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Properties of porcine liver and testicular steroid sulphotransferases: Reaction conditions and influence of naturally occurring steroids and steroid sulphates
0022-4731/83 $3.00+0.00 Copyright Q 1983Pergaman Press Ltd
J. steroid Biochem. Vol. 19, No. 2, pp. I103-1109, 1983
Printed in Great Britain. All rights reserved
PROPERTIES OF PORCINE LIVER AND TESTICULAR STEROID SULPHOTRANSFERASES: REACTION CONDITIONS AND INFLUENCE OF NATURALLY OCCURRING STEROIDS AND STEROID SULPHATES G,
Department
M. COOKE*, SUSANE. FERGUSON,E. RYTINA and D. B. GOWER~ of Biochemistry, Guy’s Hospital Medical School, London SE1 9RT, England (Received 18 October 1982)
Summary-Sulphotransfe~se activity has been assayed in porcine liver and testis cytosol using either 3’-phosphoadenosine-5’-phospho [3sS]sulphate (PAPS) or unlabelled PAPS as sulphate donors. In porcine liver the sulphotransferase for DHA was linear for up to lOmin, the optimum pH was 7.7 and optimum temperature, 37°C. The apparent Km value was found to be 91 pmol/l and the activity was inhibited non-competitively by 5a-androst-16en-3P-yl sulphate, with all concentrations used (0.02-25pmol/l) inhibiting the enzyme to the same extent. Time courses for sulphoconjugation of pregnenolone and Sa-androst-16-en-3/I-01 were linear for up to at least 10 min or up to only 5 min, respectively. The optimum pH values and temperatures were pH 8.0 and 37°C in each case. The porcine testicular sulphotransferase activity for DHA as substrate was linear with time up to lOmin, the apparent Km for the reaction was Z~mol/l and apparent V,,, lOnmoI/l/mg~min. Sff-Androst-l4-en-3~-yl sulphate (11.3-45.2 pmoljl) failed to inhibit the enzyme activity. The time-course for the reaction, when pregnenolone was used as substrate, was also linear up to 10 mm at the optimum pH 8.0 but, in contrast to the reaction when DHA was the substrate, had an apparent K,,, of 20ymol/l Sa-androst-16en-3/I-y] sulphate, DHA and and was inhibited by pregnenolone sulphate, Sc+androst-16-en-3/I-ol, but not by DHA sulphate. Sa-Androst-16en-3b-yl sulphate inhibited the reaction non-competitively and to the same extent at concentrations over the range l1.3-45.2~mol/l. These data suggest that DHA and pregnenolone may not be sulphoconjugated by the same sulphotransferase. With Se-androst-16-en-3/I-ol as substrate, the time-course for its sulphate formation was linear up to 15 min, and this reaction could explain the quantities of 5a-androst-16”en-3/3-yl sulphate that are found endogenously in porcine testis. Our results further suggest that these quantities could well inhibit the sulphation of pregnenolone in porcine testis in &JO, and the possibility of control of sulphoconjugation in this tissue is discussed. Having regard to the smaller quantities of Sa-androst-16-en-3fi-yl sulphate present in porcine liver, our results suggest that the suiphation of DHA there may not be so much affected.
INTRODUCTION Steroid sulphates, which are present in large quantities in many mammaIian tissues, are thought to be sources of biolo~cally active steroid hormones, intermediates in steroid biosynthetic pathways and also as detoxification products of hormone metabolism [I].
*Present Address: The Royal Victoria Hospital, The Women’s Pavilion, Department of Obstetrics and Gynecology, 687 Pine Avenue West, Montreal PQ, Canada H3A lA1. tAddress reprint requests to Dr. D. B. Gower. Abbreviations and trivial names: 3fi-hydroxy-5-androsten!7-one, dehydroepiandrosterone (DHA); 38-hydroxy-5pregnen-20-one, pregnenolone: 4-pregnene-3,20-dione, progesterone; 3B-hydroxy-5cl-androstan-17-one, epiandrosterone; 3-hydroxy-1,3,5(10)-oestratrien-17-one, oestrone; 5c(-pregnane-3,20-dione, Sa-pregnanedione; 3’-phosphoadenosine-5’-phosphosulphate, PAPS; 3’ph~sph~adenosine-S-phosphate, PAP. Enzyme names: 17a-hvdroxv-C,,-steroid C-17.20 lvase, C-17,20 lvase; 5-ene~3~-h~dro~ysteroid oxido-reductase (EC I.i.i.5l)t 3-oxosteroid-4,5isomerase (EC 5.3.3. l), 5-ene-3flOHSDH/4,5-isomerase. SB19/2--F
The steroid sulphotransferases of liver cytosol may be involved in the detoxification and excretory processes whereas, in males, the sulphotransferases of the testis can be considered as of biosynthetic importance. Two enzymes were separated from rabbit liver which catalysed the sulphation of DHA and oestrone, and which were distinct from phenol sulphotransferase [2]. In a more recent study, a partially purified 3P-hydroxysteroid sulphotransferase from rat liver was found to accept epiandrosterone, DHA, 5a-pregnane-3&20fi-dial and Sa-androstane3/?,178-diol as substrates [3]. Other 3/L?-hydroxysteroids, including pregnenolone and 5-androstene3&l ?‘/I-dial were not preferred. Fish and coworkers [4] observed both cytosolic and microsomal sulphotransferase activity towards 5a-androst-16-en3/I-01 in subcellular fractions of porcine liver. The microsomal enzyme exhibited an optimum pH of 7.4 and was released from the membranes by sodium dodecyl sulphate. The testis tissue of humans, boars and rats has all been shown to possess steroid suiphotransferase activity [ 1,5,6] but the intratesticular distribution of
1103
G.
1104
CHOLESTEROL + (SO,)
PREGNENOLONE (SO,) 1
-
M.
CWKE et al.
I
PROGESTERONE ( sHo; 17-OH
PROGESTERONE
1 4-ANDROSTENEDIONE It TESTOSTERONE
Fig. 1 Pathways of biosynthesis of 16-androstenes, androgens and steroid sulphates in boar testis. (a) 5,16-androstadien-3/?-ol and its sulphate; (b) 4,16-androstadien-3-one; (c) 5cc-androst-16-en-3-one; (d) 5a-androst-16-en-3a-ol and its sulphate; (e) Sa-androst-16-en-3j-ol and its sulphate; DHA, 3P-hydroxy-Sandrosten-17-one.
these enzymes remains uncertain. The testis contains large quantities of 3fi-hydroxysteroid sulphates [6,7] and, in the boar testis, one of the principal steroid sulphates is Sa-androst- 16-en-3/r’-yl sulphate. Unconjugated 5x-androst- 16-en-3B-ol is also found in large amounts in boar testis [8], but no physiological role has been determined for either the free steroid or its sulphate ester. It is believed that 16-androstene biosynthesis in boars provides two pheromonally active steroids, 5x-androst-16-en-3-one and 5xandrost-16-en-3a-ol[9, lo] and that these are produced from pregnenolone by enzymes distinct from those involved in androgenesis [ll]. The pathway is outlined in Fig. 1. Pregnenolone sulphate can be converted to 5,16-androstadien-3/3-yl sulphate in vitro [12] but this route to 16-androstenes is considered to be less significant than the unconjugated steroid pathway [13]. This may be due to the low activity of boar testicular steroid sulphatase [14] as compared to that of bovine, ovine and human testes. The present investigations were performed to gain further information about the inter-relationships between free and sulphate conjugated steroids in porcine liver and testis and also to examine the role in steroid hormone metabolism of steroid sulphates, especially 501-androst-16-en-3/I-yl sulphate, as this is quantitatively the most important 16-androstene sulphate in boar testis [8]. MATERIALS
AND METHODS
Materials
Reagents and materials were as reported previously [4] except for 3’-phosphoadenosine-5’phospho [3SS]sulphate (2.09 CJmmol) which was purchased from New England Nuclear, Dreieich, W.
Germany and stored at -20°C in ethanol-water (1: 1, v/v) prior to use. Unlabelled PAPS was obtained from Sigma Chemicals Co., Poole, Dorset, U.K. The purity of the former was checked by TLC, followed by radioautography and found to be satisfactory. To minimize decomposition, in some experiments, the lithium salt of unlabelled PAPS was used, which is more stable than the potassium salt. This was stored, desiccated, at -20°C and made up freshly for experimental studies. [4-‘4C]Pregnenolone (56.5 mC,/mmol) and [7n-3H]DHA (23 Ci/mmol) were from the Radiochemical Centre, Amersham, [5c(,6c(-3H]5cr-Androst-16-en-3-one Bucks, U.K. (21 CJmmol) was obtained from ROTOP, Isocommerz, GmbH. Kontor, Dresden, D.D.R. 8051 from which [5c(,6u-3H]5c(-androst-16-en-3,?-ol was prepared by borohydride reduction [4]. Tritium labelled and unlabelled steroid sulphates were prepared as described earlier [4]. Precoated silica gel plates for TLC were purchased from Merck (supplied by Anderman & Co. Ltd., East Molesey, Surrey, U.K.). Scintillation counting was performed using a Packard Tri-carb Liquid Scintillation Spectrometer, Model 3255. An aqueous scintillation mixture was used which consisted of 2,5-diphenyloxazole (5g) and 1,4&s 2-(5-phenyloxazolyl) benzene (0.5 g) both dissolved in a mixture (2 : 1, v/v) of toluene-Triton X-100 [ll]. Counting efficiencies for 35S, 3H and 14C were 98, 55 and 88% respectively. METHODS
Preparation of tissues
Porcine liver and testis were obtained from abbatoirs (Farmeat, Ashford, Kent, U.K. and British
Porcine liver and testis sulphotransferases Beef, Bury St. Edmunds, Suffolk, U.K. respectively). After transportation in ice to the Laboratory, 25% homogenates were prepared by homogenizing tissues in Tris-HCl buffer (Tris lOmmol/l, EDTA 1 mmol/l, 2-mercaptoethanol 3 mmol/l) pH 8.0, using a Waring blender. Subcellular fractions were obtained using differential centrifugation [1 l] and the cytosolic fraction was respun at 176,000 g,,, for 40 min at 4°C in a Beckman L8 Ultracentrifuge. The resulting supernatant was used for assays of steroid sulphotransferase activity and could be stored at -20°C without loss of enzyme activity for at least 8 weeks. Assay of steroid sulphotransferases
Liver sulphotransferase activity was assayed as follows: Unlabelled steroid substrate (0.1 pmol/l, final concentration) and PAPS-[3SS] (3.5 pmol/l, final concentration) were incubated at 37°C with tissue fraction in Tri-HCl buffer pH 8.0 in a final volume of 50 ~1. Because of the possibility of hydrolysis of PAPS (14) ATP (up to 4 mmol/l) was included in some incubations but was found to make little or no difference to the yield of steroid sulphate. The reaction was terminated after a suitable interval (see Results) by the addition of methanol (2.0 ml) and a known quantity (== 10,000 dpm) of 3H-labelled steroid sulphate was added. The methanol was clarified by centrifugation at 2OOOg,,, in a Gallenkamp Junior centrifuge, transferred to clean tubes and dried under reduced pressure. The residue was subjected to TLC alongside authentic steroid sulphates in benzene-ethanol-butanone-water (3:3:3:1, by vol). The region corresponding to the mobility of authentic steroid sulphate (R, approx 0.75 (4)) was removed, eluted with methanol (4 x 2 ml) and evaporated to dryness. The residue was redissolved in methanol (200 ~1) and a portion (50 ~1) taken for liquid scintillation counting. The use of tritiated steroid sulphate enabled a correction to be made for analytical losses and the ‘% activity was then corrected for radioactive decay. All assays were performed in duplicate together with substrate blanks and denatured enzyme control incubations. Protein estimations were determined as described earlier [I 51. Testis sulphotransferase assay
Testicular pregnenolone sulphotransferase activity was measured by incubating known amounts of [4-‘4C]pregnenolone (0.1 pmol/l, final concentration) and unlabelled PAPS (3.5 pmol/l, final concentration) with boar testis cytosol for 10min in Tris-HCl buffer, pH 8.0 in the same way as described for the liver. The final volume was 50 ~1. DHA and 5cr-androst-16-en-3/?-o] sulphotransferase assays involved incubating for 10min either [7n-3H]DHA or [5a,6cr-3H]5cr-androst-16-en-3/?-ol (synthesized from [5a,6a-3H]5c(-androst-16-en-3-one [4]) with cytosol and unlabelled PAPS, with the appropriate unlabelled steroid sulphate (50 pg) was added as carrier.
1105
Following TLC of the methanol extract as described for liver sulphotransferase assays, the region corresponding to the mobility of authentic steroid sulphate (R, approx 0.75 [4]), was removed, eluted with methanol, reduced to dryness and acid solvolysis performed as follows. The residue was suspended in sulphuric acid (1 mol/l) (1 ml) and 1 ml of ethyl acetate-diethyl ether (water saturated) (6: 94, v/v). After thorough mixing, the tubes were left overnight. The following day, diethyl ether (5 ml) was added, vortex mixed, centrifuged (25OOg,,,,,) for 5 min and the ether phase transferred to clean tubes. This was neutralized by washing with saturated sodium bicarbonate solution and then twice with water. The ether phase was removed, dried using anhydrous sodium sulphate, filtered, reduced to dryness and the residue benzene-methanol chromatographed first in (9: 1, v/v) followed by benzene-acetone (4: 1 v/v) in the case of [3H]DHA or benzene-ether (9: 1, v/v), run twice, in the case of the [3H]5a-androst-16-en-3/?-ol. Elution was achieved with chloroform and the purified steroids were then quantified by scintillation counting and analytical losses were corrected by GLC as described previously [ 161.
RESULTS
Properties of liver steroid sulphotransferases
Cytosolic DHA sulphotransferase activity was found to be linear with time up to 10min (Fig. 2); thereafter activity decreased, possibly due to the effect of PAP. DHA sulphotransferase was measured over a range of pH values 5.3-l 1.O, maximum activity being found at pH 7.7 (Fig. 3). However, in view of the fact that the optimum pH of both liver sulphotransferase for pregnenolone and Sa-androst-
^ 2Or .E
2 0
h
16-
\F
.
z P
12-
P E :
6-
0 ‘d4Jz -0 ii u IO 0
.
/\
.
./
/ .; I
I
I
2
6
IO
I 15
Time (min)
Fig. 2. Time course of porcine liver dehydroepiandrosterone sulphotransferase activity. Porcine liver cytosol was incubated at 37°C in the presence of dehvdroepiandrosterone (0.1 pmol/l) and PAPS [35S] (3.5 pmol;l) in Tri-HC1 Buffer (pH 8.0) in a final volume of 50~1. [‘HISteroid sulphate was isolated and purified as described in Experimental Procedure.
G. M. COOKEef al.
1106
ities were observed at pH 8.0 and at a temperature of 37°C for both enzymes.
1.5r
Properties qf porcine testicular sulphotransferases
0
I 8
I 7
I 6
5
9
IO
II
Fig. 3. pH dependence of dehydr~piandrosterone sulphotransferase activity in porcine liver cytosol. Sulphotransferase activity with dehydraepiandrosterone as substrate was measured by incubation with PAPS [“S] at 37°C in Tris-HCI buffer at different pH values. For further details see Legend to Fig. 2 and Methods.
16-en-3fi-ol are close to pH 8.0 (results not shown), this value was used for all subsequent assays, the difference in activity of DHA sulphotransferase at this pH being so small as to be unimportant. Maximal activity of the DHA sulphotransferase was found to be at 37°C (Fig. 4). These conditions were used to estimate the apparent K,,, of the sulphotransferase with DHA concentrations ranging from 0.1 to 10 ,umol/l. The apparent K,,, was found to be 91 pmol/l, compared with the rat liver enzyme, 6 pmol/l[4]. The effect of 5a-androst-16-en-3@-yl suiphate was also investigated using concentrations ranging from 0.02 to 25 pmol/l with the same substrate concentrations (0.1-10 fimol/l) as above. All concentrations of the sulphate were found to inhibit DHA sulphotransferase to the same extent, the inhibition being of the non-competitive type, as shown by Lineweaver-Burk analysis (Fig. 5). Pregnenolone and Sa-androst-16-en-38-01 sulphotransferase activities exhibited similar properties to the DHA sulphotransferase except that the time course of the sulphotransferase for the 16-androstene was only linear up to 5 min, thereafter forming a plateau. Thus, an incubation time of 4 min was used for all further assays of this enzyme. Maximal activ-
2.0
.
.E
\
t
E 1.5 .c 0 g I.0
i
I
D
/
~05fk: 0
(a) DHA su~phot~a~ferase. The optimal conditions determined for the liver enzymes above were used in these studies. Under these conditions of pH and temperature, DHA sulphotransferase activity was linear with time for at least 10min. Using three concentrations (0.2, 1.0 and 10 pmol/l) of DHA, the apparent Km was found to be 2 pmol/l and the apparent V,,, was 10 nmol/I/mg protein/min (Fig. 6). When Sa-androst-16en-3P-yl sulphate was added to identical incubations, at concentrations of 11.3 and 45.2 pmol/l, it failed to inhibit enzyme activity. (b) Pregnenolone s~~hotra~sferase. The suiphation of pregnenolone by boar testis cytosol was shown to be linear with time for at least 10min and was inhibited by pregnenolone sulphate, Sa-androst-16en-3P-yl sulphate, DHA and .Sa-androst- 16-en-3/I-01, but not by DHA sulphate (Table 1). In more detailed investigations the apparent K, was found to be 20 +umol/l (Fig. 7), with Sa-androst-l4-en-3~-yl sulphate non-competitively inhibiting the reaction to the same extent at concentrations of 11.3 and 45.2 pmol/l. The apparent K, values were in the range 5.82-19.69 pmol/l. This situation was similar to that found for DHA sulphotransferase in the liver (Fig. 5). s~lph~transfer~e. (c) ~-Androst-16-en-3P-oI Figure 8 illustrates the in t&o synthesis of Sa-androst-16-en-3P-yl sulphate by porcine testicular cytosol. Activity was linear with time up to 15 min but decreased thereafter, possibly due to inhibition by PAP. Thus, the amounts of the 16-androstene sulphate found endogenousiy [ 131 can be considered to be a consequence of testicular biosynthesis.
f
, IO
,
,
,
20 30 40 Temperature (%I
5o
Fig. 4 Temperature dependence of dehydroepiandrosterone sulphotransferase activity in porcine liver cytosol. The sulphotransferase was assayed at various temperatures as described in Methods and in legend to Frg. 2.
Fig. 5 Determination of apparent K,,, of porcine liver dehydroepiandrosterone sulphotransferase and the effect of Se-androst-16-en-3j?-yl sulphate. Concentrations of dehydroepiandrosterone ranged from 0.1 to IO ymol/l (e--a) and the apparent ii,, caIcuiated by Lineweaver-Burk anaiysis, was 91 ,umol/l. Incubation of dehydroepiandrosterone (O.l-tO~mol/l) in the presence of %-androst-l6-en-3fi-yl sulphate at concentrations ranging from 0.02 to 25 pmol/l (0-O) caused non-competitive inhibition of sulphotransferase activity to the same extent for all concentrations tested. For further details see Methods.
Porcine liver and testis sulphotransferases
Fig. 6. Determination of apparent Km for porcine testicular dehydroepiandrosterone sulphotransferase. [7r~-~H] Dehydehydroepiandroepiandrosterone plus unlabelled drosterone (to give concentrations of 0.2,l.O and 10 ~moljl) were incubated at 37°C in Tris-HCl buffer @H 8.0) for 10 min with boar testis cytosol, using PAPS as the sulphate donor. The steroid sulphate so formed was isolated and purified and the apparent i”r, (20 gmol/l) determined using the Lineweaver-Burk method.
1107
sors, then interference in this process by the presence of large amounts of k-androst-16en-3/?-yl sulphate may present the animal with a hormone imbalance, Rapid removal of steroid sulphates from the liver would therefore prevent this occurrence. The testis, however, is also responsible for the biosynthesis of the three sulphate esters considered here; in particular, the in vitro biosynthesis of S~-androst-l6-en-3~-yl sulphate is shown in Fig. 8. Such sulphotransferase activity is in keeping with the finding that the levels of DHA sulphate [8] and 5cr-androst- l6-en-3fl-yl sulphate [ 131 in boar testis are considerably greater than their free steroids but the quantities of pregnenolone sulphate are unknown at present. However, if the situation is the same as in humans, then these values are Iikely to be considerable, DHA appears to be preferred to pregnenolone as substrate for the sulphotransferase, as the apparent v,,, is five times that for the latter steroid.
DISCUSSION
In these studies, porcine liver cytosol was shown to sulphoconjugate DHA, pregnenolone and 5crandrost-16en-3/3-ol. The optimal incubation conditions were similar for the three substrates and Sa-androst-16-en-3/3-yl sulphate was found to inhibit DHA sulphotransferase activity (Fig, 5). The apparent Km remained unchanged and all the concentrations used inhibited to the same degree, suggesting that the enzyme was saturated with this inhibitor at all the concentrations used. An estimate of the endogenous levels of 5~ -androst- 16-en-38 -yl sulphate in porcine liver (1.25 pmol/kg wet weight) indicated that this steroid would be unlikely to regulate the metabolism of DHA in uiuo since the levels of DHA sulphate were much greater (813~mol/kg wet weight) (G. M. Cooke & D. B. Gower, unpublished observation). If the sulphation of steroids by the liver is necessary for the detoxification and excretion of hormone precur-
k f~mol/l) Fig. 7. Dete~ination of apparent K,,, of porcine testicular pregnenolone sulphotransferase and the effect of 5n-androst-16-en-3fi-yl sulphate. In the absence of inhibitor (a---a), the apparent Km for boar testis pregnenolone sulphotransferase was 20gmol/l, as calculated using the Lineweaver-Burk method. Incubation under the same conditions but also in the presence of 5a-androst-16-en-3b-yl sulphate at concentrations of 11.3 (0-O) and 45.2 (A-A) ~mol/l resulted in non-competitive inhibition to the same extent for both concentrations. The mathematically derived apparent Ki values were in the range 5.82 to 19.69 pmol,/l.
Table 1. Effects of free and sulphated steroids on the sulphation of pregnenolone by boar testis cytosol in vitro Incubation addition None Pregnenolone sulphate (2.7 ~mol/l) 5a-Androst-16-en-3jI-yl (I .2 pmol/l) DHA sulphate (8.1 pmol/l) Sa-Androst-16-en-3/l-01 (I .2 pmol/l) DHA (8.1 ~mol/l)
sulphate
Pregnenolone sulphate formed (nmol/mg proteinlmin)
Percentage inhibition
0.27 0.18
None 33.4
0.15
44.4
0.28
None
0.13
51.8
0.14
48.1
All additions were made at the stated ~ncentrations and were incubated with [4-‘4C]pregnenolone (63 nmol/l) and unlabelled PAPS at 37°C for 10 min at pH 8.0 (see Methods). Each value is the mean of duplicate incubations, corrected for any activity found in absence of steroid.
G. M. COOKE et al.
1108
6
Time
(min)
Fig. 8. Time course of sulphation of 5a-androst-16-en3p-01 in porcine testis in vitro. Boar testis cytosol was incubated with [5a,6a-3H]5a-androst-16-en-3fi-ol and PAPS at 37°C and pH 8.0 for the stated times. The labelled steroid was isolated and purified as described in Methods.
Furthermore, the apparent K,,, values suggest that there is a greater affinity for DHA (apparent K,,, 2 pmol/l) than for pregnenolone (apparent K,,, 20 pmol/l). Although the sulphation of pregnenolone was inhibited by DHA (Table 1), it is conceivable that these two steroids may not be conjugated by the same sulphotransferase. Pregnenolone sulphate biosynthesis was not inhibited by DHA sulphate, but both Sa-androst-16-en-3/3-ol and its sulphate were inhibitory, the latter at levels found endogenously in the testis [13]. In contrast, DHA sulphate biosynthesis was not similarly affected by Sa-androst16-en-3b-yl sulphate and we suggest that separate sulphotransferases may exist for DHA and pregnenolone, and that inhibition of pregnenolone sulphation by DHA is a consequence of competition for PAPS. Furthermore, as DHA is sulphated at a greater rate under these conditions, its presence would markedly inhibit the amount of pregnenolone sulphate formed. Using rat seminiferous tubules, the apparent K, value for DHA sulphation was found to be 3 pmol/l [ 11,a value very similar to that in the present work (Fig. 6). However, in this instance, pregnenolone inhibited competitively with an apparent K, of 0.3 pmol/l, suggesting that this steroid is the preferred substrate. This result is in contrast to that found using boar testis cytosol. The endocrinological significance of the inhibition of pregnenolone sulphotransferase by 5u -androst16-en-3/I-ol and its sulphate may be important in regulating 16-androstene biosynthesis from pregnenolone sulphate, which has been shown to occur in vitro [12, 131, although
yields
of
16-androstenes
are
lower than when unconjugated pregnenolone is used [9]. The non-competitive nature of the inhibition by Sa-androst-16-en-3B-yl sulphate (Fig. 7) would also mean that the inhibition would not be overcome
by increasing the pregnenolone concentration. In contrast to pregnenolone, DHA is known to be a poor substrate for 16-androstene biosynthesis [ 171 and this may help to explain the lack of inhibition of DHA sulphotransferase by 5a -androst- 16-en-3/I-yl sulphate. The importance of the sulphation of 16-androstene steroids was observed by Saat and coworkers [18]. Following an infusion of [5cr-3H]5a-androst-16-en-3one into the boar testis, 16-androstene sulphates were evident within a few minutes after the start of the infusion, at the same rate as the free steroids were synthesized. The in vitro observation described in this work (Fig. 8) is in keeping with this. In this instance the regulation of pregnenolone sulphate biosynthesis would help in the control of testicular 16-androstene biosynthesis in the boar. In conclusion, we have provided evidence which suggests that the biosynthesis of steroid sulphates in the boar testis may be subject to regulation by the presence of both free 3p-hydroxysteroids and their 3p-yl sulphate esters, whereas in the porcine liver this influence would be less likely. Acknowledgements-We are most grateful to Mrs D. M. Gower and Miss R. T. K. Seville for typing the manuscript. The Agricultural Research Council generously supported G. M. Cooke financially (Grant No. 35/35). REFERENCES 1. Payne A. H. and Singer S. S.: The role of steroid sulphatase and sulphotransferase enzymes in the metabolism of C,, and C,, steroids. In Steroid Biochemistry, Vol. I, (Edited by R. Hobkirk). CRC Press, Florida (1979) pp. 11 l-145. 2. Nose Y. and Lipmann F.: Separation of steroid sulphokinases. J. biol. Chem. 233 (1958) 134881351. R. A. and Carrol J.: Studies on a 3. Ryan 3fi-hydroxysteroid sulphotransferase from rat liver. Biochim. biophys. Acta 429 (1976) 391401. 4. Fish D. E.. Cooke G. M. and Gower D. B.: Insulphoconjugation of vestigation into the 5a-androst-16-en-3B-ol by porcine liver. FEBS Leit. 117 (1980) 28832. T., Laatinen E. A. and Vihko R. Secretion 5. Laatikainen of free and sulphate conjugated neutral steroids by the human testis. Effect of administration of human chorionic gonadotrophin. J. clin. Endocr. Metab. 32 ( 1969) 59-64. I. and Huis in’t Veld L. G.: 6. Baulieu E. E. Fabre-Jung Dehydroepiandrosterone sulphate, a secretory product of the boar testis. Endocrinology 81 (1967) 3448. A. and Vihko R.: Steroid metabolism in 7. Ruokonen human and boar testis tissue. Steroid concentrations and the position of the sulphate group in steroid sulphates. Steroids 23 (1974) l-16. 8. Booth W. D.: Changes with age in the occurrence of C,, steroids in the testis and submaxillary gland of the boar. J. reprod. Fert. 42 (1975) 459472. C,, steroids. A review of 9. Gower D. B.: Unsaturated their chemistry, biochemistry and possible physiological role. J. steroid Biochem. 3 (1972) 45-103. and exocrine factors in the 10. Booth W. D.: Endocrine reproductive behaviour of the pig. In Symposium qf the Zoological Society of London, No. 45. (Edited by D. M. Stoddart). Academic Press, London and New York (1980) pp. 155-162.
Porcine liver and testis sulphotransferases Il. Cooke G. M. and Gower D. B.: Investigation into the possible effects of naturally occurring steroids on biosynthesis of 16-androstenes and androgens in microsomes of boar testis. J. Endocr. 88 (1981) 409418. 12. Gasparini F., Hochberg R. and Lieberman S.: Biosynthesis of steroid sulphates by the boar testes. Biochemistry 15 (1976) 39693975. 13. Ruokonen A.: Steroid metabolism in testis tissue. The metabolism of pregnenolone, pregnenolone sulphate, dehydroepiandrosterone and dehydroepiandrosterone sulphate in human and boar testes in vitro. J. steroid Biochem. 9 (1978) 939-946. 14. Bailey-Wood
R., Dodgson K. and Rose F. A.: Purification and properties of two adenosine-S-phosphosulphate sulphohydrolases from rat liver and their possible role in the degradation of 3’-phosphoadenosine-
S-phosphosulphate.
1109 Biochim. biophys. Acta 220 (1970)
284299. 15. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall
R. J.: Protein measurements with the Folin phenol reagent. J. biol. Chem. 193 (1951) 265-275. 16. Cooke G. M. and Gower D. B.: The submicrosomal distribution in rat and boar testis of some enzymes involved in androgen and 16-androstene biosynthesis. Biochim. biophys. Acta, 498 (1977) 265-271. 17. Ahmad N. and Gower D. B.: The biosynthesis of some
androst- 16enes from C,, steroids in boar testicular tissue. Biochem. J. 108 (1968) 233-241. 18. Saat Y. A., Gower D. B., Harrison F. A. and Heap R. B.: Studies on the metabolism of Scc-androst16-en-3-one in boar testis in uiuo. Biochem. J. 144 (1974) 347-352.
J. steroid Biochem. Vol. 19, No. 2, pp. I103-1109, 1983
Printed in Great Britain. All rights reserved
PROPERTIES OF PORCINE LIVER AND TESTICULAR STEROID SULPHOTRANSFERASES: REACTION CONDITIONS AND INFLUENCE OF NATURALLY OCCURRING STEROIDS AND STEROID SULPHATES G,
Department
M. COOKE*, SUSANE. FERGUSON,E. RYTINA and D. B. GOWER~ of Biochemistry, Guy’s Hospital Medical School, London SE1 9RT, England (Received 18 October 1982)
Summary-Sulphotransfe~se activity has been assayed in porcine liver and testis cytosol using either 3’-phosphoadenosine-5’-phospho [3sS]sulphate (PAPS) or unlabelled PAPS as sulphate donors. In porcine liver the sulphotransferase for DHA was linear for up to lOmin, the optimum pH was 7.7 and optimum temperature, 37°C. The apparent Km value was found to be 91 pmol/l and the activity was inhibited non-competitively by 5a-androst-16en-3P-yl sulphate, with all concentrations used (0.02-25pmol/l) inhibiting the enzyme to the same extent. Time courses for sulphoconjugation of pregnenolone and Sa-androst-16-en-3/I-01 were linear for up to at least 10 min or up to only 5 min, respectively. The optimum pH values and temperatures were pH 8.0 and 37°C in each case. The porcine testicular sulphotransferase activity for DHA as substrate was linear with time up to lOmin, the apparent Km for the reaction was Z~mol/l and apparent V,,, lOnmoI/l/mg~min. Sff-Androst-l4-en-3~-yl sulphate (11.3-45.2 pmoljl) failed to inhibit the enzyme activity. The time-course for the reaction, when pregnenolone was used as substrate, was also linear up to 10 mm at the optimum pH 8.0 but, in contrast to the reaction when DHA was the substrate, had an apparent K,,, of 20ymol/l Sa-androst-16en-3/I-y] sulphate, DHA and and was inhibited by pregnenolone sulphate, Sc+androst-16-en-3/I-ol, but not by DHA sulphate. Sa-Androst-16en-3b-yl sulphate inhibited the reaction non-competitively and to the same extent at concentrations over the range l1.3-45.2~mol/l. These data suggest that DHA and pregnenolone may not be sulphoconjugated by the same sulphotransferase. With Se-androst-16-en-3/I-ol as substrate, the time-course for its sulphate formation was linear up to 15 min, and this reaction could explain the quantities of 5a-androst-16”en-3/3-yl sulphate that are found endogenously in porcine testis. Our results further suggest that these quantities could well inhibit the sulphation of pregnenolone in porcine testis in &JO, and the possibility of control of sulphoconjugation in this tissue is discussed. Having regard to the smaller quantities of Sa-androst-16-en-3fi-yl sulphate present in porcine liver, our results suggest that the suiphation of DHA there may not be so much affected.
INTRODUCTION Steroid sulphates, which are present in large quantities in many mammaIian tissues, are thought to be sources of biolo~cally active steroid hormones, intermediates in steroid biosynthetic pathways and also as detoxification products of hormone metabolism [I].
*Present Address: The Royal Victoria Hospital, The Women’s Pavilion, Department of Obstetrics and Gynecology, 687 Pine Avenue West, Montreal PQ, Canada H3A lA1. tAddress reprint requests to Dr. D. B. Gower. Abbreviations and trivial names: 3fi-hydroxy-5-androsten!7-one, dehydroepiandrosterone (DHA); 38-hydroxy-5pregnen-20-one, pregnenolone: 4-pregnene-3,20-dione, progesterone; 3B-hydroxy-5cl-androstan-17-one, epiandrosterone; 3-hydroxy-1,3,5(10)-oestratrien-17-one, oestrone; 5c(-pregnane-3,20-dione, Sa-pregnanedione; 3’-phosphoadenosine-5’-phosphosulphate, PAPS; 3’ph~sph~adenosine-S-phosphate, PAP. Enzyme names: 17a-hvdroxv-C,,-steroid C-17.20 lvase, C-17,20 lvase; 5-ene~3~-h~dro~ysteroid oxido-reductase (EC I.i.i.5l)t 3-oxosteroid-4,5isomerase (EC 5.3.3. l), 5-ene-3flOHSDH/4,5-isomerase. SB19/2--F
The steroid sulphotransferases of liver cytosol may be involved in the detoxification and excretory processes whereas, in males, the sulphotransferases of the testis can be considered as of biosynthetic importance. Two enzymes were separated from rabbit liver which catalysed the sulphation of DHA and oestrone, and which were distinct from phenol sulphotransferase [2]. In a more recent study, a partially purified 3P-hydroxysteroid sulphotransferase from rat liver was found to accept epiandrosterone, DHA, 5a-pregnane-3&20fi-dial and Sa-androstane3/?,178-diol as substrates [3]. Other 3/L?-hydroxysteroids, including pregnenolone and 5-androstene3&l ?‘/I-dial were not preferred. Fish and coworkers [4] observed both cytosolic and microsomal sulphotransferase activity towards 5a-androst-16-en3/I-01 in subcellular fractions of porcine liver. The microsomal enzyme exhibited an optimum pH of 7.4 and was released from the membranes by sodium dodecyl sulphate. The testis tissue of humans, boars and rats has all been shown to possess steroid suiphotransferase activity [ 1,5,6] but the intratesticular distribution of
1103
G.
1104
CHOLESTEROL + (SO,)
PREGNENOLONE (SO,) 1
-
M.
CWKE et al.
I
PROGESTERONE ( sHo; 17-OH
PROGESTERONE
1 4-ANDROSTENEDIONE It TESTOSTERONE
Fig. 1 Pathways of biosynthesis of 16-androstenes, androgens and steroid sulphates in boar testis. (a) 5,16-androstadien-3/?-ol and its sulphate; (b) 4,16-androstadien-3-one; (c) 5cc-androst-16-en-3-one; (d) 5a-androst-16-en-3a-ol and its sulphate; (e) Sa-androst-16-en-3j-ol and its sulphate; DHA, 3P-hydroxy-Sandrosten-17-one.
these enzymes remains uncertain. The testis contains large quantities of 3fi-hydroxysteroid sulphates [6,7] and, in the boar testis, one of the principal steroid sulphates is Sa-androst- 16-en-3/r’-yl sulphate. Unconjugated 5x-androst- 16-en-3B-ol is also found in large amounts in boar testis [8], but no physiological role has been determined for either the free steroid or its sulphate ester. It is believed that 16-androstene biosynthesis in boars provides two pheromonally active steroids, 5x-androst-16-en-3-one and 5xandrost-16-en-3a-ol[9, lo] and that these are produced from pregnenolone by enzymes distinct from those involved in androgenesis [ll]. The pathway is outlined in Fig. 1. Pregnenolone sulphate can be converted to 5,16-androstadien-3/3-yl sulphate in vitro [12] but this route to 16-androstenes is considered to be less significant than the unconjugated steroid pathway [13]. This may be due to the low activity of boar testicular steroid sulphatase [14] as compared to that of bovine, ovine and human testes. The present investigations were performed to gain further information about the inter-relationships between free and sulphate conjugated steroids in porcine liver and testis and also to examine the role in steroid hormone metabolism of steroid sulphates, especially 501-androst-16-en-3/I-yl sulphate, as this is quantitatively the most important 16-androstene sulphate in boar testis [8]. MATERIALS
AND METHODS
Materials
Reagents and materials were as reported previously [4] except for 3’-phosphoadenosine-5’phospho [3SS]sulphate (2.09 CJmmol) which was purchased from New England Nuclear, Dreieich, W.
Germany and stored at -20°C in ethanol-water (1: 1, v/v) prior to use. Unlabelled PAPS was obtained from Sigma Chemicals Co., Poole, Dorset, U.K. The purity of the former was checked by TLC, followed by radioautography and found to be satisfactory. To minimize decomposition, in some experiments, the lithium salt of unlabelled PAPS was used, which is more stable than the potassium salt. This was stored, desiccated, at -20°C and made up freshly for experimental studies. [4-‘4C]Pregnenolone (56.5 mC,/mmol) and [7n-3H]DHA (23 Ci/mmol) were from the Radiochemical Centre, Amersham, [5c(,6c(-3H]5cr-Androst-16-en-3-one Bucks, U.K. (21 CJmmol) was obtained from ROTOP, Isocommerz, GmbH. Kontor, Dresden, D.D.R. 8051 from which [5c(,6u-3H]5c(-androst-16-en-3,?-ol was prepared by borohydride reduction [4]. Tritium labelled and unlabelled steroid sulphates were prepared as described earlier [4]. Precoated silica gel plates for TLC were purchased from Merck (supplied by Anderman & Co. Ltd., East Molesey, Surrey, U.K.). Scintillation counting was performed using a Packard Tri-carb Liquid Scintillation Spectrometer, Model 3255. An aqueous scintillation mixture was used which consisted of 2,5-diphenyloxazole (5g) and 1,4&s 2-(5-phenyloxazolyl) benzene (0.5 g) both dissolved in a mixture (2 : 1, v/v) of toluene-Triton X-100 [ll]. Counting efficiencies for 35S, 3H and 14C were 98, 55 and 88% respectively. METHODS
Preparation of tissues
Porcine liver and testis were obtained from abbatoirs (Farmeat, Ashford, Kent, U.K. and British
Porcine liver and testis sulphotransferases Beef, Bury St. Edmunds, Suffolk, U.K. respectively). After transportation in ice to the Laboratory, 25% homogenates were prepared by homogenizing tissues in Tris-HCl buffer (Tris lOmmol/l, EDTA 1 mmol/l, 2-mercaptoethanol 3 mmol/l) pH 8.0, using a Waring blender. Subcellular fractions were obtained using differential centrifugation [1 l] and the cytosolic fraction was respun at 176,000 g,,, for 40 min at 4°C in a Beckman L8 Ultracentrifuge. The resulting supernatant was used for assays of steroid sulphotransferase activity and could be stored at -20°C without loss of enzyme activity for at least 8 weeks. Assay of steroid sulphotransferases
Liver sulphotransferase activity was assayed as follows: Unlabelled steroid substrate (0.1 pmol/l, final concentration) and PAPS-[3SS] (3.5 pmol/l, final concentration) were incubated at 37°C with tissue fraction in Tri-HCl buffer pH 8.0 in a final volume of 50 ~1. Because of the possibility of hydrolysis of PAPS (14) ATP (up to 4 mmol/l) was included in some incubations but was found to make little or no difference to the yield of steroid sulphate. The reaction was terminated after a suitable interval (see Results) by the addition of methanol (2.0 ml) and a known quantity (== 10,000 dpm) of 3H-labelled steroid sulphate was added. The methanol was clarified by centrifugation at 2OOOg,,, in a Gallenkamp Junior centrifuge, transferred to clean tubes and dried under reduced pressure. The residue was subjected to TLC alongside authentic steroid sulphates in benzene-ethanol-butanone-water (3:3:3:1, by vol). The region corresponding to the mobility of authentic steroid sulphate (R, approx 0.75 (4)) was removed, eluted with methanol (4 x 2 ml) and evaporated to dryness. The residue was redissolved in methanol (200 ~1) and a portion (50 ~1) taken for liquid scintillation counting. The use of tritiated steroid sulphate enabled a correction to be made for analytical losses and the ‘% activity was then corrected for radioactive decay. All assays were performed in duplicate together with substrate blanks and denatured enzyme control incubations. Protein estimations were determined as described earlier [I 51. Testis sulphotransferase assay
Testicular pregnenolone sulphotransferase activity was measured by incubating known amounts of [4-‘4C]pregnenolone (0.1 pmol/l, final concentration) and unlabelled PAPS (3.5 pmol/l, final concentration) with boar testis cytosol for 10min in Tris-HCl buffer, pH 8.0 in the same way as described for the liver. The final volume was 50 ~1. DHA and 5cr-androst-16-en-3/?-o] sulphotransferase assays involved incubating for 10min either [7n-3H]DHA or [5a,6cr-3H]5cr-androst-16-en-3/?-ol (synthesized from [5a,6a-3H]5c(-androst-16-en-3-one [4]) with cytosol and unlabelled PAPS, with the appropriate unlabelled steroid sulphate (50 pg) was added as carrier.
1105
Following TLC of the methanol extract as described for liver sulphotransferase assays, the region corresponding to the mobility of authentic steroid sulphate (R, approx 0.75 [4]), was removed, eluted with methanol, reduced to dryness and acid solvolysis performed as follows. The residue was suspended in sulphuric acid (1 mol/l) (1 ml) and 1 ml of ethyl acetate-diethyl ether (water saturated) (6: 94, v/v). After thorough mixing, the tubes were left overnight. The following day, diethyl ether (5 ml) was added, vortex mixed, centrifuged (25OOg,,,,,) for 5 min and the ether phase transferred to clean tubes. This was neutralized by washing with saturated sodium bicarbonate solution and then twice with water. The ether phase was removed, dried using anhydrous sodium sulphate, filtered, reduced to dryness and the residue benzene-methanol chromatographed first in (9: 1, v/v) followed by benzene-acetone (4: 1 v/v) in the case of [3H]DHA or benzene-ether (9: 1, v/v), run twice, in the case of the [3H]5a-androst-16-en-3/?-ol. Elution was achieved with chloroform and the purified steroids were then quantified by scintillation counting and analytical losses were corrected by GLC as described previously [ 161.
RESULTS
Properties of liver steroid sulphotransferases
Cytosolic DHA sulphotransferase activity was found to be linear with time up to 10min (Fig. 2); thereafter activity decreased, possibly due to the effect of PAP. DHA sulphotransferase was measured over a range of pH values 5.3-l 1.O, maximum activity being found at pH 7.7 (Fig. 3). However, in view of the fact that the optimum pH of both liver sulphotransferase for pregnenolone and Sa-androst-
^ 2Or .E
2 0
h
16-
\F
.
z P
12-
P E :
6-
0 ‘d4Jz -0 ii u IO 0
.
/\
.
./
/ .; I
I
I
2
6
IO
I 15
Time (min)
Fig. 2. Time course of porcine liver dehydroepiandrosterone sulphotransferase activity. Porcine liver cytosol was incubated at 37°C in the presence of dehvdroepiandrosterone (0.1 pmol/l) and PAPS [35S] (3.5 pmol;l) in Tri-HC1 Buffer (pH 8.0) in a final volume of 50~1. [‘HISteroid sulphate was isolated and purified as described in Experimental Procedure.
G. M. COOKEef al.
1106
ities were observed at pH 8.0 and at a temperature of 37°C for both enzymes.
1.5r
Properties qf porcine testicular sulphotransferases
0
I 8
I 7
I 6
5
9
IO
II
Fig. 3. pH dependence of dehydr~piandrosterone sulphotransferase activity in porcine liver cytosol. Sulphotransferase activity with dehydraepiandrosterone as substrate was measured by incubation with PAPS [“S] at 37°C in Tris-HCI buffer at different pH values. For further details see Legend to Fig. 2 and Methods.
16-en-3fi-ol are close to pH 8.0 (results not shown), this value was used for all subsequent assays, the difference in activity of DHA sulphotransferase at this pH being so small as to be unimportant. Maximal activity of the DHA sulphotransferase was found to be at 37°C (Fig. 4). These conditions were used to estimate the apparent K,,, of the sulphotransferase with DHA concentrations ranging from 0.1 to 10 ,umol/l. The apparent K,,, was found to be 91 pmol/l, compared with the rat liver enzyme, 6 pmol/l[4]. The effect of 5a-androst-16-en-3@-yl suiphate was also investigated using concentrations ranging from 0.02 to 25 pmol/l with the same substrate concentrations (0.1-10 fimol/l) as above. All concentrations of the sulphate were found to inhibit DHA sulphotransferase to the same extent, the inhibition being of the non-competitive type, as shown by Lineweaver-Burk analysis (Fig. 5). Pregnenolone and Sa-androst-16-en-38-01 sulphotransferase activities exhibited similar properties to the DHA sulphotransferase except that the time course of the sulphotransferase for the 16-androstene was only linear up to 5 min, thereafter forming a plateau. Thus, an incubation time of 4 min was used for all further assays of this enzyme. Maximal activ-
2.0
.
.E
\
t
E 1.5 .c 0 g I.0
i
I
D
/
~05fk: 0
(a) DHA su~phot~a~ferase. The optimal conditions determined for the liver enzymes above were used in these studies. Under these conditions of pH and temperature, DHA sulphotransferase activity was linear with time for at least 10min. Using three concentrations (0.2, 1.0 and 10 pmol/l) of DHA, the apparent Km was found to be 2 pmol/l and the apparent V,,, was 10 nmol/I/mg protein/min (Fig. 6). When Sa-androst-16en-3P-yl sulphate was added to identical incubations, at concentrations of 11.3 and 45.2 pmol/l, it failed to inhibit enzyme activity. (b) Pregnenolone s~~hotra~sferase. The suiphation of pregnenolone by boar testis cytosol was shown to be linear with time for at least 10min and was inhibited by pregnenolone sulphate, Sa-androst-16en-3P-yl sulphate, DHA and .Sa-androst- 16-en-3/I-01, but not by DHA sulphate (Table 1). In more detailed investigations the apparent K, was found to be 20 +umol/l (Fig. 7), with Sa-androst-l4-en-3~-yl sulphate non-competitively inhibiting the reaction to the same extent at concentrations of 11.3 and 45.2 pmol/l. The apparent K, values were in the range 5.82-19.69 pmol/l. This situation was similar to that found for DHA sulphotransferase in the liver (Fig. 5). s~lph~transfer~e. (c) ~-Androst-16-en-3P-oI Figure 8 illustrates the in t&o synthesis of Sa-androst-16-en-3P-yl sulphate by porcine testicular cytosol. Activity was linear with time up to 15 min but decreased thereafter, possibly due to inhibition by PAP. Thus, the amounts of the 16-androstene sulphate found endogenousiy [ 131 can be considered to be a consequence of testicular biosynthesis.
f
, IO
,
,
,
20 30 40 Temperature (%I
5o
Fig. 4 Temperature dependence of dehydroepiandrosterone sulphotransferase activity in porcine liver cytosol. The sulphotransferase was assayed at various temperatures as described in Methods and in legend to Frg. 2.
Fig. 5 Determination of apparent K,,, of porcine liver dehydroepiandrosterone sulphotransferase and the effect of Se-androst-16-en-3j?-yl sulphate. Concentrations of dehydroepiandrosterone ranged from 0.1 to IO ymol/l (e--a) and the apparent ii,, caIcuiated by Lineweaver-Burk anaiysis, was 91 ,umol/l. Incubation of dehydroepiandrosterone (O.l-tO~mol/l) in the presence of %-androst-l6-en-3fi-yl sulphate at concentrations ranging from 0.02 to 25 pmol/l (0-O) caused non-competitive inhibition of sulphotransferase activity to the same extent for all concentrations tested. For further details see Methods.
Porcine liver and testis sulphotransferases
Fig. 6. Determination of apparent Km for porcine testicular dehydroepiandrosterone sulphotransferase. [7r~-~H] Dehydehydroepiandroepiandrosterone plus unlabelled drosterone (to give concentrations of 0.2,l.O and 10 ~moljl) were incubated at 37°C in Tris-HCl buffer @H 8.0) for 10 min with boar testis cytosol, using PAPS as the sulphate donor. The steroid sulphate so formed was isolated and purified and the apparent i”r, (20 gmol/l) determined using the Lineweaver-Burk method.
1107
sors, then interference in this process by the presence of large amounts of k-androst-16en-3/?-yl sulphate may present the animal with a hormone imbalance, Rapid removal of steroid sulphates from the liver would therefore prevent this occurrence. The testis, however, is also responsible for the biosynthesis of the three sulphate esters considered here; in particular, the in vitro biosynthesis of S~-androst-l6-en-3~-yl sulphate is shown in Fig. 8. Such sulphotransferase activity is in keeping with the finding that the levels of DHA sulphate [8] and 5cr-androst- l6-en-3fl-yl sulphate [ 131 in boar testis are considerably greater than their free steroids but the quantities of pregnenolone sulphate are unknown at present. However, if the situation is the same as in humans, then these values are Iikely to be considerable, DHA appears to be preferred to pregnenolone as substrate for the sulphotransferase, as the apparent v,,, is five times that for the latter steroid.
DISCUSSION
In these studies, porcine liver cytosol was shown to sulphoconjugate DHA, pregnenolone and 5crandrost-16en-3/3-ol. The optimal incubation conditions were similar for the three substrates and Sa-androst-16-en-3/3-yl sulphate was found to inhibit DHA sulphotransferase activity (Fig, 5). The apparent Km remained unchanged and all the concentrations used inhibited to the same degree, suggesting that the enzyme was saturated with this inhibitor at all the concentrations used. An estimate of the endogenous levels of 5~ -androst- 16-en-38 -yl sulphate in porcine liver (1.25 pmol/kg wet weight) indicated that this steroid would be unlikely to regulate the metabolism of DHA in uiuo since the levels of DHA sulphate were much greater (813~mol/kg wet weight) (G. M. Cooke & D. B. Gower, unpublished observation). If the sulphation of steroids by the liver is necessary for the detoxification and excretion of hormone precur-
k f~mol/l) Fig. 7. Dete~ination of apparent K,,, of porcine testicular pregnenolone sulphotransferase and the effect of 5n-androst-16-en-3fi-yl sulphate. In the absence of inhibitor (a---a), the apparent Km for boar testis pregnenolone sulphotransferase was 20gmol/l, as calculated using the Lineweaver-Burk method. Incubation under the same conditions but also in the presence of 5a-androst-16-en-3b-yl sulphate at concentrations of 11.3 (0-O) and 45.2 (A-A) ~mol/l resulted in non-competitive inhibition to the same extent for both concentrations. The mathematically derived apparent Ki values were in the range 5.82 to 19.69 pmol,/l.
Table 1. Effects of free and sulphated steroids on the sulphation of pregnenolone by boar testis cytosol in vitro Incubation addition None Pregnenolone sulphate (2.7 ~mol/l) 5a-Androst-16-en-3jI-yl (I .2 pmol/l) DHA sulphate (8.1 pmol/l) Sa-Androst-16-en-3/l-01 (I .2 pmol/l) DHA (8.1 ~mol/l)
sulphate
Pregnenolone sulphate formed (nmol/mg proteinlmin)
Percentage inhibition
0.27 0.18
None 33.4
0.15
44.4
0.28
None
0.13
51.8
0.14
48.1
All additions were made at the stated ~ncentrations and were incubated with [4-‘4C]pregnenolone (63 nmol/l) and unlabelled PAPS at 37°C for 10 min at pH 8.0 (see Methods). Each value is the mean of duplicate incubations, corrected for any activity found in absence of steroid.
G. M. COOKE et al.
1108
6
Time
(min)
Fig. 8. Time course of sulphation of 5a-androst-16-en3p-01 in porcine testis in vitro. Boar testis cytosol was incubated with [5a,6a-3H]5a-androst-16-en-3fi-ol and PAPS at 37°C and pH 8.0 for the stated times. The labelled steroid was isolated and purified as described in Methods.
Furthermore, the apparent K,,, values suggest that there is a greater affinity for DHA (apparent K,,, 2 pmol/l) than for pregnenolone (apparent K,,, 20 pmol/l). Although the sulphation of pregnenolone was inhibited by DHA (Table 1), it is conceivable that these two steroids may not be conjugated by the same sulphotransferase. Pregnenolone sulphate biosynthesis was not inhibited by DHA sulphate, but both Sa-androst-16-en-3/3-ol and its sulphate were inhibitory, the latter at levels found endogenously in the testis [13]. In contrast, DHA sulphate biosynthesis was not similarly affected by Sa-androst16-en-3b-yl sulphate and we suggest that separate sulphotransferases may exist for DHA and pregnenolone, and that inhibition of pregnenolone sulphation by DHA is a consequence of competition for PAPS. Furthermore, as DHA is sulphated at a greater rate under these conditions, its presence would markedly inhibit the amount of pregnenolone sulphate formed. Using rat seminiferous tubules, the apparent K, value for DHA sulphation was found to be 3 pmol/l [ 11,a value very similar to that in the present work (Fig. 6). However, in this instance, pregnenolone inhibited competitively with an apparent K, of 0.3 pmol/l, suggesting that this steroid is the preferred substrate. This result is in contrast to that found using boar testis cytosol. The endocrinological significance of the inhibition of pregnenolone sulphotransferase by 5u -androst16-en-3/I-ol and its sulphate may be important in regulating 16-androstene biosynthesis from pregnenolone sulphate, which has been shown to occur in vitro [12, 131, although
yields
of
16-androstenes
are
lower than when unconjugated pregnenolone is used [9]. The non-competitive nature of the inhibition by Sa-androst-16-en-3B-yl sulphate (Fig. 7) would also mean that the inhibition would not be overcome
by increasing the pregnenolone concentration. In contrast to pregnenolone, DHA is known to be a poor substrate for 16-androstene biosynthesis [ 171 and this may help to explain the lack of inhibition of DHA sulphotransferase by 5a -androst- 16-en-3/I-yl sulphate. The importance of the sulphation of 16-androstene steroids was observed by Saat and coworkers [18]. Following an infusion of [5cr-3H]5a-androst-16-en-3one into the boar testis, 16-androstene sulphates were evident within a few minutes after the start of the infusion, at the same rate as the free steroids were synthesized. The in vitro observation described in this work (Fig. 8) is in keeping with this. In this instance the regulation of pregnenolone sulphate biosynthesis would help in the control of testicular 16-androstene biosynthesis in the boar. In conclusion, we have provided evidence which suggests that the biosynthesis of steroid sulphates in the boar testis may be subject to regulation by the presence of both free 3p-hydroxysteroids and their 3p-yl sulphate esters, whereas in the porcine liver this influence would be less likely. Acknowledgements-We are most grateful to Mrs D. M. Gower and Miss R. T. K. Seville for typing the manuscript. The Agricultural Research Council generously supported G. M. Cooke financially (Grant No. 35/35). REFERENCES 1. Payne A. H. and Singer S. S.: The role of steroid sulphatase and sulphotransferase enzymes in the metabolism of C,, and C,, steroids. In Steroid Biochemistry, Vol. I, (Edited by R. Hobkirk). CRC Press, Florida (1979) pp. 11 l-145. 2. Nose Y. and Lipmann F.: Separation of steroid sulphokinases. J. biol. Chem. 233 (1958) 134881351. R. A. and Carrol J.: Studies on a 3. Ryan 3fi-hydroxysteroid sulphotransferase from rat liver. Biochim. biophys. Acta 429 (1976) 391401. 4. Fish D. E.. Cooke G. M. and Gower D. B.: Insulphoconjugation of vestigation into the 5a-androst-16-en-3B-ol by porcine liver. FEBS Leit. 117 (1980) 28832. T., Laatinen E. A. and Vihko R. Secretion 5. Laatikainen of free and sulphate conjugated neutral steroids by the human testis. Effect of administration of human chorionic gonadotrophin. J. clin. Endocr. Metab. 32 ( 1969) 59-64. I. and Huis in’t Veld L. G.: 6. Baulieu E. E. Fabre-Jung Dehydroepiandrosterone sulphate, a secretory product of the boar testis. Endocrinology 81 (1967) 3448. A. and Vihko R.: Steroid metabolism in 7. Ruokonen human and boar testis tissue. Steroid concentrations and the position of the sulphate group in steroid sulphates. Steroids 23 (1974) l-16. 8. Booth W. D.: Changes with age in the occurrence of C,, steroids in the testis and submaxillary gland of the boar. J. reprod. Fert. 42 (1975) 459472. C,, steroids. A review of 9. Gower D. B.: Unsaturated their chemistry, biochemistry and possible physiological role. J. steroid Biochem. 3 (1972) 45-103. and exocrine factors in the 10. Booth W. D.: Endocrine reproductive behaviour of the pig. In Symposium qf the Zoological Society of London, No. 45. (Edited by D. M. Stoddart). Academic Press, London and New York (1980) pp. 155-162.
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