Oestrogen Sulphotransferase Research Articles

Enzymic synthesis of steroid sulphates: XI. Study of the oestrogen binding site of oestrogen sulphotransferase by affinity labelling with 4-Mercuri-17β-oestradiol

Oestrogen sulphotransferase (3′-phosphoadenylylsulphate: oestrone sulphotransferase, EC 2.8.2.4) contains a single sulphydryl group thought to be at, or near, the oestrogen-binding site. 4-Mercuri-17β-oestradiol, synthesised by Chin and Warren (Chin, C-C. and Warren, J.C. (1968) J. Biol. Chem. 243, 5056–5062) as an affinity label for oestrogen-binding sites on proteins, was used to gain further information. Upon reaction of enzyme with 4-mercuri-17β-oestradiol, the activity of the enzyme decreased with increasing concentration of the oestrogen derivative. However, some 40% of the activity remained when all the sulphydryl had reacted to form mercaptide. Formation of mercaptide was only marginally decreased in the presence of the substrate 17β-oestradiol. Other steroids, such as 11-deoxycorticosterone and testosterone, which are non-substrates for the enzyme, were more effective than 17β-oestradiol in inhibiting mercaptide formation. Bovine serum albumin also reacted with 4-mercuri-17β-oestradiol and the effects of various steroids on mercaptide formation by the affinity label closely paralleled those found for the enzyme. It is concluded that the single sulphydryl group in the enzyme is not directly involved in the binding of oestrogen at the active site but is perhaps in closer proximity to a second site capable of binding certain non-substrate steroids.

Biosynthesis of 17beta-oestradiol in human breast carcinoma tissue and a novel method for its characterization.

Conversion of 7alpha3H-testosterone to 17beta-oestradiol by human mammary carcinoma tissue in vitro has been demonstrated. It was characterized unequivocally by conversion to 17beta-oestradiol-3-sulphate upon incubation with adenosine-3'-phosphate-5'-phosphosulphate and the highly specific enzyme oestrogen sulphotransferase.

Enzymic synthesis of steroid sulphates: X. Isolation of oestrogenn sulphotransferase from bovine placenta and comparison of its properties with adrenal oestrogen sulphotransferase

Oestrogen sulphotransferase (3′-phosphoadenylylsulphate-oestrone sulphotransferase, EC 2.8.2.4) was isolated from bovine placenta by the same methods developed for the purification of bovine adrenal oestrogen sulphotransferase (Adams, J. B., Ellyard, R. K. and Low, J., (1974) Biochim. Biophys. Acta 370, 160–188). The isoenzyme patterns, substrate specificity, molecular weight, amino acid composition, kinetic behaviour and effect of thiols were studied. All of these properties were identical to oestrogen sulphotransferase of adrenal origin. Evidence was provided that oestrone was bound to the purified placental enzyme.

Enzymic synthesis of steroid sulphates: IX. Physical and chemical properties of purified oestrogen sulphotransferase from bovine adrenal glands, the nature of its isoenzymic forms and a proposed model to explain its wave-like kinetics

A method is described for the preparation of oestrogen sulphotransferase (3′-phosphoadenylylsulphate:oestrone sulphotransferase, EC 2.8.2.4) in pure form from bovine adrenal glands. Analytical ultracentrifuge studies gave s 0 20. w = 4.92. The molecular weight, as determined by centrifugation methods, was 74 000 ± 700. No evidence of the presence of subunits, or of an association-dissociation system, could be found. The presence of substrates had no effect on the sedimentation properties. After reduction and alkylation, 23 residues of S-carboxymethyl cysteine per mole were found. In the native enzyme only 0.2 SH groups per mole could be titrated; this value being increased to approximately 1.0 upon addition of sodium dodecylsulphate. Addition of thiols resulted in activation. Dithiothreitol activated when low oestrogen levels were used, but inhibited when high estrogen levels were used in assays, p-Hydroxymercuribenzoate, at 0.1 mM, was inhibitory only when the enzyme was assayed at low oestrogen concentrations. Higher concentrations (0.5 mM) inhibited at all oestrogen levels. Wave-like kinetics were obtained when oestrogen was varied at constant 3′-phosphadenylsulphate but normal Michaelis-Menten kinetics were obtained when 3′-phosphoadenylylsulphate was the variable substrate. Inhibition studies with adenine nucleotides indicated that both 3′- and 5′-phosphate groups are probably involved in the binding of the substrate to the enzyme. Four isoenzymes have been isolated and their properties compared. Each isoenzyme consists of a single polypeptide chain of molecular weight about 74 000. Amino acid analyses were similar, but significant differences were observed particularly in Lys, Asp, His and Met. The specific activities differed and partial resolution of the isoenzymes, which occurred upon chromatography on ion-exchange columns, resulted in the formation of multi-peaks in the elution profiles. Each isoenzyme exhibited wave-like kinetics on varying oestrogen at constant 3′-phosphoadenylylphosphate. The existence of a number of enzyme conformers is proposed which differ in the accessibility of the single SH group and in their affinity for oestrogen; the existence of these low- and high- K m species then accounting for the wave-like kinetics exhibited by the enzyme.

Enzymic synthesis of steroid sulphates: VIII. Inhibition of estrogen sulphotransferase by retinoic acid and free fatty acids

1. 1. The possibility that vitamin A or its derivatives might act as coenzymes for sulphotransferases was investigated using the enzyme estrogen sulphotransferase (3'-phosphoadenylylsulphate:estrone sulphotransferase, E.C. 2.8.2.4). The purified enzyme did not contain vitamin A, or its derivatives, as judged by lack of absorption in the 360-nm region. Retinol had no effect on enzyme activity at a concentration of o.i mM but retinoic acid, and chemically similar substances such as unsaturated fatty acids, were powerful inhibitors at this concentration. 2. 2. Within the homologous series of straight-chain fatty acids, studied at pH 8.2 in Tris buffer, inhibition became evident at C 11, was maximal at C 14, declined again at C 16 and was minimal at C 18. The corresponding methyl esters in the series were without effect. Inhibitory activity was then broadly related to detergency. Cationic detergents were inhibitory but non-ionic detergents were ineffective. 3. 3. The inhibition by myristate (C 14) was related to the ratio of fatty acid to enzyme protein rather than to the absolute concentration of fatty acid. Inhibition by myristate was non-competitive with respect to the substrate estrone.

Enzymic synthesis of steroid sulphates: III. Isolaton and properties of estrogen sulphotransferase of bovine adrenal glands

1. 1.|Estrogen sulphotransferase (3′-phosphoadenylysulphate:estrone sulphotransferase, EC 2.8.2.4) has been isolated from bovine adrenal glands free of other types of sulphotransferase enzymes. 2. 2.|Two forms of the enzyme were isolated by chromatography on DEAE-cellulose and the properties of the more stable form were studied. 3. 3.|A pH optimum of 8 was found. The enzyme was activated by cysteine but was only moderately sensitive to SH-blocking agents. 4. 4.|Although an absolute requirement for metal ions was not shown, the enzyme was activated by Mg 2+, Ca 2+ and Mn 2+ but strongly inhibited by Zn 2+, Co 2− and Ni 2−. EDTA at concentrations up to 20 mM did not show inhibition of activity measured in the absence of metal ions. 5. 5.|The enzyme was specific for natural estrogens, yielding a monosulphate of the phenolic hydroxyl group. Simple phenols, 2-naphthylamine and 3 β-hydroxysteroids were not sulphated. Stilbestrol and hexestrol, but not dienestrol, were sulphated at very low rates. 6. 6.|Kinetic studies revealed that the mechanism was of the sequential type. K m values for 17 β-estradiol and adenosine-3′-phosphate-5′-phosphosulphate were 14 and 70 μM respectively. The K m value for the nucleotide was unchanged in the presence of Mg 2− suggesting that the metal ion effect involved the v max , rather than the binding of the nucleotide to the enzyme.

Enzymic synthesis of steroid sulphates: IV. The nature of the two forms of estrogen sulphotransferase of bovine adrenals

1. 1. The nature of the two forms (A and B) of estrogen sulphotransferase (3′- phospoadenylylsulphate: estrone sulphotransferase, EC 2.8.2.4) isolated by DEAE-cellulose chromatography, has been investigated. In contrast to the A form, the B form gave non-linear double-reciprocal kinetic plots. Similar plots were obtained when cysteine was added to the A form. 2. 2. Form B was shown to be converted to Form A on standing. 3. 3. Only the B form was obtained when the enzyme was isolated in the presence of mercaptoethanol. 4. 4. Form B was shown to be related to A as trimer (or perhaps tetramer) to monomer, by thin-layer chromatography on Sephadex G-200. The molecular weight of the monomer was estimated to be about 67 000 by this method. 5. 5. The ability to associate to the trimer is suggested to be related to the possession of a conformation dependent on the maintenance of a SH group, or groups, in the reduced state. Non-linear kinetic curves are explained by interaction of binding sites on the associated protein. 6. 6. Allosteric properties exhibited by the fully-associated enzyme are discussed in terms of control mechanisms which may operate at the level of steroid sulphotransferase in endocrine organs. 7. 7. Both forms showed three to four closely migrating protein bands on acrylamide gel electrophoresis which persisted throughout the purification procedures. Evidence was presented that these bands represented individual isoenzymes. 8. 8. The amino acid composition of the highly purified enzyme has been determined.

Enzymic synthesis of steroid sulphates: V. On the binding of estrogens to estrogen sulphotransferase

1. 1. Estrone has been shown to be bound to the estrogen sulphotransferase (3′-phosphoadenylylsulphate: estrone sulphotransferase, EC 2.8.2.4) of bovine adrenals following purification steps involving (NH 4) 2SO 4 precipitations and DEAE-cellulose chromatography. Incubation of the purified enzyme with [ 35S]adenosine-3′-phosphate-5′-phosphosulphate led to the liberation of estrone [ 35S]sulphate which was characterized by chromatography and co-crystallisation with authentic material. Other estrogens were not detected. 2. 2. The binding of estrogens to the A form of the enzyme has been examined by a determination of K m and v max. values. 17-Deoxyestrone acted as a substrate with a K m of 14μM which was identical to the value for other estrogens containing hydroxyl groups on the β-side of ring D. Variation in v max . values amongst this group did, however, occur. 3. 3. Introduction of α-hydroxyl groups into ring D decreased the K m values. 4. 4. An overall fit of the steroid via the rear or α-side to the protein surface is judged to be the best interpretation of these results which is also in keeping with other studies on steroid binding to proteins. Multi-point attachment by way of Van Der Waals forces could be assisted by polar attachments via α-hydroxyl groups on ring D.