Enzymic studies of sulphatases in tissues of the normal human and in metachromatic leukodystrophy with multiple sulphatase deficiencies: arylsulphatases A, B and C, cerebroside sulphatase, psychosine sulphatase and steroid sulphatases

Abstract— Several sulphatases (arylsulphatases A, B and C, cholesterol sulphatase, dehydroepiandroster-one sulphatase, cerebroside sulphatase and psychosine sulphatase) were deficient in various tissues from two patients with a variant form of metachromatic leukodystrophy. Deficient activities of cerebroside sulphatase and psychosine sulphatase, using physiological substrates, in tissues from metachromatic leukodystrophy with multiple sulphatase deficiencies provided another example that these enzymes may be identical to arylsulphatase A. β-Galactosidase activity was reduced to about 30-50 per cent of normal in brain and liver. Other lysosomal enzyme activities were found to be normal or elevated five to eight times. Arylsulphatase B isolated from the liver of one patient was abnormal, with respect to pi (70) and enzyme kinetics. In mixing experiments with normal enzymes the reduced activities of arylsulphatases A. B and C, cerebroside sulphatase and steroid sulphatases were shown not to be due to the presence of endogenous inhibitors. No arylsulphatase A or B activity in the brain specimen from the patient with multiple sulphatase deficiencies could be detected on isoelectric focussing. In normal brain tissue arylsulphatase A had a pi of 4-6-4-8 while arylsulphatase B had a pi of 7-8 and 8-1. When 4-methylumbelliferyl sulphate was used as a substrate the elution patterns of normal brain and liver arylsulphatase B were more heterogeneous and showed more variation than that when p-nitrocatechol sulphate was used. Arylsulphatase C and steroid sulphatases (cholesterol sulphatase, dehydroepiandrosterone sulphatase and oes-trone sulphatase I were solubilized by the addition of lysolecithin and Triton X-100 and subjected to isoelectric focussing. The pi of cholesterol sulphatase, oestrone sulphatase and arylsulphatase C was 6-8, and the elution patterns of the activities of these enzymes were similar. The pattern of dehydroepiandrosterone sulphatase was more heterogeneous and two major peaks were observed at pi 6 5 and 70. Residual enzyme activities of arylsulphatase C and steroid sulphatases from the brain of the patient with multiple sulphatase activities were not detectable by isoelectric focussing. Simultaneous deficiencies of arylsulphatase C and steroid sulphatases plus isoelectric focussing findings in tissues suggest that these enzymes are closely related in regard to their function. The nature of the genetic defect in metachromatic leukodystrophy with multiple sulphatase deficiencies is discussed.     

Activity of sterol-sulphate sulphohydrolase in rat brain: characterization, localization and change with age

Abstract— Homogenates of rat brain hydrolysed the sulphate esters of dehydroepiandrosterone, oestrone and pregnenolone to free steroids. The pH optimum was 6.6 for all three steroid sulphates. Under similar conditions, cholesterol sulphate was not hydrolysed to a significant extent. Unlike sterol sulphatases (EC 3.1.6.2) from extraneural tissues, most of the activity in brain was found in the crude nuclear fraction. The remainder of the activity was found in the crude mitochondrial fraction and almost none was detected in microsomal or cytosol fractions. Sterol sulphatase activity was present in the foetal brain and increased rapidly with increasing postnatal age to a plateau at approx. 25 days of postnatal age. The enzymic activity did not differ significantly with the sex of the animal. The sulphatase activity was found throughout the brain, with cerebellum and brain stem exhibiting a slightly higher activity per wet wt. of tissue than other regions. Inhibition of enzymic activity occurred in the presence of sodium deoxycholate, Triton X-100, sodium dodecyl sulphate and inorganic phosphate or sulphate.     

Studies of the oestrogen sulphatase and arylsulphatase C activities of rat liver

Detailed studies on the hydrolysis of p-acetylphenyl sulphate and oestrone sulphate by rat liver preparations strongly indicate that arylsulphatase C and oestrogen sulphatase are the same enzyme. Liver is the richest source of both enzymes, which have identical intracellular distributions, being localized mainly in the microsomal fraction. Low oestrogen sulphatase and arylsulphatase C activities were present in foetal liver and these increased at a similar rate after birth. The activities of the enzymes in an ethionine-induced hepatoma were similarly low. Results of heat inactivation, mixed-substrate and competitive-inhibition experiments employing liver microsomal fractions were also consistent with one enzyme being involved. Oestradiol-17beta 3-sulphate was also hydrolysed by microsomal preparations and activity towards both this substrate and oestrone sulphate was inhibited by oestrone and oestradiol-17beta. The physiological significance of this inhibition is discussed.     

Sulphatase activities are regulated by the interaction of sulphatase-modifying factor 1 with SUMF2

Sulphatases undergo a unique post-translational modification that converts a highly conserved cysteine located within their active site into formylglycine. This modification is necessary for the catalytic activities of the sulphatases, and it is generated by the protein product of sulphatase-modifying factor 1 (SUMF1), the gene mutated in multiple sulphatase deficiency (MSD). A paralogous gene, SUMF2, was discovered through its sequence similarity to SUMF1. We present evidence that SUMF2 colocalizes with SUMF1 within the endoplasmic reticulum and that the two proteins form heterodimers. SUMF1 and SUMF2 also form homodimers. In addition, SUMF2 is able to associate with the sulphatases with and without SUMF1. We have previously shown that co-transfection of SUMF1 with the sulphatase complementary DNAs greatly enhances the activities of the overexpressed sulphatases. Here, we show that SUMF2 inhibits the enhancing effects of SUMF1 on sulphatases, suggesting that the SUMF1-SUMF2 interaction represents a further level of control of these sulphatase activities.     

Ultrastructural localization of steroid sulphatase in cultured human fibroblasts by immunocytochemistry: A comparative study with lysosomal enzymes and the mannose 6-phosphate receptor

Summary Immunocytochemistry was used to study the subcellular localization of steroid sulphatase in cultured human fibroblasts. Ultra-thin cryosections were incubated with antibodies raised against steroid sulphatase purified from human placenta and immune complexes were visualized with gold probes as electron dense markers. Steroid sulphatase was found in rough endoplasmic reticulum, Golgi cisternae and in the trans-Golgi reticulum, where it co-distributes with lysosomal enzymes and the mannose 6-phosphate receptor. The enzyme was not detected in lysosomes. Steroid sulphatase was also found at the plasma membrane and in the endocytic pathway (i.e. coated pits, endosomes and multivesicular endosomes). These may be the sites where sulphated oestrogen precursors are hydrolysed. Also here, it co-localizes with lysosomal enzymes and the mannose 6-phosphate receptor. It is concluded that microsomal steroid sulphatase and lysosomal enzymes share several cellular compartments.     

Comparative kinetics of the sulphatases A

The kinetic behaviour of the sulphatase A from kangaroo liver is that of a simple hysteretic system involving a substrate-modified form of the enzyme. The equilibrium between the native and substrate-modified forms is influenced by one of the reaction products, sulphate. The behaviour of the system differs markedly from that involving the ox enzyme and a generalised model is presented to account for the behaviour of the sulphatases A from ox, human, rat and kangaroo livers.     

Simultaneous azo-coupling method for steroid acetate hydrolyzing enzyme

A simultaneous azo-coupling method for histochemical localization of steroid acetate hydrolyzing enzyme is described. It is based on the observation that d-equilenin, a natural oestrogenic steroid hormone, forms deeply coloured insoluble reaction products with diazonium salts under reaction conditions suitable for histochemical purposes. An acetate at position 3 of d-equilenin is rapidly hydrolysed by tissue esterase and the liberated d-equilenin couples with a diazonium salt to form a coloured precipitate. Steroid acetate hydrolyzing enzyme activity was observed in various tissues of the rat; a comparison with nonspecific esterase activity using alpha-naphthyl acetate as substrate suggested that steroid acetate hydrolyzing enzyme activity represents the activity of one or several isozymes of classical nonspecific esterase. This conclusion has also been drawn previously from biochemical studies using esters of other steroids.     

Comparison of the cerebroside sulphatase and the arylsulphatase activity of human sulphatase A in the absence of activators.

A cerebroside sulphatase (cerebroside-3-sulphate 3 sulphohydrolase, EC 3.1.6.8) assay based on radio thin-layer chromatography is described. The substrate was labelled by the catalytic addition of tritium to cerebroside sulphate. Using this assay the cerebroside sulphatase activity of sulphatase A (Aryl-sulphate sulphohydrolase, EC 3.1.6.1) from human liver and kidney in the absence of activators was investigated. The pH optimum of this reaction depends on the buffer concentration, being pH 4.5 at 50 mM and 5.3 at 10 mM sodium formate. With the latter concentration the apparent Km for cerebroside sulphate is 0.06 mM; SO2-4 and nitrocatechol sulphate inhibit noncompetitively with a Ki of 4.51 mM for Na2SO4 and 0.43 mM for nitrocatechol sulphate. The cerebroside sulphatase activity of sulphatase A is highly dependent on the ionic strength. The optimum sodium formate concentration is 10 mM, and the cerebroside suophatase activity decreases rapidly with increasing buffer concentration. The same concentration dependence is observed in the inhibitory effect of cerebroside sulphate on the arylsulphatase reaction. The inhibition decreases at increasing buffer concentrations, becoming an activation at 70 mM sodium formate. The progress curve of the cerebroside sulphatase reaction shows a deviation from linearity similar to that of the arylsulphatase reaction. Investigation of the effect of preincubation with cerebroside sulphate on the arylsulphatase activity of the enzyme shows that cerebroside sluphatase activity and inactivation of the enzyme by cerebroside sulphate occur simultaneously. These observations are interpreted as supporting the assumption that cerebroside suophate and arylsulphates are degraded at an identical active site on the same enzyme. Differences in the properties of the cerebroside sulphatase and the arylsulphatase reaction of the enzyme may be attributed to the differences in the physiocochemical state of the two substrates.     

Assay and subcellular localization of the arylsulphatases in rat brain

—The properties and subcellular localization of type I (nitrophenyl) and type II (nitrocatechol) arylsulphatases were investigated in brain tissue of the rat, and optimal assay conditions were established. Sulphate, phosphate and sulphite ions inhibited the nitrocatechol sulphatases; nitrophenyl sulphatase was inhibited only by sulphite. The presence of latent enzyme activity was demonstrated for the nitrocatechol sulphatases, beta-glucuronidase, and beta-glycerophosphatase in rat and mouse brain homogenates. These hydrolases were highly sensitive to mechanical and osmotic damage; and Triton X-100 was very effective in releasing their latent (bound) activities, a finding suggestive of a lysosomal localization. Activity of nitrophenyl sulphatase was unaffected by osmotic changes or Triton X-100, characteristics suggesting a membranous association for this enzyme. Total activity of nitrophenyl sulphatase was approximately twice as great in canine gray matter as in canine white matter; the converse obtained for beta-glucuronidase activity. Values for total enzymic activity of the nitrocatechol sulphatases in canine white and gray matter were similar. Fractionation of homogenates from rat brain by differential centrifugations and separation of crude mitochondrial fractions by sucrose density gradient centrifugations revealed the following: (1) most of the nitrocatechol sulphatase activity (93 per cent) and all of the nitrophenyl sulphatase activity were sedimentable; (2) crude mitochondrial fractions exhibited the highest relative specific activity (RSA = 1·38) for the nitrocatechol sulphatases, whereas microsomal fractions displayed the highest RSA for nitrophenyl sulphatase (1·89); (3) the lightest fraction (A + B) and the densest fraction (E) from the sucrose density gradient contained most of the activity for both the type I and type II arylsulphatases, whereas the RSA of cytochrome oxidase was greatest in the intermediate density regions (fractions C and D); (4) the highest RSA for beta-glucuronidase and beta-glycerophosphatase occurred in gradient fraction C; (5) appreciable activity of beta-glycerophosphatase was found in a nerve ending fraction (M₃). It is suggested that the hydrolases in heterogeneous tissue like brain might be associated with lysosomal particles of differing enzyme compositions and varying populations, and that the data on distribution lend credence to the concept of bimodal and possible trimodal particle affinity for the hydrolases of brain tissues.     

The Development of A-ring Modified Analogues of Oestrone-3-O-sulphamate as Potent Steroid Sulphatase Inhibitors with Reduced Oestrogenicity

Steroid sulphatases regulate the formation of oestrogenic steroids which can support the growth of endocrine-dependent breast tumours. The development of potent steroid sulphatase inhibitors could therefore have considerable therapeutic potential. Several such inhibitors have now been developed of which the most potent to date is oestrone-3-O-sulphamate (EMATE). Unexpectedly, this inhibitor proved to be a potent oestrogen. In an attempt to reduce the oestrogenicity, whilst retaining the potent sulphatase inhibitory properties associated with this type of molecule, a number of A-ring modified derivatives were designed and synthesized. A-ring modified compounds included the 2-methoxy, 2/4-nitro, 2/4-n-propyl and 2/4-allyl EMATE analogues. The ability of these derivatives to inhibit oestrone sulphatase activity was examined using placental microsomes. The allyl-substituted EMATE derivatives were more potent inhibitors than the propyl analogues but were all considerably less potent than EMATE. In contrast, the 2-methoxy and 2/4-nitro analogues were potent sulphatase inhibitors with 4-nitro EMATE being 5 times more active than EMATE. The 4-nitro, 2-methoxy, 4-n-propyl and 4-allyl derivatives were also tested in vivo for their oestrogenicity and ability to inhibit sulphatase activity. While both 4-nitro and 2-methoxy EMATE were potent inhibitors in vivo, 2-methoxy EMATE had no stimulatory effect on uterine growth in ovariectomized rats. The identification of a potent steroid sulphatase inhibitor lacking any oestrogenicity, such as 2-methoxy EMATE, should be of considerable value in evaluating the potential of steroid sulphatase inhibition for breast cancer therapy.     

Human liver N-acetylglucosamine-6-sulphate sulphatase. Catalytic properties.

Kinetic parameters (Km and kcat.) of the two major forms (A and B) and a minor form (C) of human liver N-acetylglucosamine-6-sulphate sulphatase [Freeman, Clements & Hopwood (1987) Biochem. J. 246, 347-354] were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin, heparan sulphate and keratan sulphate. Enzyme activity is highly specific towards glucosamine 6-sulphate or glucose 6-sulphate residues. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are hydrolysed with catalytic efficiencies up to 3900 times above that observed for the monosaccharide substrate N-acetylglucosamine 6-sulphate. Forms A and B both desulphate substrates derived from keratan sulphate and heparin. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue 6-carboxy and 2-sulphate ester groups for heparin-derived substrates and penultimate-residue 6-sulphate ester groups for keratan sulphate-derived substrates. The 4-hydroxy group of the N-acetylglucosamine 6-sulphate or the 2-sulphaminoglucosamine 6-sulphate under enzymic attack is involved in the catalytic mechanism. The presence of a 2-amino group in place of a 2-acetamido or a 2-sulphoamino group considerably decreases the catalytic efficiency of the sulphatase, particularly in the absence of a penultimate-aglycone-residue 6-carboxy group. Both forms A and B are exo-enzymes, since activity towards internal sulphate ester bonds was not observed. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone 2-sulphate ester, 6-carboxy group and 6-sulphate ester groups on the glucosamine 6-sulphate residue under attack considerably affects the pH response. Sulphate and phosphate ions are potent inhibitors of enzyme activity.     

Immunochemical recognition of phosphate ester derivatives of phenol rings is dependent upon the electronic charge of the ester group

The recognition of phosphate and sulphate esters of tyrosine residues has been studied employing antisera with specificity for tyrosine phosphate, and the enzymes aryl sulphatase, and acid and alkaline phosphatases. The ability of tyrosine phosphate, and of phosphate esters of phenol, to inhibit the antiserum was pH dependent. The capacity to effect inhibition appeared to correlate with alterations in the ionisation of the inhibitor. Moreover, the antisera with reactivity for tyrosine phosphate had no reactivity with tyrosine sulphate or sulphate esters of phenol at any pH value studied. The enzymes alkaline phosphatase, acid phosphatase, and aryl sulphatase were also studied. The phosphatases were found not to hydrolyse sulphate ester containing substrate analogues at any pH value in the range 5.0–9.0. In contrast, aryl sulphatase appeared to hydrolyse phosphate esters at pH 5.0 and 7.0, but not at pH 9.0.Abbreviations ABP Azobenzyl phosphonate - KLH-ABP Keyhole limpet haemocyanin derivatised with azobenzyl phosphonate groups - OVA-ABP Ovalbumin derivatised with azobenzylphosphonate groups     

The Diversity and Functions of Choline Sulphatases in Microorganisms

Choline sulphates have two putative roles in microorganisms: as a reservoir of C, N and S and as osmoprotectants. Although there is no established connection to date regarding the relative distribution of these two functions in microbial communities, this information is crucial in determining the role of choline sulphate in soils, particularly in cultivated soils where S is limiting. Therefore, in order to establish such a connection, the diversity of choline sulphatase (betC) genes was investigated in this study using numerous fully sequenced microbes available in GenBank. Our genomic analyses revealed unequivocally that the betICBA operon is restricted to Rhizobiaceae family members, which live under symbiotic conditions that prevent elemental depletion. Together with the uniform genetic organisation of the betICBA operon in Rhizobiaceae, BetC appears to be both utilised for osmoprotection or S replenishment. In contrast, betC in a wide variety of free-living microbes (including fungi, archaea and bacteria) was found in a cassette encoding only BetC and a choline sulphate transporter, a configuration that appears to be responsible for fulfilling elemental S requirements. Lastly, the relatively high number of BetC sequences available allowed the identification of a specific signature sequence that discriminates between these two functions and also globally defines some conserved motifs in microbial choline sulphatases. Due to the widespread presence of BetC in microbes and the wide repartition of the betC cassette system, the potential importance of choline sulphatase in global S recycling requires further clarification.     

The sulphatase of ox liver. XXV. Sulphatase A as a hysteretic enzyme

The kinetic behaviour of the system native--substrate-modified sulphatase A (arylsulphate sulphohydrolase, EC 3.1.6.1) has been investigated and it has been shown that the progress curve of the complete reaction, including both the inactivation and reactivation stages, can be treated as that of a simple hysteretic system in which the substrate-modified enzyme is activated by a product of the reaction. It has been concluded that the early suggestions that the modification of sulphatase A was accompanied by the exposure of a second ligand-binding site are incorrect. It has been shown that, in the absence of sulphate, the rate of reversion of substrate-modified to native sulphatase A is increased by 4-nitrocatechol but not by the same concentration of 2-nitrophenol. A detailed reaction sequence is proposed. This explains the kinetic behaviour of sulphatase A with nitrocatechol sulphate or 2-nitrophenyl sulphate as substrate and in the presence or absence of sulphate.     

3',5'-Cyclic nucleotide phosphodiesterase activity of the sulphatase A of ox liver

The sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver hydrolyses adenosine 3',5'-monophosphate (cyclic AMP) to adenosine 5'-phosphate at an optimum pH of approx. 4.3, close that for the hydrolysis of cerebroside sulphate, a physiological substrate for sulphatase A. The Km is 11.6 mM for cyclic AMP. On polyacrylamide gel electrophoresis sulphatase A migrates as a single protein band which coincides with both the arylsulphatase and phosphodiesterase activities, suggesting that these are due to a single protein. Cyclic AMP competitively inhibits the arylsulphatase activity of sulphatase A, showing that both activities are associated with a single active site on the enzyme. sulphatase A also hydrolyses guanosine 3',5'-monophosphate, but not uridine 3',5'-monophosphate nor adenosine 2',3'-monophosphate.     

Purification and properties of N-acetylgalactosamine 6-sulphate sulphatase from human placenta

1. N-Acetylgalactosamine 6-sulphate sulphatase was purified about 20000-fold from the soluble extract of human placenta with N-acetylgalactosamine 6-sulphate-glucuronic acid-N-acetyl[1-(3)H]galactosaminitol 6-sulphate as substrate in the activity assay. The enzyme appears to be a glycoprotein with a mol.wt. of about 100000 as determined by gel filtration. On gel electrophoresis in the presence of sodium dodecyl sulphate the major protein band had a mol.wt. of 78000. Variable charge heterogeneity was observed in several enzyme preparations. 2. The purified enzyme released up to one sulphate molecule from the disulphated trisaccharide. It was active towards N-acetylgalactosamine 6-sulphate and exhibited no measurable N-acetylglucosamine 6-sulphate sulphatase or any other known lysosomal sulphatase activity. Hydrolysis of [1-(3)H]galactitol 6-sulphate was achieved by incubation neither with a crude nor with a purified enzyme preparation. Chondroitin 6-sulphate and keratan sulphate, as well as heparin and heparan sulphate, served as competitive inhibitors of the enzyme. 3. Purified N-acetylgalactosamine 6-sulphate sulphatase activity was optimal at pH4.9 and 4.4 when assayed in 0.02m-sodium acetate buffer and at pH4.2 and 5.2 in 0.1m-sodium acetate buffer. A single pH-optimum at pH4.8 was observed for the crude enzyme and for the purified enzyme after mild periodate treatment. The sulphatase activity was inhibited by a variety of anions and cations and activated by thiol-specific and thiol reagents.     

Genetic complementation of steroid sulphatase after somatic cell hybridization of X-linked ichthyosis and multiple sulphatase deficiency

Summary Complementation studies of steroid sulphatase were carried out in the heterokaryon population of fibroblasts derived from patients with X-linked ichthyosis and multiple sulphatase deficiency. The activity of steroid sulphatase of the fused cell population was constantly higher (approximately 2–5 fold) than that of the unfused cocultivated cells. The occurrence of complementation further supports the hypothesis that at least two different loci control the expression of steroid sulphatase in the human genome.     

Aromatase activity of steroid sulphatase-deficient placentae

Aromatase activity was measured in homogenates from steroid sulphatase deficient and steroid sulphatase positive placentae. The activity of the aromatase complex was determined from the rate of formation of [14C] oestrone plus [14C] oestradiol when [14C] testosterone was incubated with a rate-limiting quantity of homogenate. A reduced level of aromatase activity was found in vitro in 70% of steroid sulphatase-deficient placentae tested, but the deficiency was much less complete than that of steroid sulphatase. The mean (+/- SD) aromatase activity of steroid sulphatase-deficient placentae was 380 +/- 180 pmol product/h/g tissue (n = 10), significantly lower (P less than 0.001, t-test) than the mean aromatase activity of steroid sulphatase positive placentae (701 +/- 70 pmol product/h/g tissue, n = 10). Seventy per cent of the steroid sulphatase deficient placentae showed lower aromatase activity than that of normal placentae stored for a comparable period of time. Assay imprecision and the sex of the foetus were excluded as reasons for the diminished aromatase activity found. It may arise through an abnormal gene product and consequent alterations in the structure of the microsomal membrane in which both aromatase complex and steroid sulphatase enzymes are retained.     

Recent advances in the development of steroid sulphatase inhibitors

Inhibition of steroid sulphatase is now an important target for the development of new drugs for the treatment of women with endocrine-dependent breast tumours. The first potent sulphatase inhibitor identified, oestrone-3-O-sulphamate (EMATE) proved, unexpectedly, to be oestrogenic. A number of strategies have therefore been adopted to design and synthesize a non-oestrogenic inhibitor. For this, a number of modifications have been made to the A and D rings of the oestrone nucleus. 2 Methoxyoestrone-3-O-sulphamate, while having similar in vitro and in vivo sulphatase inhibitory potency to that of EMATE, was devoid of oestrogenic activity when tested at 2 mg/kg in an ovariectomised rat uterine weight gain assay. 17-Deoxyoestrone-3-O-sulphamate was also a potent steroid sulphatase inhibitor and while it was devoid of oestrogenic activity when tested at 0.1 mg/kg, did stimulate uterine growth at 1.0 mg/kg. As an alternative approach to the use of steroid-based inhibitors a number of single ring, bicyclic non-fused ring, and two fused ring sulphamate analogues were designed, synthesized and tested for their ability to inhibit steroid sulphatase activity. In general, although the single ring and bicyclic non-fused ring sulphamate analogues could inhibit sulphatase activity, they were considerably less potent than EMATE. The mono- and bis-sulphamate derivatives of 5,7-dihydroxyisoflavone were relatively potent, inhibiting in vivo steroid sulphatase activity by 62 and 81% respectively at a single oral dose of 10 mg/kg. A study of the structure-activity relationship of a series of coumarin-based sulphamates has led to the development of a number of potent non-steroidal inhibitors, one of which has a similar potency to that of EMATE. The identification of potent steroid- and non-steroid-based sulphatase inhibitors will enable the therapeutic value of this therapy to be examined in the near future.     

Inhibition of steroid sulphatase activity by steroidal methylthiophosphonates: Potential therapeutic agents in breast cancer

The hydrolysis of steroid sulphates, by steroid sulphatase, is an important source of oestrogenic steroids (oestrone, oestradiol and 5-androstene-3β,17β-diol) which are found in tumours. In the present study, we have examined the effect of dehydroepiandrosterone-3-O-methylthiophosphonate (DHA-3-MTP), pregnenolone-3-O-methylthiophosphonate (pregnenolone-3-MTP) and cholesterol-3-O-methylthiophosphonate (cholesterol-3-MTP) on the inhibition of oestrone sulphatase as well as DHA sulphatase activities in intact MCF-7 breast cancer cells and in placental microsomes. All three methylthiophosphonates significantly (P< 0.01) inhibited the hydrolysis of oestrone sulphate (E₁ S) in intact MCF-7 cells (31–85% inhibition at 1 μM and 53–97% inhibition at 10 μM). Significant inhibition of DHA sulphatase was also achieved. At a concentration of 50 μM, all three compounds inhibited the hydrolysis of dehydroepiandrosterone sulphate (DHAS) by > 95%. Using human placental microsomes, the K_m and V_max of E₁S were determined to be 8.1 μM and 43 nmol/h/mg protein. The corresponding K_i values for DHA-3-MTP, pregnenolone-3-MTP and cholesterol-3-MTP were found to be 4.5, 1.4 and 6.2 μM, respectively. Such inhibitors which are resistant to metabolism may have considerable potential as therapeutic agents and may have additional advantage over aromatase inhibitors in also reducing tumour concentrations of the oestrogenic steroid, 5-androstene-3β,17β-diol, by inhibiting the hydrolysis of DHAS.