated by the addition of 0.3 ml of absoluteethanol. Following the addition of 5.0 ml of spectroscopic-grade methylene chloride,the flasks were vigorously shaken for I min. The contents were transferred to conicalcentrifuge tubes. The tubes were centrifuged at 600 X g for 10 min at room tem­perature. The aqueous phase and protein formed at the interface were removed bysuction. Anhydrous Na2S04 (0.5 g) was added to each of the methylene chlorideextracts. The methylene chloride extracts were read in a Beckman spectrophotometerat 241 m,U.

Two controls and one blank mixture were carried out simultaneously. Onecontrol system was a tissue blank in which 0.03 ml of methanol was used in place ofthe steroid solution. The second control system was used to assay for the non­enzymic isomerization in buffer alone in which I ml of crystalline bovine albumin(5 mg) was substituted for the enzyme preparation. A third blank containedmethanol and bovine albumin solution in place of the steroid solution and enzymepreparation, respectively. This sample was used as blank for the spectroscopicreadings. The readings of the enzymic reaction samples were corrected by sub­tracting those obtained from the tissue blanks and non-enzymic isomerization. Themolar absorbancy index for progesterone at 241 mt' was taken as 17000 (ref. 10).

Procedure for tissue fractionationHuman term placenta was obtained immediately upon delivery and perfused

with 0.9% saline solution. All subsequent steps were performed at o-z". The cotely­dons were dissected away from vessels and connective tissues and cut into smallpieces. So g of tissue was homogenized with ISO ml of a.z5 M sucrose-O.OI M phos­phate buffer (pH 7.5) in a Waring Blendor at full speed for 30 sec and filtered throughcheese-cloth. The filtrate was centrifuged at 600 X g for IS min and the pelletwas discarded. The supernatant solution was centrifuged at 7800 X g for I h. Thepellet was resuspended in 75 ml of 0.z5 M sucrose-O.OI M phosphate buffer (pH 7.5)in a Teflon homogenizer. The supernatant solution was centrifuged at IOS 000 X g forI h. The pellet was resuspended in 75 ml of 0.25 M sucrose-O.OI M phosphate buffer(pH 7.5) in a Teflon homogenizer. The supernatant solution was designated as thesoluble fraction. The centrifugation was repeated with both resuspended solutions.The pellets obtained after the second centrifugation at 7800 X g for I h were resus­pended in zS ml of 0.25 M sucrose-O.OI M phosphate buffer (pH 7.5), and weredesignated as the mitochondrial and microsomal fractions, respectively.

RESULTS

The specific activities of the 3-p-hydroxysteroid dehydrogenase in the sub­cellular fractions of human term placenta with various 3-p-hydroxysteroids areshown in Table 1. The greatest activity resided in the particulate fractions and wasobserved with s-p-androstane-3-p-ol-I7-one as substrate. The least activity was ob-

Biochim, Biophys. Acta, 105 (1965) II5-120

u8

TABLE I

S. S. KOIDE, M. T . TORRES

SPECIFIC ACTIVITIES OF 3-j3-HYDROXYSTEROID DEHYDROGENASE IN THE SUBCELLULAR FRACTIONSOF HUMAN TERM PLACENTA WITH VARIOUS 3-~-HYDRaXYSTEROIDS

The final pH of the assay system was IO.a.

Substrate Specif ic actiuity (mpt12oleslminlmg protein)

S oluble f racti on Mitochondrialfraction

Microsoma l fraction

- - - - - _.NA D NA DP HA D NA DP NAD NA DP

5-f3-Andrcst ane-g-d-ol-I7-a n e 0 ·3 0 1.6 1.4 1.7 1.4.5-a-Andrastane-3-,8-o1-I 7- a11C 0 .1 0 0 ·5 0·5 o.o 0·3Dehydroepiandrosterone 0 .1 0 0.1 0·3 0 0· 35(10) -Estrene-j-ji-o l- I 7-one a 0 0 0 0.1 0

tained with 5{IO)-estrene -3-tJ-ol-17-one, In general the NAD-linked activity wasgre ater than the NADP-linked activity. No NADP-linked activity was observedin the soluble fraction with any of the substrates tested.

The specific activity of the 3-.B-hydroxysteroid dehydrogenase in the sub­cellular fractions with s -p-androstane-3-tJ-ol-17-one at various pH values are shownin Table II. The optimum pH appeared t o be in the vicinity of 10-10.5 . Glycine­NaOH buffers were used in this study. Somewhat lower act ivit ies were obtainedwith pyrophosphate-sulfate or pyrophosphate-H'Cl buffers. In a control study t odetermine non-enzymic dehydrogenation, cry stalline bovine albumin was used inplace ofthe enzyme preparations at various pH values, Non-enzymic dehydrogenationdid not occur at any of the pH values tested.

The mitochondrial and microsomal fr actions were incubated at 37° for I hwith snake venom (Crotalus adamanieus} , ribonuclease, lipase, trypsin, deoxy­cholat e, taurocholat e, cholate or subjected to sonic oscillation at 20 kcycles for 5 min

T ABLE II

SPECIFIC ACTIVITIES OF 3-,8-HYDROXYSTEROID DEHYDROGENASE IN THE SUBCELLULAR l'RACTIONSOF HUMAN TERM PLACENTA AT VARIOUS pH VALUES

The method is described in the text. The concentration of s-f3-androstane-3-f3-o1-17-0nC was3.3 ' 10-6 M. Glycine-NaOH buffers at the designated pH v alu es were used in the assay sy st em ,The pH values were checked after com p let ion of the assay.

pH Specific activity (mWrtOleslminlmg prote-in)

Soluble fraction Mitochon dri al 1\1icrosomalf raction fraction

NA D NAD? NA D N ADP NAD NA DP

g .o a 0 0 0 0 · 4 0.29 ·5 0.1 a 1.0 La 1. 1 1.2

10. 0 0·3 0 1.9 log 1.5 1.S10·5 0 ·3 0 3,9 1,5 2·5 1.7II .O 0 a 0 0 0· 4 0

Biochi m: B iophys , A cta. l0S (1965) II5-120

HYDROXYSTEROID DEHYDROGENASE AND ISOMERASE IIg

to solubilize the 3-~-hydroxysteroiddehydrogenase activity. These methods failedto yield any soluble material after centrifugation at lOS 000 X g for I h.

The results of the isomerizing activity in the subcellular fractions of humanterm placenta with 5-androstene-3,17-dione as assayed according to Procedure I areshown in Table III. The highest specific activity was found with the particulate

TABLE IIILl5_3-0XOSTEROID ISOMERASE ACTIVITY IN THE SUBCELLULAR FRACTIONS OF HUMAN TERM PLACENTA

WITH 5-ANDROSTENE-3,I7-DIONE

The assay was performed according to Procedure I as described in the text. The concentrationof y-androstene-g.f y-dione was 5' 10-5 M. The results are averages of 8 separate experiments.Thc values in parentheses are the range of the determinations.

Fractions

SolubleMitochondrialMicrosomal

Specific activity(m,tmoleslminimg protein)

2.0 (0.5-3.6)

33 (28-7 1)

32 (23-75)

fractions and lowest in the soluble fraction. No reaction was observed when S(lO)­estrene-rv-ri-ol-j-one was assayed with any of the subcellular fractions.

The results of the ,15-3-oxosteroid isomerase activity with 5-pregnene-3,20­dione and s-androstene-3,17-dione as substrate in the various subcellular fractionsof human term placenta as assayed according to Procedure 2 are presented inTable IV. The greatest specific activity resided in the particulate fractions. Theresults of the determinations with fresh preparations of the fractions obtainedwithin 48-72 h after delivery are presented in Table IV. Because of the variations inactivities with different preparations the results of a representative experiment arepresented with the ranges of activities enclosed in parentheses. The ratios of theisomerase activities of s-androstene-3,I7-dione to s-pregnene-3,20-dione are shownin Table IV. They ranged from 4-4 to 6.S. It was observed that the isomerase activitieswere labile when kept at 0-4°. In a representative experiment the isomerase activitiesof the soluble, mitochondrial and microsomal fractions with s-pregnene-3,2o-dioneas substrate were I.2, 13.8 and II.7 mzzmoles per 10 min per mg protein, respectively,

TABLE IVLl5_3-OXOSTEROJD ISOMERASE ACTIVITY WITH 5-PREGNENE-3,20-DlONE AND 5-ANDROSTENE-3,I7­

DIONE IN THE SUBCELLULAR FRACTIONS OF HUMAN TERM PLACENTA

The assay was performed according to Procedure 2 in the text. The results of a representativeexperiment is presented. The values in parentheses are ranges of 4 separate experiments. Theassay systems with 5-pregnene-3,20-dione contained ro, 4-4, and 3.4 mg of protein in the soluble,mitochondrial and microsomal fractions, respectively. The assay systems for 5-androstene-3,17­clione contained 2, 0.9 and 0.7 mg protein for the respective fractions.

Specific activity (m,umoleslIO minlmg protein)Fractions

SolubleMitochondrialMicrosomal

with 5-pregnene-3,2o­diane (P)

r.z (0.9-2.1)

14 (8-15)

12 (ro-14)

with 5-androstene-3,I7­dione (A)

7-9 (S·6-7·9)61 (30-75)7 2 (40 - 7 3)

RatioAlP

6·S4·46.0

Biochim, Biophys. Acta, 105 (196S) IIS-120

120 S. S. KOIDE, M. T. TORRES

which decreased to 0.1, 5.8 and 7.1 m,umoles per 10 min per mg protein after 3 days.The activities of these fractions with s-androstene-3,I7-dione decreased from 9.9,61 and 72 mjzmoles per 10 min per mg protein to 7.6,40 and 9.1 m,umoles per 10 minper mg protein, respectively.

DISCUSSION

The results of this study suggest that the conversion of Ll5-3-,B-hydroxysteroidsto the ,d4-3-oxosteroids is mediated by a 3-,B-hydroxysteroid dehydrogenase andisomerase. This finding is in accord with that observed with homogenates frombovine adrenal glands and many rat organs 2-4. The dehydrogenating and isomerizingactivities were found in all subcellular fractions assayed. Similar distribution wasreported in beef adrenal tissue with the greatest concentration in the particulatefraction 2,3. It is noteworthy that the optimum pH for the 3-,B-dehydrogenatingactivity was in the vicinity of pH 10-10.5. The isomerizing activity was assayed atpH 7.0. At this pH no dehydrogenating activity could be detected in any of thefractions. Whether the dehydrogenating and isomerizing activities are due to separateenzymes or at different sites of the same enzyme was not established in this study.This point can only be clarified by further purification of the activities.

The L15-3-oxosteroid isomerases in bovine adrenal glands were reported to besubstrate-specificw-" In this study the isomerase activities with 5-androstene-3,I7­dione and s-pregnene-3,2o-dione were greatest in the particulate fraction. Theratios of the isomerase activities in the fresh preparation of subcellular fractionsvaried slightly. However, with aged preparations the activities declined and theirratios varied more widely. This finding is similar to the microsomal Ll4-3-oxosteroidhydrogenases ofrat liver reported by MCGUIRE AND TOMKINSll. Whether the placentalisomerase for the two substrates are distinct and separate can only be clarified by theseparation and purification of the isomerase(s).

ACKNOWLEDGEMENT

The author is grateful to Dr. R. W. RAWSON for his interest and encourage­ment in this project, and to Mrs. J. STIEFEL for technical assistance. This investiga­tion was supported by a U.S. Public Health Service Research Career ProgramAward No. I-K3-AM-5517-01 from The National Institute of Arthritis and MetabolicDiseases, and Grant C-3809 from the National Cancer Institute, U.S. Public HealthService.

REFERENCES

I K. F. BEYER AND L. T. SAMUELS, ]. Bioi. Chem., 219 (1956) 69.2 W. EWALD, H. WERBIN AND I. L. CHAIKOFF, Steroids, 3 (1964) 505.3 H. L. KR0sKEMPER, E. FORCHIELLI AND H. G. RINGOLD, Steroids, 3 (1964) 295.4 W. EWAI-D, H. WER13IN AND I. L. CHAIKOFF, Biochsm. Biophys, Acta, 81 (1964) I99.5 C. DJERASSI, R. R. ENGLE AND A. BOWERS, j. Org: Chem., 21 (1956) 1547·6 W. R. NES, E. LOESER, R. KIRDANI AND J. MARSH, Tet-rahedron, (1963) 299.7 a.H. LOWRY, N. J.ROSEBROUGH,A.L. FARR AND R. J. RANDALL, j. Bioi. Chem., 193 (1951) 265.8 B. L. HORECKER AND A. KORNBERG, I. Bioi. cie«; 175 (1948) 385.9 F. S. KAWAHARA, S. WANG AND P. TALAI-AY, j. Bioi. Chem., 237 (1961) 1500.

10 L. DORFMAN, Chem. Rev., 53 (19.'i3) 47.II J. S. McGUIRE AND G. M. TOMKINS, Arch. Biochim, Biopiiys., 82 (1959) 476.

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