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Dehydroepiandrosterone sulfatase in the prostate and seminal vesicles of the rat

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148 nnA 55763 BIOCHIMICA ET BIOPHYSICA ACTA DEHYDROEPIANDROSTERONE SULFATASE” IN THE PROSTATE AND SEMINAL VESICLES OF THE RAT WILLIAM GILL AND CHIADAO CHEN Department of Surgery (Urology), University of Chicago, Department of Biochemistry, Northwestern University, Chicago, Ill. (U.S.A.) (Received May 25th, 1970) SUMMARY I. The object of this investigation was to determine the presence or absence of dehydroepiandrosterone sulfatase in the accessory sex glands of the male rat. This search was prompted by the known influence of the adrenal cortex on the prostate gland and the demonstrations of the secretion of dehydroepiandrosterone sulfate by the adrenal gland. 2. Dehydroepiandrosterone sulfatase activity was demonstrated in the rat prostate gland and seminal vesicles. The tissue specificity of dehydroepiandrosterone sulfatase in this investigation was confined primarily to the prostate and the seminal vesicles. The epididymal fat pad could not effect this enzymatic hydrolysis and muscle (thigh and abdominal wall) had lower levels of activity. The substrate specificity for steroid sulfates was confined to dehydroepiandrosterone sulfate, pregnenolone sulfate, and estrone sulfate. Androsterone sulfate and testosterone sulfate were not hydrolyzed. The apparent K, for dehydroepiandrosterone sulfate in the seminal vesicles at 5% tissue concentration was found to be 2.1.10-s M. 3. In view of the tissue and substrate specificity of the prostate and seminal vesicle sulfatases for dehydroepiandrosterone sulfate, it would seem reasonable to suggest that at least part of the hormonal influence of the adrenal gland on the male accessory sex glands might be by way of dehydroepiandrosterone sulfate which can be hydrolyzed in these target tissues to free dehydroepiandrosterone, a known an- drogenic compound. INTRODUCTION The hormonal influence of the adrenal cortex on the prostate gland has been * The following abbreviations and trivial names are used: dehpdroepiandrosterone sulfatase, or 17-oxo-androst-5-en-3B_yl sulfate sulfatase, for sterol sulfate sulfohydrolase (EC 3.r.6.2) ; dehy- droepiandrosterone, for 3p-hydroxyandrost-5-en-r7-one; dehydroepiandrosterone sulfate, for r7- oxo-androst-5-en-3/%yl sulfate; androsterone sulfate, for r7-oxo-gwandrostan-3cr-yl sulfate; preg- nenolone sulfate, for zo-oxo-pregn-5-en-3P-yl sulfate; testosterone sulfate, for 3-oxo-androst-q-en- 17@-yl sulfate; estrone sulfate, for r7-oxo-cstra-r,3,5(ro)-trien-3-y1 sulfate. Hiochinz. Bio/+hys. Arta, 218 (1970) 148~154
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Page 1: Dehydroepiandrosterone sulfatase in the prostate and seminal vesicles of the rat

148

nnA 55763

BIOCHIMICA ET BIOPHYSICA ACTA

DEHYDROEPIANDROSTERONE SULFATASE”

IN THE PROSTATE AND SEMINAL VESICLES OF THE RAT

WILLIAM GILL AND CHIADAO CHEN

Department of Surgery (Urology), University of Chicago, Department of Biochemistry, Northwestern University, Chicago, Ill. (U.S.A.)

(Received May 25th, 1970)

SUMMARY

I. The object of this investigation was to determine the presence or absence of dehydroepiandrosterone sulfatase in the accessory sex glands of the male rat. This search was prompted by the known influence of the adrenal cortex on the prostate gland and the demonstrations of the secretion of dehydroepiandrosterone sulfate by the adrenal gland.

2. Dehydroepiandrosterone sulfatase activity was demonstrated in the rat prostate gland and seminal vesicles. The tissue specificity of dehydroepiandrosterone sulfatase in this investigation was confined primarily to the prostate and the seminal vesicles. The epididymal fat pad could not effect this enzymatic hydrolysis and muscle (thigh and abdominal wall) had lower levels of activity. The substrate specificity for steroid sulfates was confined to dehydroepiandrosterone sulfate, pregnenolone sulfate, and estrone sulfate. Androsterone sulfate and testosterone sulfate were not hydrolyzed. The apparent K, for dehydroepiandrosterone sulfate in the seminal vesicles at 5% tissue concentration was found to be 2.1.10-s M.

3. In view of the tissue and substrate specificity of the prostate and seminal vesicle sulfatases for dehydroepiandrosterone sulfate, it would seem reasonable to suggest that at least part of the hormonal influence of the adrenal gland on the male accessory sex glands might be by way of dehydroepiandrosterone sulfate which can be hydrolyzed in these target tissues to free dehydroepiandrosterone, a known an- drogenic compound.

INTRODUCTION

The hormonal influence of the adrenal cortex on the prostate gland has been

* The following abbreviations and trivial names are used: dehpdroepiandrosterone sulfatase, or 17-oxo-androst-5-en-3B_yl sulfate sulfatase, for sterol sulfate sulfohydrolase (EC 3.r.6.2) ; dehy- droepiandrosterone, for 3p-hydroxyandrost-5-en-r7-one; dehydroepiandrosterone sulfate, for r7- oxo-androst-5-en-3/%yl sulfate; androsterone sulfate, for r7-oxo-gwandrostan-3cr-yl sulfate; preg- nenolone sulfate, for zo-oxo-pregn-5-en-3P-yl sulfate; testosterone sulfate, for 3-oxo-androst-q-en- 17@-yl sulfate; estrone sulfate, for r7-oxo-cstra-r,3,5(ro)-trien-3-y1 sulfate.

Hiochinz. Bio/+hys. Arta, 218 (1970) 148~154

Page 2: Dehydroepiandrosterone sulfatase in the prostate and seminal vesicles of the rat

PROSTATIC DEHYDROEPIANDROSTERONE SULFATASE I49

established by several types of studies. BURRILL AND GREENE’ found that the ventral

prostate developed in immature, castrate rats but not in adrenalectomized, immature,

castrate rats. TULLNER~ described a z-fold increase in the weight of the ventral

prostate following ACTH administration to immature, castrate rats, but ACTH had

no effect on the ventral prostate of adrenalectomized, immature, castrate rats. PRICE

AND INGLI? demonstrated local stimulation of the prostate by adrenal autotransplan-

tation into the ventral prostate of adult castrate rats.

The hormones mediating the influence of the adrenal cortex on the prostate

have not been established. Corticosterone, cortisone and hydrocortisone were without

effect on the weight of the ventral prostates in hypophysectomized, castrated adult

rat+. GRAYHACK et al.5 also found cortisone ineffective on the weight of ventral

prostates in hypophysectomized castrates.

Dehydroepiandrosterone sulfates has recently been found to be a major secre-

tory product of the adrenal cortex. The level of dehydroepiandrosterone sulfate in the

adrenal vein is significantly higher than in the peripheral venous blood7, which indi-

cates an adrenal secretion rather than an exclusive liver origin. In adrenal cortical

tumor9, dehydroepiandrosterone sulfate has been found in significant quantities,

whereas, little or no free dehydroepiandrosterone was present. Adrenal cortical homo-

genates can synthesize dehydroepiandrosterone sulfate from free dehydroepian-

drosteroneO, as well as from cholesterol sulfate and pregnenolone sulfatelo.

The foregoing literature indicates that the adrenal cortex secretes dehydro-

epiandrosterone sulfate and that there is a hormonal influence of the adrenal cortex

on the prostate. The unanswered query than is: does dehydroepiandrosterone sulfate

mediate the hormonal effects of the adrenal cortex on the prostate and other male

accessory sex glands ?

In evaluating the effects of dehydroepiandrosterone sulfate on the presumed

target organs, the male accessory sex glands, we decided to first investigate the possi-

bility that dehydroepiandrosterone sulfate is metabolized by the prostate. Meta-

bolism could occur in one of two general ways: (I) dehydroepiandrosterone sulfate

could be converted to other steroid sulfates, or (2) dehydroepiandrosterone sulfate

could be hydrolyzed to free dehydroepiandrosterone, a known androgenic compoundll

with approx. I/IO the activity of testosterone. This second possibility, hydrolysis of

steroid sulfates to free steroids, was investigated in this paper.

The ability of the rat prostate and seminal vesicles to hydrolyze dehydroepian-

drosterone sulfate as well as estrone sulfate to the corresponding free steroids is re-

ported in this paper. The tissue specificity of the enzymatic hydrolysis was demon-

strated by the failure of the epididymal fat pad to hydrolyze dehydroepiandrosterone

sulfate and the lower levels of activity in thigh and abdominal wall musculature. The

substrate specificity was shown by the absence of hydrolysis of androsterone sulfate,

and testosterone sulfate by the prostate and the seminal vesicles.

METHODS

Preparation of substrates and tissues Steroid sulfate substrates were synthesized by the sulfamic acid method of

JOSEPH et aLla. Purity of the synthesized substrates was determined by: homogeneity

on thin-layer chromatography of silica gel with several solvent systems (chloroform,

Biochim. Biophys. Acta, 218 (1970) 148-154

Page 3: Dehydroepiandrosterone sulfatase in the prostate and seminal vesicles of the rat

150 W. GILL, C. CHEN

methanol, chloroform-methanol (g5:5, by vol.), followed by homogeneity on gas- liquid chromatography of the free steroids following solvolysisr3.

Tissues were prepared from 250-300 g Sprague-Dawley male rats, which were exsanguinated (venously) under nembutal anesthesia. The ventral, dorsolateral, and coagulating lobes of the prostate and the seminal vesicles were separately dissected out. After the seminal vesicles and coagulating glands were removed, the secretions were gently expressed. Epididymal fat pad, thigh muscle, and abdominal wall muscle were also removed for use as controls as presumed non-target tissues. All tissues were immediately placed in 0.25 M sucrose at 4”. After drying by filter paper blotting and recording of wet weights, tissues were homogenized in distilled deionized water {I g/IO ml water) in an all glass Potter-Elvehjem homogenizer.

Incubations

Incubations were carried out in 3o-ml centrifuge tubes at 37’ in a Dubnoff shaking incubator with the following additions: 2.5 ml homogenate, 0.5 ml Tris buffer (pH 7.4, 0.2 M), 2.0 ml water, I .IO-~-I 'IO-" M steroid sulfate in water giving a total volume of 5.2-5.4 ml. Each reaction vessel contained the equivalent of 0.25 g of tissue (wet weight), which represented a 5% tissue concentration.

Extractions and separations

Incubations were terminated by adding 5 vol. (25 ml) of 95q{> ethanol (doubly redistilled). After vigorous shaking and overnight storage at 4’, the precipitated material was separated by centrifugation at 800 xg for IO min. After decantation of the ethanol extract the solid residue was vigorously resuspended in 20 ml carbon dichloride (doubly redistilled) and again centrifuged at 800 xg for IO min. The carbon dichloride extract was aspirated free from the solid phase.

The pooled 95% ethanol and carbon dichloride extracts were evaporated to dryness with a rotary evaporator at 40’. The residues were then redissolved in 8 ml water and 25 ml carbon dichloride and partitioned in separatory funnels. The aqueous residue was extracted a second time with 25 ml carbon dichloride. This partition be- tween an aqueous and a carbon dichloride phase gave a preliminary separation of steroid sulfate substrate from free steroid product. A completely quantitative separa- tion of steroid sulfate from free steroid could not be effected merely by solvent sepa- ration alone. In the presence of buffer ions, some steroid sulfate was always extracted into the organic solvent (regardless of whether the solvent was carbon dichloride, chloroform, ether, benzene or hexane). A thin-layer chromatographic step was there- fore necessary to insure complete separation of steroid sulfate substrates from free steroid products. Gelman ITLC (fiber glass impregnated with silica gel) was used with a pure chloroform solvent system. All steroid sulfates remained at the origin but the free steroids moved with an RF of o.qo-0.98.

Quantitation and identification The free steroid fronts from thin-layer chromatography were eluted and divided

into three aliquots. One part was gas chromatographed straight without derivatiza- tion. The second portion was silylatedl4 with bis-(trimethylsilyl) trifluoroacetamide, and the third was acetylatedls with acetic anhydride-pyridine.

Identification of the free steroids was determined by matching retention time

Bzochiin. Biophys. Acta, 218 (1970) 148-154

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PROSTATIC DEHYDROEPIANDROSTERONE SCLFATASE 151

of the free, acetylated, and silylated forms on three different columns, 5% OV-I, 394 OV-25, 2% QF-I (non selective, non polar to selective, polar liquid phases). Routine quantitation was usually carried out with the t~methylsilyi derivatives on 27;

QF-I and 5% OV-I.

RESULTS

An enzyme system which hydrolyzes dehydro~piandrosterone sulfate, as well as certain other steroid sulfates, has been found in the rat prostate gland and seminal vesicles (Table I). The enzymatic nature of this dehydroepiandrosterone sulfate sul- fatase was established by the failure of boiled tissue controls to effect this hydrolysis, and the linearity of the reaction rate with respect to tissue (enzyme) concentration. Boiled tissue controls @us substrates were always incubated the same length of time as the enzymatic groups to check the absence of non-enz~rmatic hydrolysis in the systems.

TABLE I

SUBSTRATE SPECIFICITY OF STEROID SIJLFATASE(S) IS THE PROSTATE AND SEMIPiAL VESICLES OF THE RAT

Whole tissue homogenates (0.25 g wet tissue weight) were incubated in 5.2 ml (total volume) of 0.05 M Tris buffer (pH 7.4) with 5. row5 M steroid sulfate substrate for 2 h at 37”. The extractions, separations, identifica- tions, and gas chromatographic quantitation of the liberated free steroids were as described in the text. p-Nitro- catechol sulfate hydrolysis was determined by the method of Rorz3. Data represent mean :t_ S.E. (number of determinations). ~~~ -

IO-~~ moles steroid sulfate hydrolyzed peg min per fi tissw

I’entral Dovsolateval Coaeulatine Seminal prostate pvostate g&d - oesicles

_---.- -- _ - ____I -- ._I__ Dehydroepiandrosterone sulfate (38 sul-

fate, C,,) 0.63 i_ 0.03 (8) 1.24 + o.rr (5) 2.38 i o.r4 (4) 3.31 i 0.08 (5) Androsterone sulfate (3~ sulfate, C,,) o (5) o

8 O (3) o

Pregnenolone sulfate (38 sulfate, C,,) 1.05 1.83 3.95 Testosterone sulfate (17j? sulfate, C,,) o 1:; (f) o (4) o Estrone sulfate (3-aromatic sulfate, C,,) 3.82 (2) Z.65 (2) 8.35 (2) 17.10 $-Nitrocatechol sulfate (aromatic

sulfate) 621 i 36 (5)

The substrate specificity for steroid sulfate hydrolysis (Table I) was confined to steroids sulfated at the 3 position of &, C,,, and C,, steroids. Dehydroepian- drosterone sulfate (3/3 sulfate, C,,) pregnenolone sulfate (38 sulfate, C,,), and estrone sulfate (3-aromatic sulfate, C,,) were all hydrolyzed by the prostate and seminal vesicles. The 178 sulfate of testosterone, however, was not attacked by either the prostate or the seminal vesicles. The 3 position specificity for sulfate hydrolysis was confined to the @ (equatorial), or the aromatic configurations. The 3 CE (axial) sulfate of androsterone sulfate was not hydrolyzed.

The tissue specificity of dehydroepiandrosterone sulfatase in selected tissues of the rat is presented in Table II. The sulfatase activities, expressed as 10-l~ moles dehydroepiandrosterone sulfate hydrolyzed per min per g of wet tissue weight, in- creased from the ventral prostate (0.63) through the dorsolateral prostate (1.24) and coagulating gland (2.38) to the highest specific activity, 3.31, which was found in the seminal vesicles. In contrast, other tissues, which were presumably non-target or not major target tissues, had either no detectable activity (epididymal fat pad) or low levels (thigh muscle, 0.21 and abdominal wall muscle, 0.17).

Biochim. Biophys. Acta, 218 (1970) 148-154

Page 5: Dehydroepiandrosterone sulfatase in the prostate and seminal vesicles of the rat

152 W. GILL, C. CHEN

TABLE II

TISSUE SPECIFICITY OF DEHYDROEPIANDROSTERONE SULFATASE Ii,- THE RAT

Whole tissue homogenates (0.25 g wet tissue weight) were incubated in 5.2 ml (total volume) of 0.05 M Tris buffer (pH 7.4) with 5.10~~ M dehydroepiandrosterone sulfate substrate for 2 h at 37’. The analysis of liberated free dehydrocpiandrosterone was as described in the text. Date represent mean * S.E. (number of determinations).

_ __ ~. TiSSUC 10-l~ wzoles dehydroepiandvosterone sulfate

hydvolyzrd per min PEY g tissue _ Ventral prostate 0.63 $_ 0.03 (8) Dorsolateral prostate I.24 3: 0.11 (5) Coagulating gland 2.38 TV 0.14 (4) Seminal vesicles 3.3’ 1 0.08 (5) Epididymal fat pad o (4) Muscle (thigh) 0.21 4: 0.05 (5) Muscle (abdominal wall) 0.1, (2)

A time study of total product (dehydroepiandrosterone) formation with respect to time (Fig. I) was found to be essentially linear with time points at o, 0.5, I, 2,325,

and 5 h. This observed failure of product to level off or plateau with time was not surprising, because incubation conditions were made optimal for hydrolysis. Also the

92 1 2 3 4 5 Time(h)

Fig. I. Ventral prostate dehydroepiandrosterone sulfate sulfatase. Time study of hydrolysis. Whole tissue homogenates (0.25 g met tissue weight) mere incubated in 5.2 ml (total volume) of 0.05 M Tris buffer (pH 7.4) with 5’ IO-~ M dehydroepiandrosterone sulfate at 37”. The determination of the liberated free dehydroepiandrosterone was as described in the text.

Fig. 2. Plot of [Sj iv, V~YSUS [S] (Woolf plot). Effect of substrate concentration on initial rate (uO) of dehydroepiandrosterone sulfate hydrolysis by seminal vesicle homogenates (57; tissue concen- tration). Incubations were carried out in 5 ml of 0.05 M Tris buffer (pH 7.4) at 37” for I h. vO was calculated as 10-l” moles dehydroepiandrosterone sulfate hydrolyzed per g tissue per min.

free dehydroepiandrosterone formed did not inhibit the further change of dehydro- epiandrosterone sulfate. Interconversions of either free steroids or sulfated steroids was not expected in view of the fact that neither NADPH or a NADPH-generating system were added to the incubation system. A steady state of dehydroepiandrosterone + dehydroepiandrosterone sulfate was also not expected, because sulfurylation re- quirements (ATP, Mg2+, sulfate) were not added to the reaction vessels.

DISCUSSION

Sulfatases capable of hydrolyzing steroid sulfates have been extensively studied in the liver, but very few investigations of these enzymes have been made in the testis, and essentially none in the prostate. Mammalian steroid sulfate sulfatase was

Biochzm. Biophys. Acta, 218 (1970) 148-154

Page 6: Dehydroepiandrosterone sulfatase in the prostate and seminal vesicles of the rat

PROSTATIC DEHYDROEPIANDROSTERONE SULFATASE I53

first demonstrated in the liver by GIBIAN AND BRATFISCH’~. Roy’7 subsequently

localized the dehydroepiandrosterone sulfate sulfatase activity in ox liver to the microsomal subcellular fraction. RoyI also found that p-nitrophenyl sulfate was hydrolyzed at rates from 5 to 15 times greater than that for dehydroepiandrosterone sulfate. In our studies with the prostate a similar rate differential was found with an

increase from dehydroepiandrosterone sulfate (3,!?-steroid sulfate) through estrone sulfate (3-aromatic steroid sulfate) to the maximal rate with p-nitrocatechol sulfate

(a single ring aromatic sulfate). Stereochemical specificity was demonstrated by ROY’* for the steroid sulfate

sulfatase in Patella vul,aata which hydrolyzed only the 3-sulfates of the 5a-3fi-hydroxy-

or of the A5-3,!-hydroxy steroids. A similar limitation of activity to the 38 and 3-aro- matic sulfates was found in our studies with the rat prostate and seminal vesicles in that the gee sulfate of androsterone sulfate was not hydrolyzed. Also the 178 sulfate of testosterone was not attacked by either the prostate or seminal vesicles. This lack of testosterone-170 sulfate sulfatase is especially significant in view of the recent

demonstration by SAEZ et aLlo of approx. 0.1 pg/roo ml testosterone sulfate in human peripheral plasma and as high as I pg/Ioo ml in seminal vein plasma.

Testicular steroid sulfatase (rat and guinea pig) was studied in 1963 by BUR- STEIN AND DORFMAN~~ using [3H]dehydroepiandrosterone sulfate and measuring the toluene-extractable radioactivity as a function of time. Their assay was complicated

by the finding of extractable tritium at zero time, which was substrate that was ex- tracted into the toluene in the presence of tissue preparations. Lowering the ionic strength of their aqueous alkali extracts reduced the blank values but did not eliminate them. Similarly in our studies we found that an organic extraction alone did not give complete separation of free from sulfated steroids. We used an additional thin-layer chromatographic separation rather than relying solely on subtraction of o-h blanks.

NOTATION AND UNGAR~~, using rat testis homogenates, reported on the inhibi- tion of cleavage of dehydroepiandrosterone sulfate by dehydroepiandrosterone. Also

pregnenolone sulfate hydrolysis was decreased by pregnenolone in these studies. Pregnenolone sulfate also inhibited dehydroepiandrosterone sulfate hydrolysis. The kinetic data in all cases were compatible with competitive inhibition. No steroid sul- fate substrates other than dehydroepiandrosterone sulfate and pregnenolone sulfate

were used by BURNSTEIN AND DORFMAN~~ and NOTATION AND UNGAR~~, and no other reports on steroid sulfatases in the testis or the prostate are available.

The observations of this study support the hypothesis that dehydroepian- drosterone sulfate (secreted by the adrenal) and other steroid sulfates in the plasma form a reservoir from which the male accessory sex glands can obtain sulfated steroids (presumably hormonally inactive) and hydrolyze them to free steroids with known androgenic activity (dehydroepiandrosterone) or estrogenic activity (estrone). Furthermore, in view of the known influence of the adrenal cortex on the prostate gland and the secretion of dehydroepiandrosterone sulfate by the adrenal cortex, it would seem reasonable to suggest that at least part of the hormonal influence of the adrenal gland on the prostate gland might be by way of dehydroepiandrosterone sulfate, which can be hydrolyzed to free dehydroepiandrosterone, which has andro- genie activity. The possibility also exists that the free dehydroepiandrosterone might be converted to other more androgenic steroids such as the conversion of testosterone

to dihydrotestosterone in the prostatez2.

Biochim. Biophys. Acta, 218 (1970) 118-154

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I.54 W. GILL, C. CHEN

ACKNOWLEDGEMENTS

This work was supported in part by U.S. Public Health Service General Research Support Grant FR 5367 and U.S. Public Health Service NIH Grant No. Ro-I, National Institutes of Health AM 09685 and the generous contributions of Mr. Sydney Stein, Jr.

I M. W. BURRILL AND R. R. GREENE, Pvoc. Sot. Exptl. Biol. Med., 40 (1939) 327. 2 W. TULLXER, Biology of the Prostate and Related Tissues, Monograph No. 12, National Cancer

Institute, Bethesda, Md., 1963, p. 211. 3 D. PRICE ANI) D. J. INGLE, Rev. Suisse Zool., 64 (1957) 743. A A. LOSTROH AXD C. LI. Acta Endocrinol., 25 (1957) I. i J. GRAYHACK, P. BUNCE, J. KEARXS AND W. SCOTT, Bull. JohnsHopkinsHosp.,96(1955) 154. 6 JZ. E. BAULIEU. C. CORPECHOT, F. DRAY, R. EMILIOZZI, M. LEBEAU, P. MAUVAIS-JARVIS ASD

P. ROBEL, Rec.‘Prcgr. Hormone Res., 21 (1965) 411. 7 E. E. BAULIECT. 1. Clin. Endocvinol. Metab., 22 (1962) 501. ii E. E. BAULIEU, 'i-. E~L~ILIOZZI AND C. CORPECHOT,~EXpevientia, I7 (1961) IIO. 9 E. WALLACE AND S. LIEBERMAN, J. Clin. Endocrinol. Metab., 23 (1963) 90.

IO Ii. ROBERTS, L. BANDI, H. CALVIX, W. DRUCKER AND S. LIEBERMAN, J. Am. Chenz..Soc., 86 (‘964) 958.

II L. F. FIESER AND 111. FIESER, Strvoids, Reinhold I’ubl., Xew York, 1959, p. 519. IL T. TOSEPH. 1. DUSZA AND S. BERNSTEIN, Steroids, 7 (1966) 577. 13 ~."HUDSON-AND G. OERTEL, Anal.Biochem.. 2 (1966) 248. 14 K. SJOVAL, J.SJOVALL, K. MADDOCK AND E. HORNING, Anal.Biochem., 14(1966) 337 15 S. CHATTORAJ AND A. SCOMMENGNA, Steroids, 9 (1967) 327. 16 H. GIBIAX AND G. BRATFISCH, Z. Physzol. Chem., 305 (1956) 265. 17 A. ROY, Biochem. J., 66 (‘957) 700. IX A. ROY, Biochrm. ,I., 62 (1956) 41. 19 J, SAEZ, S. SAEZ AND C. MIGEOS, Steroids, 9 (1967) I. LO S. BURXSTEIX AND R. DORPMAN, J. Riol. Chem., 238 (1963) 1656. 21 A. NOTATION AILD F. UNGAR, Can. J, Biochem., 46 (1968) 1185. 22 L. ANDERSON; AND S. LIAO, Naturr,z19(rg68) 277. 23 A. ROY, Biochcm. J., 53 (1953) 12.

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