J.B. Adams

Enzymic synthesis of steroid sulphates

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 Mg2+, Ca2+ and Mn2+ but strongly inhibited by Zn2+, Co2− and Ni2−. 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. Km values for 17 β-estradiol and adenosine-3′-phosphate-5′-phosphosulphate were 14 and 70 μM respectively. The Km value for the nucleotide was unchanged in the presence of Mg2− suggesting that the metal ion effect involved the vmax, rather than the binding of the nucleotide to the enzyme.

Hormone-sensitive lipase is involved in the hydrolysis of lipoidal derivatives of estrogens and other steroid hormones

Long-chain fatty acid esters of 17 beta-estradiol and other steroid hormones, which are formed in hormone-sensitive tissues, can be regenerated to the free hormone by the action of an esterase present in the cytosol. This esterase has now been examined in bovine placenta cotyledons. Activity towards steroid fatty acid esters was accompanied by activity towards a diacylglycerol analogue and cholesteryl oleate. During purification procedures, the ratio of activities towards the diacylglycerol analogue and estradiol 17 beta-oleate remained approximately constant. Activity towards these two substrates was inhibited by increasing concentrations of HgCl2 and phenylmethanesulfonyl fluoride in a parallel manner. Upon treatment with [3H]diisopropyl fluorophosphate, a major labelled species of Mr approx. 84,000 was formed. Activation by ATP and the catalytic subunit of cAMP-dependent protein kinase occurred. These properties were very similar to those of the hormone-sensitive lipase of bovine adipose tissue previously reported and run in parallel in this study. A highly purified preparation of this latter enzyme was found to hydrolyse steroid fatty acid esters and relative activities towards such substrates, diacylglycerol analogue and cholesteryl oleate, were similar to the placenta esterase. When the two esterases were phosphorylated with [gamma-32P]ATP, a labelled species of Mr 84,000 was isolated in both cases by use of an antibody raised against purified hormone-sensitive lipase of bovine adipose tissue. It is concluded that hormone-sensitive lipase is very likely the enzyme responsible for hydrolysis of steroid fatty acid esters in bovine placenta and possibly steroid hormone target tissues in general.

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

Abstract 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 XII. Isolation of dehydroepiandrosterone sulphotransferase from human adrenals by affinity chromatography

Abstract Steroid alcohol sulphotransferases (EC 2.8.2.—) have not previously been obtained in a pure state possibly because of their inherent instability. A rapid isolation procedure involving affinity chromatography was developed and initially applied to the isolation of dehydroepiandrosterone sulphotransferase from human adrenals, since the sulphate ester of this steroid is quantitatively (along with unconjugated cortisol) the most important secretory product of the human adrenal cortex. By use of the coupled product of dehydroepiandrosterone-17-( O -carboxymethyl) oxime and AH Sepharose 4B, the enzyme was isolated in one step from an (NH 4 ) 2 SO 4 cut derived from the cytosol of human adrenal glands. Enzyme activity, employing dehydroepiandrosterone or etiocholanolone as substrates, was associated with the major band revealed on acrylamide gel electrophoresis. One or two very minor bands, lacking enzyme activity, were also usually present. On sodium dodecyl sulphate gels, a band having a molecular weight of 34 500 was obtained. By sucrose gradient ultracentrifugation the active enzyme was found to have a molecular weight of 68 000 and thus contains two subunits. These appear to be identical, as judged by fingerprint data of the tryptic peptides and the amino acid composition. Steroids other than dehydroepiandrosterone acted as substrates. The decreasing order of sulphurylation rates were: epiandrosterone, 1.6; androst-5-ene-3β,17α-diol, 1,4; dehydroepiandrosterone and pregnenolone, 1.0; etiocholanolone, 0.89; androst-5-ene-3β,17β-diol, 0.75; androsterone, 0.44; testosterone, 0.15; estradiol-17β, 0.17; 11-deoxycorticosterone, 0.10. Complex wave-like curves were obtained when either substrate, i.e. dehydroepiandrosterone or 3′-phosphoadenosine-5′-phosphosulphate, was varied in the presence of a fixed concentration of the cosubstrate. In marked contrast, enzyme isolated from bovine liver using the same affinity gel, yielded normal Michaelis-Menten kinetics.

Adrenal androgens and human breast cancer: A new appraisal

A clearer picture of the role of adrenal androgens in the etiology of breast cancer is beginning to emerge. Women who develop breast cancer in premenopausal years tend to have subnormal serum levels of adrenal androgens, while subjects who develop the disease in postmenopausal years have supranormal levels of these hormones. Androgens, by acting via the androgen receptor, oppose estrogen-stimulated cell growth in premenopausal years. In postmenopausal women, elevated adrenal androgen levels stimulate cell growth by the action of the unique adrenal androgen 5-androstene-3β,17β-diol, also termed hermaphrodiol, via its combination with the estrogen receptor in a hormone milieu lacking, or having low concentrations of, the classical estrogen 17β-estradiol.

Steroid hormones and human breast cancer. An hypothesis.

The likely occurrence of two distinct types of human breast cancer is discussed. In the development of the “Western‐environmental” or “adrenal” type, it is proposed that dehydroepiandrosterone sulphate (DHEAS), secreted by the adrenal, may be implicated. This steroid is metabolized by mammary tumors to active androgens and estrogens. That these metabolic pathways are very similar to that in skin is emphasized and this is possibly explained by evolution of the mammary gland from primitive sweat glands. Formation of DHEAS from DHEA in mammary tumors, claimed to be correlated with an individual subjects prognosis and response to hormone ablation, is discussed in the light of a possible regulatory role of the sulphotransferase. It is proposed furthermore that a specific control of DHEAS secretion in the adrenal may exist at the level of the sulphurylation step. Nutritional factors are also possibly implicated here and these are discussed in the light of: 1) a possible higher secretion rate of DHEAS in obese subjects; and 2) the low breast cancer incidence in the Japanese and the accompanying low blood DHEAS levels. Cancer 40:325–333, 1977.

Products of dehydroepiandrosterone metabolism by human mammary tumors and their influence on estradiol receptor binding

The high concentrations of dehydroepiandrosterone and its 3beta-sulfate in the blood are potential preocursors for further metabolism by normal and tumorous human mammary tissue. In vitro metabolism of 7n-3H- and 1,2,6,7(n)-3H-dehydroepiandrosterone by fifteen mammary tumors was examined. Some 10--50% of the radioactivity recovered was in the form of 7-oxygenated derivatives: the major metabolite being 7alpha-hydroxydehydroepiandrosterone accompanied by lesser amounts of the 7beta-epimer. 5-Androstene-3beta,17beta-diol was formed in all but one case. Evidence showed that the high yield of 7alpha-hydroxy derivative resulted from direct action of a 7alpha-hydroxylase capable of using both dehydroepiandrosterone and 5-androstene-3beta,17beta-diol as substrates. Although 5-androstene-3beta,17beta-diol competed with estradiol-17beta for the estrogen receptor, this property was considerably reduced as a consequence of the introduction of a 7alpha-hydroxyl or 16alpha-hydroxyl group. Dehydroepiandrosterone, which competed less effectively for the estrogen receptor site, showed almost no affinity for the site upon the introduction of a 7alhpa-hydrocyl group. A regulatory role for the 7alpha-hydroxylase is outlined.

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

Abstract 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.

Properties of fatty acyl-coenzyme A: estradiol-17β acyltransferase in bovine placenta microsomes

The properties of the enzyme catalyzing the formation of non-polar derivatives of estradiol-17 beta (E2) esterified to long-chain fatty acids have been investigated in microsomal preparations from bovine placenta cotyledons. A rapid enzyme assay has been developed which involves simple solvent partitioning. The membrane-bound enzyme showed a pH optimum of 5.0 and addition of fatty acyl-coenzymes A (CoAs), such as oleoyl-CoA, palmitoyl-CoA and palmitoleoyl-CoA, increased [3H]E2-fatty acyl ester formation from [3H]E2 by some 7-fold. Linoleoyl-CoA, linolenoyl-CoA and arachidonoyl-CoA were much less effective as acyl donors. Only 17 beta-fatty acyl monoesters were synthesized in each instance. Similar results were obtained with microsomes or mitochondria from bovine endometrium. The apparent Km for E2 employing placenta microsomes was 8.0 +/- 2.2 (SD) microM. Steroids such as testosterone, dehydroepiandrosterone and 5-androstene-3 beta, 17 beta-diol acted as competitive inhibitors (Ki values 79, 46 and 39 microM, respectively). These, and other data to be reported separately, which showed that these steroids were substrates for the enzyme, demonstrate that the latter is not specific for E2. The [3H]E2-fatty acyl ester fractions biosynthesized from [3H]E2 and bovine placental or endometrial tissue were analyzed by high pressure liquid chromatography (HPLC) and were found to have similar compositions characterized by a high percentage of unsaturated fatty acids.

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

Abstract 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.