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Formation of steroid sulphates by extracts of human adrenals
PRELIMINARY NOTES
243
PX 1236
Formation of steroid sulphates by extracts of human adrcn~|s E n z y m e s which can con\'ert ster~ficts t . their water-soluble sulphate esters h a v e to d a t e been flmnd only in m a m m a l i a n lix-erL Conjugation of steroids to form the g ] u c . ronides also takes place in the ll,.cr but the kidney of t h e mouse has recently been shown to be capable of forming glucur, mides with certain steroids 2. By contrast. k i d n e y does not contain steroid sulph.kinase~", a. Results described below shiny t h a t cell-free e x t r a c t s of l m m a n adrenals form steroid sulphates from adenosine 3"-phosp h a t e 5 ' - p h o s p h o s u l p h a t e and ".,str~gens a n d androgens but not with the cortic~,-steroids tested u n d e r the e x p . r i m " n t a l c~mditions employed. Fresh h u m a n female a d r e n a l tissue was o b t a i n e d fronl the o w r a t i n g t h e a t r e and i m m e d i a t e l y homog:mised in the cold with 2 vol. of o.oi M phosphate in normal saline (pH 7.4). T h e homc.g~,nate was then centrifuged at IOOOOO ~ for I h and t h e supern a t a n t stored at - - 4 o~ until used. Steroid sulphokinase a c t i v i t y was retained fi~r at least 2 m o n t h s at this temperatur~.
0
---.,
2~
Origin ~ ~ HUT:\ NOL - AMMONIA
Fig. i. Two-dimensional chromatogram of an aliquot of an incubation mixture comprising: 0.03 mt of 0.2 M phosphate buffer (pH 7.o), adenosine 3'-phosphate 3'-phospho-~SS:sulphate (Iooooo c o u n t s / m i n ) in o.o~ ml, and 0.05 ml high-speed human adrenal extract. T h e area outlined indicates the position of a new radioactive zone formed when a steroid (0.05 ymole) was added to t h e tube as an alcoholic solution and the solvent removed prior to addition of the other components. T h e t i m e of incubation was 1.5 h at 37 °. Autoradiographs were developed after a pe,riod o~ -" w e e k s in contact with Kodak Blue-Brand X-ray film.
Fig. I d e m o n s t r a t e s t h e c o m p l e x p a t t e r n of ester sulphates formed b v sulphokinases a c t i n g on endogenous acceptors present in t h e extract. W h e n i n c u b a t e d in t h e presence of a d d e d steroid, a new zone a p p e a r i n g ill t h e Vo.~itio~J outgi~ed wa_. Biochim. Biophys, Acta, 7 x (i903) --43-243
244
PRELIMINARY
NOTES
apparent with estrone, dehydroepiandrosterone, androsterone, pregnan-3a, eoa-diol, testosterone and aetiocholan-3a-ol-17-one but not with cortisone, hydrocortisone or aldosterone. The position outlined in Fig. I is the one to which most authentic steroid sulphates, and the 3sS-labelled steroid sulphates formed by extracts of rat liver and exogenous steroids, migrate in the solvent system used 4. When sulphation of exogenous steroids occurred by the adrenal extracts, some of the ester sulphates in Fig. I were either considerably reduced or completely missing. In the case of dehydroepiandrosterone, proof that the corresponding 3-sulphate ester was formed is shown in Table I. The specific activity of the enzymically synthesised species eluted from two-dimensional paper chromatogtams and mixed with authentic m0terial was constant after three recrystallisations. This procedure could not be applied to estrone sulphate due to its marked instability in hot organic solvents~; indeed complete rupture of the ester sulphate group occurred on attempted recrystallisation of the potassium salt from hot isopropanol. However, when the enzymically synthesised [s~S~sulphate esters, formed from dehydroepiandrosterone and estrone, were isolated from chromatograms and co-chromatographed in two dimensions with potassium dehydroepiandrosterone-3-sulphate and potassium estrone-3-sulphate, respectively, the zones revealed by autoradiography were "fingerprints" of zones demonstrated by chemical staining with Zimmerman reagent. Furthermore, in these two cases the enzymically synthesised steroid sulphates had identical RF values in three different solvent systems (designed for separating steroid sulphates ~) to the authentic substances. TABLE 1 SPECIFIC ACTIVITY OF DEHYDROEPIANDROSTERONE ~SS~SULPHATE ISOLATED FROM T~A'O-DIMENSIONAL CHROMATOGRAMS AND MIXED WITH A U T H E N T I C MATERIAL
The incubation in this case contained 1.5 itrnoles ATP, 3 pmoles MgCIv 20 pmoles Tris buffer (pH 8.3), 4° pC carrier-free Naz35SO4, o.1 pmole dehydroepiandrosterone and o.I 7 ml adrenal extract in a total volume of 0.35 ml, Chromatography as in Fig. i and after locating the steroid ~S!sulphate by autoradiography it was eluted from the paper with water. No. of cryslallisations froat water
I
2 3
Counts/rain at infinite thickness
IO0 x6 400 I8 2OO I8
It is apparent then that the cell-free adrenal extracts can sulphate 3a- and 3flhydroxyl groups and also secondary ITa-hydroxyl groups (testosterone) but not tert. ~-hydroxyl (cortisone) nor Ilfl-hydroxyl groups (hydrocortisone and aldosterone). These results closely parallel those described for the coniugation Gf steroids by soluble enzymes of rabbit liver ~. It should be emphasised, however, that corticosteroids normally undergo transformation in the liver to the "tetrahydro" derivatives (of Ring A) followed by subsequent conjugation. Such transformations evidently do not occur in unfortified, cell-free extracts of liveff. In the vicinity of the area outlined in Fig. I, a number of weak zones are present Biod~.~m. Biophys. Acta, 71 (x963) 243-245
PRELIMINARY .NOTES
245
which are p r o b a b l y formed by sulphation of endogenous steroids. P h e n y l sulphates, a n d o t h e r sutphates formed from sulphate acceptors of low molecular weigh~; &~ n*;t m i g r a t e into this region on chromatography~. T h e presence of steroid ~,flphokinases in a tissu~ responsible for steroid biosynthesis poses the question as to t h d r biological significance. Conjugates m a y be the t r a n s p o r t form of steroids, or p o s s M y because of an increased polarity, this form of the steroid m a y p l a y a specific, ; r i d as yet u n k n o w n role. in their m o d e of action. T h e a u t h o r w;shes to t h a n k Dr. E. GOULSTO.X a n d Dr. R. MELVlLLI~. for their generous help in p r o v i d i n g surgica.t m a t e r i a l and to Dr. K. STARR for his encouragem e n t a n d support.
New South IVales State Cancer Council. Special Unit, Prince of Wales Hospital,
J . B . ADA.XIS
t A. B. RoY, ::,h,e.~:. Enzrmol., 2 z (196o) 205. 2 W. STEPHENS, D. L. BERLINGERANDT. F. |)OUGHERTY,Endocrinol., 6~ (:961) '573. a y. Nose A,'~DF. L[P.~IAN.'%J. Biol. Chem., 233 (1958) 1348. * H. BOSTR6M,Acta Endocrinol., 37 (1961) 405 • b A. BUTENANOTAND H. HOFSTETTER,Z. Physiol. Chem., 259 (1939) 222. J. SCttI~E!D~RAND M. LEWBART,J. Biol. Chem., 2z2 (1956) 787 . 7 D. BERLINER AND T. F. DOUGHERTY,Pharmacol. Rev., 13 (196I) 320. R e c e i v e d J a n u a r y 281h, I963
BiochDn. Biophys..-lcla, 71 (1903) 243-245
243
PX 1236
Formation of steroid sulphates by extracts of human adrcn~|s E n z y m e s which can con\'ert ster~ficts t . their water-soluble sulphate esters h a v e to d a t e been flmnd only in m a m m a l i a n lix-erL Conjugation of steroids to form the g ] u c . ronides also takes place in the ll,.cr but the kidney of t h e mouse has recently been shown to be capable of forming glucur, mides with certain steroids 2. By contrast. k i d n e y does not contain steroid sulph.kinase~", a. Results described below shiny t h a t cell-free e x t r a c t s of l m m a n adrenals form steroid sulphates from adenosine 3"-phosp h a t e 5 ' - p h o s p h o s u l p h a t e and ".,str~gens a n d androgens but not with the cortic~,-steroids tested u n d e r the e x p . r i m " n t a l c~mditions employed. Fresh h u m a n female a d r e n a l tissue was o b t a i n e d fronl the o w r a t i n g t h e a t r e and i m m e d i a t e l y homog:mised in the cold with 2 vol. of o.oi M phosphate in normal saline (pH 7.4). T h e homc.g~,nate was then centrifuged at IOOOOO ~ for I h and t h e supern a t a n t stored at - - 4 o~ until used. Steroid sulphokinase a c t i v i t y was retained fi~r at least 2 m o n t h s at this temperatur~.
0
---.,
2~
Origin ~ ~ HUT:\ NOL - AMMONIA
Fig. i. Two-dimensional chromatogram of an aliquot of an incubation mixture comprising: 0.03 mt of 0.2 M phosphate buffer (pH 7.o), adenosine 3'-phosphate 3'-phospho-~SS:sulphate (Iooooo c o u n t s / m i n ) in o.o~ ml, and 0.05 ml high-speed human adrenal extract. T h e area outlined indicates the position of a new radioactive zone formed when a steroid (0.05 ymole) was added to t h e tube as an alcoholic solution and the solvent removed prior to addition of the other components. T h e t i m e of incubation was 1.5 h at 37 °. Autoradiographs were developed after a pe,riod o~ -" w e e k s in contact with Kodak Blue-Brand X-ray film.
Fig. I d e m o n s t r a t e s t h e c o m p l e x p a t t e r n of ester sulphates formed b v sulphokinases a c t i n g on endogenous acceptors present in t h e extract. W h e n i n c u b a t e d in t h e presence of a d d e d steroid, a new zone a p p e a r i n g ill t h e Vo.~itio~J outgi~ed wa_. Biochim. Biophys, Acta, 7 x (i903) --43-243
244
PRELIMINARY
NOTES
apparent with estrone, dehydroepiandrosterone, androsterone, pregnan-3a, eoa-diol, testosterone and aetiocholan-3a-ol-17-one but not with cortisone, hydrocortisone or aldosterone. The position outlined in Fig. I is the one to which most authentic steroid sulphates, and the 3sS-labelled steroid sulphates formed by extracts of rat liver and exogenous steroids, migrate in the solvent system used 4. When sulphation of exogenous steroids occurred by the adrenal extracts, some of the ester sulphates in Fig. I were either considerably reduced or completely missing. In the case of dehydroepiandrosterone, proof that the corresponding 3-sulphate ester was formed is shown in Table I. The specific activity of the enzymically synthesised species eluted from two-dimensional paper chromatogtams and mixed with authentic m0terial was constant after three recrystallisations. This procedure could not be applied to estrone sulphate due to its marked instability in hot organic solvents~; indeed complete rupture of the ester sulphate group occurred on attempted recrystallisation of the potassium salt from hot isopropanol. However, when the enzymically synthesised [s~S~sulphate esters, formed from dehydroepiandrosterone and estrone, were isolated from chromatograms and co-chromatographed in two dimensions with potassium dehydroepiandrosterone-3-sulphate and potassium estrone-3-sulphate, respectively, the zones revealed by autoradiography were "fingerprints" of zones demonstrated by chemical staining with Zimmerman reagent. Furthermore, in these two cases the enzymically synthesised steroid sulphates had identical RF values in three different solvent systems (designed for separating steroid sulphates ~) to the authentic substances. TABLE 1 SPECIFIC ACTIVITY OF DEHYDROEPIANDROSTERONE ~SS~SULPHATE ISOLATED FROM T~A'O-DIMENSIONAL CHROMATOGRAMS AND MIXED WITH A U T H E N T I C MATERIAL
The incubation in this case contained 1.5 itrnoles ATP, 3 pmoles MgCIv 20 pmoles Tris buffer (pH 8.3), 4° pC carrier-free Naz35SO4, o.1 pmole dehydroepiandrosterone and o.I 7 ml adrenal extract in a total volume of 0.35 ml, Chromatography as in Fig. i and after locating the steroid ~S!sulphate by autoradiography it was eluted from the paper with water. No. of cryslallisations froat water
I
2 3
Counts/rain at infinite thickness
IO0 x6 400 I8 2OO I8
It is apparent then that the cell-free adrenal extracts can sulphate 3a- and 3flhydroxyl groups and also secondary ITa-hydroxyl groups (testosterone) but not tert. ~-hydroxyl (cortisone) nor Ilfl-hydroxyl groups (hydrocortisone and aldosterone). These results closely parallel those described for the coniugation Gf steroids by soluble enzymes of rabbit liver ~. It should be emphasised, however, that corticosteroids normally undergo transformation in the liver to the "tetrahydro" derivatives (of Ring A) followed by subsequent conjugation. Such transformations evidently do not occur in unfortified, cell-free extracts of liveff. In the vicinity of the area outlined in Fig. I, a number of weak zones are present Biod~.~m. Biophys. Acta, 71 (x963) 243-245
PRELIMINARY .NOTES
245
which are p r o b a b l y formed by sulphation of endogenous steroids. P h e n y l sulphates, a n d o t h e r sutphates formed from sulphate acceptors of low molecular weigh~; &~ n*;t m i g r a t e into this region on chromatography~. T h e presence of steroid ~,flphokinases in a tissu~ responsible for steroid biosynthesis poses the question as to t h d r biological significance. Conjugates m a y be the t r a n s p o r t form of steroids, or p o s s M y because of an increased polarity, this form of the steroid m a y p l a y a specific, ; r i d as yet u n k n o w n role. in their m o d e of action. T h e a u t h o r w;shes to t h a n k Dr. E. GOULSTO.X a n d Dr. R. MELVlLLI~. for their generous help in p r o v i d i n g surgica.t m a t e r i a l and to Dr. K. STARR for his encouragem e n t a n d support.
New South IVales State Cancer Council. Special Unit, Prince of Wales Hospital,
J . B . ADA.XIS
t A. B. RoY, ::,h,e.~:. Enzrmol., 2 z (196o) 205. 2 W. STEPHENS, D. L. BERLINGERANDT. F. |)OUGHERTY,Endocrinol., 6~ (:961) '573. a y. Nose A,'~DF. L[P.~IAN.'%J. Biol. Chem., 233 (1958) 1348. * H. BOSTR6M,Acta Endocrinol., 37 (1961) 405 • b A. BUTENANOTAND H. HOFSTETTER,Z. Physiol. Chem., 259 (1939) 222. J. SCttI~E!D~RAND M. LEWBART,J. Biol. Chem., 2z2 (1956) 787 . 7 D. BERLINER AND T. F. DOUGHERTY,Pharmacol. Rev., 13 (196I) 320. R e c e i v e d J a n u a r y 281h, I963
BiochDn. Biophys..-lcla, 71 (1903) 243-245