Comparison of estrogen sulfotransferase and pregnenolone sulfotransferase of guinea pig.

Guinea pig adrenal estrogen sulfotransferase from either sex was eluted as a single peak, irrespective of buffer salt concentration, when subjected to fast protein liquid chromatography on gel filtration columns. The same enzyme was consistently eluted in two distinct peaks during chromatofocusing. Adrenal pregnenolone sulfotransferase was eluted during gel filtration in a heterogeneous pattern, dependent on salt concentration. These properties have made possible almost complete separation of the two sulfotransferases in one step, although adrenal estrogen sulfotransferase may possess a minute intrinsic ability to catalyze sulfation of pregnenolone. Pregnenolone sulfotransferase had no measurable activity toward estrone. Pregnenolone sulfotransferase from both sexes yielded variable elution patterns during chromatofocusing. Estrogen sulfotransferase from the adrenal, as well as that of guinea pig chorion, was strongly inhibited by N-ethylmaleimide and to a lesser degree by iodoacetamide and iodoacetate. Adrenal and chorion estrogen sulfotransferases were thermolabile and were activated, although not protected from the effect of heat, by binding to 3'-phosphoadenosine 5'-phosphosulfate. Adrenal pregnenolone sulfotransferase was inhibited only by high concentrations of N-ethylmaleimide and not at all by iodoacetamide or iodoacetate. It was more thermostable than the estrogen sulfotransferase and was not activated by binding to 3'-phosphoadenosine 5'-phosphosulfate.     

Heterogeneity of guinea pig chorion and liver estrogen sulfotransferases

The presence of two forms of estrogen sulfotransferase (EST) in 105,000 g cytosols of guinea pig chorion and liver has been established by chromatofocusing via a fast protein liquid chromatographic (FPLC) procedure. The chorion EST forms were eluted at pH 6.2 and 5.4, and the liver forms at 6.1 and 5.3. Each has been further purified by an affinity column step using Agarose-hexane-adenosine-3',5'-diphosphate (PAP-Agarose) gel to achieve up to 386-fold and 77-fold specific activity (SA) increases over cytosol for chorion and liver, respectively. The most highly purified preparations were extremely unstable unless protected by the addition of serum albumin of high purity. Each EST form exhibited an estimated molecular weight of 48-52 KDa by FPLC gel filtration and each acted upon both estrone (E1) and estradiol (E2). Each of these steroids inhibited sulfation of the other. A departure from Michaelis-Menten kinetics occurred, particularly in the case of chorion EST, at steroid substrate concentrations above 0.1-0.15 microM. E2 caused strong substrate inhibition of the most highly purified chorion EST. Chorion EST possessed considerable affinity for E1 and E2.     

Sulfation by guinea pig chorion and uterus: Differential action towards estrone and estradiol

The activities of estrogen sulfotransferase, estrogen sulfatase and estradiol 17β-dehydrogenase change considerably in the guinea pig uterine compartment during gestation. This study was undertaken to enquire if the chorion membrane could influence the pattern of estrogen resulting when substrates were applied to the fetal surface of the chorion while it was attached, late in gestation, to the uterine wall. This tissue system resulted in a differential handling of estrone and estradiol. Estrone was largely excluded from the tissue, remaining mainly in free steroidal form. Estradiol was considerably converted to its 3-sulfate which was mainly retained by the chorion. Parallel experiments with chorion and uterus separately failed to discriminate between the two substrates. Hydrolysis of estrone sulfate and estradiol 3-sulfate was similar in all three tissue systems. It appears that the interaction of chorion with uterus late in gestation causes a difference in tissue action towards the two steroid substrates of closely related structure. The results suggest a limitation in tissue uptake of estrone compared with estradiol, or a much greater sulfotransferase activity towards estradiol. Whole cytosols of late gestational chorion catalyzed sulfation of estradiol at about double the velocity of estrone. This may only partly account for the difference in the intact chorion-uterine tissue system.     

Characteristics and behavior during partial purification of estrogen sulfotransferase of guinea pig liver and chorion

Some characteristics of estrogen sulfotransferases from guinea pig liver and chorion were compared. Liver cytosolic activity was stimulated 10-fold by 25 mM monothiolglycerol and 2-fold by 15 mM MgCl2 or CaCl2, similar to that found previously for chorion. Liver and chorion activities were each eluted as a single peak from fast protein liquid chromatography (FPLC) gel filtration columns at apparent molecular weights of 52,300 and 50,000, respectively. Each was eluted during FPLC anion exchange under single, wide peaks with low recoveries. Liver sulfotransferase activity was eluted from Affi-gel Blue columns in the form of several peaks whereas the chorion activity behaved as a single species. The enzymes from both tissues, when partially purified by gel filtration followed by anion exchange, acted upon estrone and estradiol at the 3-position but activity toward dehydroepiandrosterone and testosterone was minimal or undetectable. Affi-gel Blue chromatography followed by FPLC gel filtration resulted in increases in specific activity of 26- and 90-fold for liver and chorion, respectively. Both enzymes were eluted from agarose-hexane-adenosine 3',5'-diphosphate (PAP-agarose) columns as single peaks. Average increases in specific activity for this column step were 40-fold and 96-fold for the entire eluted peaks of liver and chorion enzyme, respectively. Individual fractions from the PAP-agarose column indicated a specific activity increase of as much as 60-fold for liver and 208-fold for chorion. These latter were markedly unstable and it was not possible to obtain further purification by additional steps. Velocity versus substrate concentration curves for the partially purified enzymes showed complex kinetics, particularly with estradiol as substrate.     

Unconjugated and sulfoconjugated steroids in plasma and zones of the adrenal cortex of the guinea pig

The following steroids were measured in their unconjugated and sulfoconjugated forms in plasma and in the outer and inner zones of the adrenal cortex of the guinea pig: pregnenolone, 17-hydroxypregnenolone, 21-hydroxypregnenolone, dehydroepiandrosterone and deoxycorticosterone. In plasma, pregnenolone and 21-hydroxypregnenolone were the predominant unconjugated steroids with concentrations 10-30 times higher than the other three steroids. Among the sulfoconjugated steroids, pregnenolone sulfate had a concentration 25-50 times higher than the other sulfoconjugates. For each steroid except 21-hydroxypregnenolone the sulfoconjugated form was present in a concentration 2-7 times higher than the unconjugated form. In the adrenal cortex, the content of 21-hydroxypregnenolone was significantly higher in the outer zone than in the inner zone and was present in amounts 3-100 times greater than the other unconjugated steroids in the outer zone. On the other hand, the content of pregnenolone was significantly greater in the inner zone than the outer zone, and was present in amounts 3-80 times greater than the other unconjugated steroids in the inner zone. With the exception of 21-hydroxypregnenolone and deoxycorticosterone, the steroid sulfoconjugates were significantly higher in the inner cortical zone. As in plasma, pregnenolone sulfate was the most abundant sulfoconjugated steroid. This report also describes preliminary studies concerning sulfurylated hydroxyl groups in different positions of 21-hydroxypregnenolone. The sulfoconjugate was prepared by using partially purified steroid sulfotransferase from the guinea pig adrenal. The results obtained indicated that of the total 21-hydroxypregnenolone conjugate formed, approximately 40% was the 21-sulfate and 20% the 3-sulfate, whereas 40% was non-hydrolyzable with the techniques used and was not further characterized.     

Testosterone sulfotransferase: evidence in the guinea pig that this reaction is carried out by 3 alpha-hydroxysteroid sulfotransferase.

During the course of isolating, characterizing, and cloning estrogen and 3-hydroxysteroid sulfotransferases from the guinea pig adrenal gland, it was noted that cytosolic preparations from this tissue would also sulfonate testosterone. Therefore, we set out to isolate and clone the enzyme that performs this reaction. Testosterone sulfotransferase (TST) was isolated from the guinea pig adrenal by using the standard procedures of ion exchange, affinity, and high-performance liquid chromatography. When purified, TST was examined by liquid-phase nondenaturing isoelectric focusing, it was found that the TST activity profile completely overlapped with the activity profile of the 3alpha-hydroxysteroid sulfotransferase (3alphaHST) isoform, but not the 3beta-hydroxysteroid sulfotransferase (3betaHST) isoform. This finding was further investigated by overexpressing the cDNAs for 3alphaHST and 3betaHST in Escherichia coli and examining the expressed proteins for TST activity. This experiment confirmed that 3alphaHST does indeed function as a TST. In addition, 3alphaHST was also found to sulfonate estradiol but not estrone, a finding that further suggested that 3alphaHST may function as a general 17beta-hydroxysteroid sulfotransferase.     

Enzymatic sulfation of bile salts: III. Enzymatic sulfation of taurolithocholate in human and guinea pig fetuses and adults

The activities of bile salt sulfotransferase, the enzyme responsible for the sulfation of bile salts, were determined in fetal and adult livers of humans and guinea pigs. Fetal enzyme activities in guinea pigs were approximately one-tenth of the adult and increased gradually as the gestation progressed. The bile salt sulfotransferase activities were found in human fetal livers but only 14% of the adult fatty liver activity. The result indicates human or guinea pig fetuses are capable of sulfating lithocholate derived from the mother.     

Estrogen sulfatase and steroid sulfatase activities in intrauterine tissues of the pregnant guinea pig

The possible role of intrauterine estrogen sulfatase and steroid sulfatase around the time of parturition in the guinea pig was investigated. [3H]Estrone sulfate or [3H]pregnenolone sulfate was incubated with intrauterine tissues. Estrogen sulfatase was found in placenta, endometrium, decidua basalis, amnion and chorion. The presence of steroid sulfatase was established in endometrium and decidua basalis but not in placenta or the fetal membranes. Examination of activities in early (days 32-35), mid (days 44-46) and late (within 5 days of parturition) gestation revealed no significant change in estrogen sulfatase specific activity in decidua basalis. However, in chorion and endometrium this activity was seen to increase approx. 12-fold (P less than 0.001) and 2.8-fold (P less than 0.001), respectively, from early to late gestation. In placenta, estrogen sulfatase activity appeared to increase 2.4-fold (P less than 0.001) and in amnion it decreased 2.8-fold (P less than 0.002). Steroid sulfatase activity in decidua basalis did not change during gestation, while activity in endometrium was found to increase by a factor of 5.3 (P less than 0.001), from early to late gestation. The increases, both in estrogen sulfatase activity in chorion, endometrium and placenta and in steroid sulfatase activity in endometrium, occurred primarily within the final 3 weeks of gestation. In contrast, the decrease in estrogen sulfatase activity in amnion occurred principally between the fifth and sixth weeks of gestation. Analysis of radiolabelled metabolites indicated that estradiol and progesterone could be produced via estrogen sulfatase and steroid sulfatase activities in certain tissues. Subcellular fractionation of tissues revealed that the greatest specific activity and total activity, in all cases, was associated with the 105,000 g pellet. Significant activity was also detected in the 750 and 10,000 g pellets but not in the 105,000 g supernatant. Radioimmunoassay of endogenous estradiol-17 beta (estradiol) in chorion extracts revealed a 6.3-fold increase in the hormone from mid to late gestation. Estradiol levels in endometrium and myometrium did not appear to change during this time. It was concluded that increased estrogen sulfatase activity in guinea pig chorion in late gestation occurs along with elevated levels of the hormone estradiol which may be important for parturition in this species.     

Production and secretion of C-19 steroids by rat and guinea pig adrenals

The concentrations of C-19 steroids were measured in guinea pig and rat adrenals before and after castration as well as after stimulation with adrenocorticotropin hormone (ACTH). Characterization of adrenal C-19 steroids was also carried out by isolation with high-performance liquid chromatography and gas chromatography/mass spectrometry (GC/MS). From radioimmunoassay (RIA) data, androstenedione (4-DIONE) and 11 beta hydroxyandrostenedione (11 beta-DIONE) were the major C-19 steroids found in guinea pig adrenals, and castration induced a decrease of 4-DIONE levels only while all other C-19 steroids remained unchanged. In rat adrenals, the major C-19 steroids were 4-DIONE and testosterone, and they were also markedly inhibited after castration. With the exception of 11 beta-DIONE, all other C-19 steroids in circulation were eliminated after castration in both animals species. After ACTH administration in the guinea pig, adrenal 4-DIONE and 11 beta-DIONE levels were markedly stimulated, while an increase of only 11 beta-DIONE was observed in plasma. In the rat, ACTH had a small stimulatory effect on adrenal 52-androstane-3 alpha, 17 beta-diol (3 alpha-DIOL) and plasma 11 beta-DIONE levels. Analysis of guinea pig adrenal steroids by GC/MS confirmed the presence of C-19 steroids in adrenals (namely, 4-DIONE and 11 beta-DIONE) while, in the rat, this could not be confirmed. Our data indicate that production of C-19 steroids occurs in guinea pig adrenals, and 11 beta-DIONE is the major C-19 steroid as well as the only C-19 steroid secreted into the circulation. In the rat, the production of C-19 steroids detected by RIA is not supported by GC/MS data.     

Estrogen sulfotransferase activity in guinea pig uterus and chorion

An estrogen sulfotransferase (ST) is detectable in high speed supernatants of pregnant guinea-pig uterus and shows maximum activity between about 47 and 55 days of gestation, with a decrease toward term. No appreciable activity was apparent in the non-pregnant state or before at least 43 days of pregnancy. A considerably higher ST activity is present in chorion as early as 30 days of gestation, and this also decreases toward term. The two ST's exhibit similar KM (0.1-0.13 microM with estrone as substrate) and pI (5.8) values, as well as similar specificities. Estradiol-17 beta and estriol are sulfurylated 82 and 6% that of estrone at equimolar concn. Neither p-nitrophenol nor several neutral steroids are substrates for the enzymes. Enzyme activity is poorly expressed in the absence of thiol groups, the presence of monothioglycerol stimulating uterine and chorion enzymes by 5- and 15-fold, respectively. Stimulation is also observed in the presence of Mg2+, Ca2+ or Mn2+. Chromatofocusing on a poly buffer ion-exchanger from pH 7.4 to 4.0 resulted in elution of a sharp peak of enzyme activity, at pH = 5.8, from both tissues provided that the eluting buffer contained thiol groups and 0.25 M sucrose. This single step resulted in at least a 35- to 100-fold increase in specific activity. The partially purified enzyme from chorion exhibited a KM for estrone of 0.13 microM.     

Characterization of the guinea pig 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase expressed in the adrenal gland and gonads

The guinea pig adrenal gland, analogous to the human, possesses the capacity to synthesize C(19) steroids. In order to further understand the control of guinea pig adrenal steroidogenesis we undertook the characterization of the guinea pig 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD) expressed in the adrenal gland. A cDNA clone encoding guinea pig 3beta-HSD isolated from a guinea pig adrenal library is predicted to encode a protein of 373 amino acid residues and 41,475Da. Ribonuclease protection assay suggests that this cDNA corresponds to the predominant, if not the sole, mRNA species detectable in total RNA from the guinea pig adrenal gland, ovary and testis. The guinea pig 3beta-HSD shows a similar affinity for both pregnenolone and dehydroepiandrosterone, and in addition, a 17beta-HSD type II-like activity was also observed. A phylogenetical analysis of the 3beta-HSD gene family demonstrates that the guinea pig is in a parallel branch to the myomorpha group supporting the hypothesis that the guinea pig lineage has branched off after the divergence among primates, artiodactyls and rodents, suggesting the paraphyly of the order rodentia.     

Bile salt sulfotransferase in guinea pig liver

Bile salt sulfotransferase from guinea pig liver is purified by the procedures of ammonium sulfate fractionation, Sephadex G-100 column chromatography, agarose-hexane-adenosine 3′,5′-diphosphate affinity chromatography and polyacrylamide gel electrophoresis. The purified enzyme exhibits a pH optimum of 6.8, an isoelectric point of 5.6 and a molecular weight of 7600 estimated by gel filtration technique. The apparent K_m values of the enzyme are 7.7·10^?5 M for taurolithocholate and 1.4·10^?6 M for 3′-phosphoadenosine 5′-phosphosulfate. It requires Mg²⁺ and free sulfohydryl group(s) for activity. The enzyme reacts with hydroxy groups of bile salts at both 3α and 3β positions. No activity is found in the kidney of guinea pig. The purified enzyme does not react with estrone, estradiol, testosterone, dehydroepiandrosterone, cholesterol, phenol, tyramine, and serotonin. The results indicate that bile salt sulfotransferase is distinct from other hepatic sulfotransferases.     

Biosynthesis of delta-7-cholesten-3-beta-ol, delta-5,7-cholestadien-3-beta-ol, and delta-5-cholesten-3-beta-ol by guinea pig intestinal mucosa in vitro

Methods were developed for the separation and determination of the various 27-carbon sterols of intestinal mucosa by means of thin-layer chromatography. Scrapings of the mucosa of the small intestine of guinea pig and rat were shown to incorporate isotope from (14)C-labeled acetate and mevalonate into sterols in vitro. For each substrate this activity was lowest in mucosa from the proximal third of the small intestine and greatest in mucosa from the more distal regions of the small intestine. The total 27-carbon sterol content of guinea pig mucosa varied only slightly along the length of the small intestine, but the concentration of cholesterol was highest distally. More than 95% of the radioactivity incorporated from acetate-2-(14)C into 27-carbon sterols by guinea pig mucosa in 4 hr was recovered as lathosterol and 7-dehydrocholesterol; less than 5% was in cholesterol. The specific activities of the 27-carbon sterols were consistent with the concept that synthesis proceeds from lathosterol to 7-dehydrocholesterol to cholesterol.     

Sulfurylation of deoxycorticosterone in human fetal tissues

We determined the specific activity of 21-hydroxysteroid sulfotransferase in a number of human fetal tissues and in tissues of a prepubertal boy (5 years of age). In fetal tissues, the highest specific activities of this enzyme were found in adrenal gland, liver, kidney, intestine, aorta, and testis. In the tissues of the prepubertal boy, 21-hydroxysteroid sulfotransferase activity was demonstrable only in adrenal and liver. Thus, 21-hydroxysteroid sulfotransferase activity is present in some fetal tissues in which DOC may be formed by 21-hydroxylation of progesterone, as steroid 21-hydroxylase activity has been demonstrated previously in adrenal, kidney, and testis. We speculate that sulfurylation of DOC in some tissue sites of DOC formation and action may regulate the action of this mineralocorticosteroid.     

Distribution in tissues and effects by acute hypoxia on angiotensin I converting enzyme activity in guinea pig and chicken

Angiotensin I converting enzyme activity was measured in homogenates of guinea pig and chicken organs (lung, kidney, heart, ileum, diaphragm and liver), using a spectrophotometric assay for hydrolysis of hippuryl-L-histidyl-L-leucine. High specific activities were found in lung, kidney and diaphragm, but the highest corresponded to guinea pig lung and chicken kidney. Acute hypoxia decreased angiotensin I converting enzyme activity in guinea pig lung and chicken diaphragm, but the changes in kidney were considered non-significant in both the guinea pig and chicken.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig.

Studies were carried out to investigate the effects of prostaglandins (PG) in vitro on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE1, PGE2, PGA1, PGF1 alpha or PGF2 alpha to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (delta A385-420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11 beta-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Study of the fetal and maternal renin-angiotensin system and chorio-placental steroids in the guinea pig]

1.) Total renin, active renin, prorenin, angiotensin II, estradiol and progesterone were measured in maternal, placental and fetal blood and in trophoblastic and uterine tissues of the guinea pig. Furthermore, membrane angiotensin II receptors were measured in trophoblastic tissues. 2.) Blood and tissue concentrations of total renin, active renin, angiotensin II and steroids are shown to increase with gestational age. At the full term of pregnancy (70th post-coital day), tissue concentrations of total renin in chorion (23,900 +/- 2,752 ng/g of tissue/h), maternal placenta (14,210 +/- 1,131), fetal placenta (12,475 +/- 927) and uterus (7,677 +/- 798) are 100 time higher than those observed in placental, fetal and maternal blood. Distribution of blood and tissue prorenin (inactive renin) is similar to that found for total renin. Active renin/Total renin ratio reaches 1% in uterine, placental and chorion tissues and 9.3 +/- 1.0% in maternal, placental and fetal blood. 3.) Angiotensin II levels in systemic maternal blood (690 +/- 99 pg/ml) and in uterine blood (467 +/- 84) are higher than those found in placental blood (266 +/- 39) and in different trophoblastic tissues (between 200 and 400 pg/g). Angiotensin II receptor concentrations are highest in chorion. 4.) Regarding the steroid hormones, it is noted that placental and maternal blood contain more progesterone than trophoblastic tissues. The highest concentrations of estradiol are found in chorion tissue and uterine blood. 5.) A positive correlation is observed between angiotensin II and estradiol in uterine blood (r = 0.69, P less than 0.01) and in chorion (r = 0.71, P less than 0.01). These findings indicate that angiotensin II and estradiol could, by their interactions, play an important role in the physiology of pregnancy.