Development of (p-O-sulfamoyl)-N-alkanoyl-phenylalkyl amines as non-steroidal estrone sulfatase inhibitors

Estrogen levels in breast tumors of postmenopausal women are as much as 10 times higher than estrogen levels in plasma, presumably due to in situ formation of estrogen. The major source of estrogen in breast cancer cells may be conversion of estrone sulfate to estrone by the enzyme estrone sulfatase. Thus, inhibitors of estrone sulfatase are potential agents for treatment of estrogen-dependent breast cancer. Several steroidal compounds have been developed that are potent estrone sulfatase inhibitors, most notably estrone-3-O-sulfamate. However, these compounds and their metabolites may have undesired effects, including estrogenicity. To avoid the problems associated with a potentially active steroid nucleus, we designed and synthesized a series of nonsteroidal estrone sulfatase inhibitors, the (p-O-sulfamoyl)-N-alkanoyl phenylalkyl amines. The compounds synthesized vary in the length of their alkanoyl chain and in the number of carbons separating the phenyl ring and the carbonyl carbon. The ability of these compounds to inhibit estrone sulfatase activity was tested using human placental microsomes and intact cultured human breast cancer cells. Estrogenicity was also evaluated, using growth of estrogen-dependent human breast cancer cells. All of the test compounds inhibited estrone sulfatase activity of human placental microsomes to some extent, with the most effective compound having an IC50 value of 72 nM. In general, compounds with longer alkanoyl chains (12-14 carbons) were more effective than those with shorter chains. The test compounds also inhibited estrone sulfatase activity in intact cultures of MDA-MB-231 human breast cancer cells. Again, the longer chain compounds were more effective. In both the placental and breast cancer cell sulfatase assays, the optimal distance between the phenyl ring and the carbonyl carbon was 1-2 carbons. The MCF-7 cell proliferation assay revealed that estrone and estrone-3-O-sulfamate were both estrogenic, but the (p-O-sulfamoyl)-N-alkanoyl phenylalkyl amines were not. Our data indicate the utility of (p-O-sulfamoyl)-N-alkanoyl phenyl alkylamines for inhibition of estrone sulfatase activity. Furthermore, our data support the concept that nonsteroidal estrone sulfatase inhibitors may be useful as therapeutic agents for estrogen-dependent breast cancers.     

An estimate of unique DNA sequence heterozygosity in the human genome

Summary Patients with recessive X-linked ichthyosis Patients with recessive X-linked ichthyosis (RXLI), one hereditary form of scaly skin, lack activity of the enzyme steroid sulfatase in all tissues studied. To investigate the molecular defect underlying the lack of enzyme activity, we prepared antisera against normal enzyme by injecting normal placental microsomal suspensions or partially purified steroid sulfatase into rabbits. Antibody activity was assessed by immunoprecipitation of detergent solubilized steroid sulfatase. In addition, we prepared rabbit antisera against RXLI placental microsomal suspensions. To detect immunologically cross-reactive material in patients' placentas, extracts were studied by immunoblot techniques and by competition with normal enzyme for antibody binding. Patients' extracts did not contain immunoreactive material co-migrating on electrophoresis with purified enzyme nor did they inhibit immunoprecipitation of normal enzyme. Sera from rabbits immunized with RXLI placental microsomes contain no antibodies to normal steroid sulfatase, as judged by their failure to immunoprecipitate normal enzyme or to react with normal steroid sulfatase on immunoblot. Thus the mutation in RXLI appears to reduce steroid sulfatase enzyme protein as well as enzyme activity. Portions of this material have appeared in abstract form in Clinical Research 31:564A, 1983 and 32:138A, 1984     

Purification and properties of steroid sulfatase from human placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.     

Origin of deoxycorticosterone and deoxycorticosterone sulfate in human pregnancy: absence of steroid 21-sulfatase activity in sulfatase-deficient placenta

The activity of steroid 21-sulfatase, the enzyme that catalyzes the hydrolysis of deoxycorticosterone sulfate (DOC-SO4) is demonstrable in human placenta. Thus, it is possible that this placental enzyme, by way of the hydrolysis of either DOC-SO4 or 21-hydroxypregnenolone mono- or di-sulfate of fetal origin, may be important in the biosynthesis of DOC, which is present in the plasma of pregnant women in high concentration. To investigate this issue further, we evaluated steroid 21-sulfatase activity in microsomal preparations of a sulfatase-deficient placenta. Immediately after delivery, at term, of a living male fetus with sulfatase deficiency, a microsome-enriched fraction of placental tissue was prepared; sulfatase activity was evaluated by use of three substrates, viz. dehydroisoandrosterone sulfate (DS), estrone sulfate (E1-SO4), and DOC-SO4, in various concentrations. Similar incubations were conducted with aliquots of a microsome-enriched fraction prepared from placental tissue of a normal fetus that was delivered, at term, within minutes of the time of delivery of the infant with sulfatase deficiency. In microsomal fractions from the normal placenta, each of the steroid sulfates was hydrolyzed. In the absence of microsomes, and in the presence of microsomal fractions from the sulfatase-deficient placenta, the hydrolysis of DOC-SO4 and DS was not detected. Moreover, in microsomes prepared from the sulfatase-deficient placenta, E1-SO4 was hydrolyzed at a rate that was only 10% of that in incubations with microsomal preparations of the normal placenta. We conclude that with sulfatase deficiency, the placenta is deficient not only in sulfatase activity for steroid-3-sulfates but for steroid 21-sulfates, e.g. DOC-SO4, as well.     

Characterization of arylsulfatase C isozymes from human liver and placenta

Arylsulfatase C and steroid sulfatase were thought to be identical enzymes. However, recent evidence showed that human arylsulfatase C consists of two isozymes, s and f. In this study, the biochemical properties of the s form partially purified from human placenta were compared with those of the f form from human liver. Only the placental s form has steroid sulfatase activity and hydrolyses estrone sulfate, dehydroepiandrosterone sulfate and cholesterol sulfate. The liver f form has barely detectable activity towards these sterol sulfates. With the artificial substrate, 4-methylumbelliferyl sulfate, both forms demonstrated a similar KM but the liver enzyme has a pH optimum of 6.9 while the placental form displayed two optima at 7.3 and 5.5. The molecular weight of the native enzyme determined with gel filtration was 183,000 for the s form and 200,000 for the f form and their pI's were also similar at 6.5. However, the T50, temperature at which half of the enzyme activity was lost, was 49.5 degrees C for the f form and 56.8 degrees C for the s form. Polyclonal antibodies raised against the placental form reacted specifically against the s and not the f form. They immuno-precipitated concomitantly greater than 80% of the total placental arylsulfatase C and steroid sulfatase activities while less than 20% of the liver enzyme was immuno-precipitable. In conclusion, the two isozymes s and f of arylsulfatase C in humans purified from placenta and liver, respectively, have similar KM, pI' and native molecular weight. However, they are distinct proteins with different substrate specificity, pH optima, heat-lability and antigenic properties. Only the s form is confirmed to be steroid sulfatase.     

Lack of inhibition of placental estrone sulfatase and aromatase enzymes by vitamin D3 and its analogs

The aromatase and estrone sulfatase enzymes are important sources of biologically active estrogens in postmenopausal women with breast cancer. Promising initial results in the treatment of endocrine-responsive breast cancer have been exhibited by 125-dihydroxyvitamin D₃ and the synthetic vitamin D analogues MC903 and EB1089. However, these compounds together with vitamin D₃ and vitamin D₃ sulfate did not inhibit the human placental aromatase enzyme when assayed up to 20 μm. Only vitamin D₃ sulfate and 125-dihydroxyvitamin D inhibited the estrone sulfatase activity in human placental microsomes, albeit at high concentration (32 and 37% inhibition, respectively with 50 μm each inhibitor). It is unlikely that inhibition of aromatase or estrone sulfatase enzymes contribute to the inhibitory effect of this group of compounds on breast cancer cells in vivo.     

Immunochemical specificity of placental NADPH cytochrome c (P-450) reductase in neoplastic and non-neoplastic human tissue.

NADPH cytochrome c (P-450) reductase was purified from human placental microsomes using a combination of affinity and gel filtration chromatography. Affinity chromatography using agarose-hexane-adenosine 2'5 diphosphate resulted in two protein bands being detected by SDS-PAGE of approximate MwS 68 and 75 kDa. Fractions containing the two proteins were pooled, and then resolved using Sephacryl S-200. Both of the purified proteins displayed enzyme activity, measured by their ability to reduce cytochrome c. The 75 kDa protein obtained was used to immunize three female New Zealand white rabbits. The IgG fraction was partly purified from rabbit sera which suppressed placental microsomal NADPH cytochrome c reductase activity by > 80% using 33% ammonium sulphate. The procured antibody suppressed androstenedione aromatase activity in microsomal preparations of human placental and breast adipose tissue, and NADPH cytochrome c reductase activity in prostate (benign and malignant), MDA-MB-231 breast cancer cells, breast adipose, Hep G2 hepatoma cells and placental microsomal preparations. The extent of NADPH cytochrome c reductase inhibition varied in the order of malignant prostate < benign prostate < MDA < breast adipose < Hep G2 < placenta. The results suggest that human placental NADPH cytochrome c (P-450) reductase shares common antigenic epitopes pertinent to its capability of reducing cytochrome c in all of the above-mentioned tissues. In attempting to associate possible changes in NADPH cytochrome c reductase activity imposed by neoplasia to the obtained immunochemical cross reactivity and enzyme activity results, it was noted that microsomes obtained from MDA cells exhibited enzyme activity significantly less than that of breast adipose microsomes (1.6 and 8.1 nmol/min/mg protein, respectively) and by comparison showed 6% less homology towards the placental antibody. The results obtained for benign and malignant prostate showed no significant difference between the neoplastic states as adjudged by enzyme activity and immunochemical assays.     

Comparison of arylsulfatase C and steroid sulfatase from human placenta and liver

Human placental and hepatic arylsulfatase C (ASC) were purified to homogeneity and about 1,000-fold, respectively. Placental ASC hydrolyzed sterol sulfates at the same active site, whereas the major hepatic ASC did not. This major hepatic ASC isozyme was more thermolabile than placental ASC and steroid sulfatase from both placenta and liver. It was not precipitated by anti-bovine ASC IgG which quantitatively precipitated both placental ASC and steroid sulfatase activities from placenta and liver. A minor hepatic ASC isozyme with similar electrophoretic mobility to the placental enzyme copurified with the major hepatic ASC and is likely responsible for the steroid sulfatase activity in this organ. Hence, placental ASC and steroid sulfatase are biochemically and antigenically identical to hepatic steroid sulfatase. In contrast, the major hepatic ASC is a distinct protein whose catalytic and structural properties differ from all the above enzymes.     

Lack of correlation between steroid sulfatase activities and lipid content in uterus and liver microsomes of guinea pigs

Lipid content and steroid sulfatase activities were determined in liver and uterus microsomes of non-pregnant guinea pigs. The results were compared with values obtained in pregnant and cortisol-treated animals. Steroid sulfatase activities were always higher in pregnant animals, and we supposed that the increase in circulating cortisol in pregnant guinea pigs before parturition has an influence on the membrane-bound sulfatase activities. Sulfatase activities were identical in cortisol-treated and untreated non-pregnant females, although cortisol induced changes in microsomal lipid composition. These results lead us to three conclusions: in intact female guinea pigs, cortisol induces variations in the lipid content of uterus and liver microsomes, especially in the cholesteryl sulfate to phospholipid ratios; the variations of the lipid composition in pregnant animals do not appear to be cortisol-dependent; membrane-bound steroid sulfatase activities are not directly influenced by the lipid composition of microsomes.     

Human placental steryl-sulfatase. Enzyme purification, production of antisera, and immunoblotting reactions with normal and sulfatase-deficient placentas

The steryl-sulfatase of normal human placental microsomes was solubilized and enriched about 350-fold. Chromatography on Sepharose 6B of the purified enzyme preparation revealed a single protein peak which eluted according to an apparent molecular mass of 270 +/- 30 kDa; when electrophorized on sodium dodecyl sulfate polyacrylamide gel the sulfatase migrated according to a molecular mass of 64 +/- 4 kDa. Estrogensulfatase activity was co-purified with the steryl-sulfatase activity; obviously, both activities belong to the same enzyme species. The purified sulfatase was injected into three rabbits. Antisera produced by the rabbits yielded a single sharp immunoprecipitation line in Ouchterlony double diffusion experiments when tested with the isolated sulfatase or with a solubilized microsomal fraction of normal placentas. The activity of sulfatase preparations incubated with antiserum was precipitated by addition of polyethylene glycol followed by centrifugation; none of the antibodies reacting with the sulfatase therefore appeared to interfere with its enzymatic activity. Using these antisera, steryl-sulfatase protein could be detected by immunoblotting analysis in solubilized microsomal fractions of normal placentas but not in solubilized microsomal fractions of three steryl-sulfatase activity-deficient placentas. This finding argues in favour of human placental steryl-sulfatase deficiency being due to extremely diminished or absent enzyme protein in the placenta.     

人骨形成蛋白1-cDNA基因工程表达产物抗体的制备及鉴定

应用在大肠杆菌中表达的人骨形成蛋白(human bone morphogenetie protein,HBMP-1)的N端片段作为免疫原,免疫新西兰大白兔,成功地制备了抗人骨形成蛋白的抗血清,经免疫转移电泳证实该抗血清有较好的特异性,用ABC免疫组织化学方法在正常和恶性肿瘤骨的石腊切片上检测抗血清效价为1:100～1:1000,免疫组化染色结果表明,HBMP存在于人胎下颌骨新生的骨质和骨细胞中,颌骨骨肉瘤和软骨肉瘤的瘤细胞呈BMP强阳性反应,这一结果表明,骨的生长、形成和骨肿瘤的发生与BMP密切相关。     

Inhibition of steryl sulfatase activity in LNCaP human prostate cancer cells

The enzyme steryl sulfatase may help support the growth of hormone-dependent tumors, including prostate cancers, by facilitating the conversion of circulating precursor steroids to active hormones. We sought to determine the presence of steryl sulfatase activity in the androgen-dependent human prostate cancer cell line LNCaP, and to determine if this activity was inhibited by known steryl sulfatase inhibitors. Intact LNCaP cultures had steryl sulfatase activity, as determined by conversion of [3H]estrone sulfate (E(1)S) to unconjugated steroids. The level of steryl sulfatase activity was relatively low (4.6 pmol/18 h/million cells) compared to MDA-MB-231 breast cancer cells (284.0 pmol/18 h/million cells). The observed activity in both cell lines was blocked by addition of 1 microM estrone sulfamate (EMATE), an active-site-directed, steroidal inhibitor of steryl sulfatase. Steryl sulfatase activity was also inhibited by Danazol, and by (p-O-sulfamoyl)-tetradecanoyl tyramine (C2-14), a non-steroidal inhibitor. Microsomes prepared from LNCaP cultures also showed steryl sulfatase activity, as determined by hydrolysis of [3H]E(1)S and [3H]dehydroepiandrosterone sulfate (DHEAS) to unconjugated forms. LNCaP and MDA-MB-231 microsomes both hydrolyzed E(1)S about two times faster than DHEAS. Hydrolysis of E(1)S in LNCaP and MDA-MB-231 microsomes was blocked by steryl sulfatase inhibitors with the following relative potencies: EMATE>C2-14>Danazol. These data demonstrate that LNCaP prostate cancer cells contain a steryl sulfatase with properties similar to that found in human breast cancer cells, and that the activity of this enzyme can be blocked by known steryl sulfatase inhibitors. Steryl sulfatase inhibitors may be useful as an adjuvant to androgen deprivation therapy for prostate cancer.     

Clinical and biochemical investigations on patients with partial deficiency of placental steroid sulfatase

Summary We report on three independent cases with a partial deficiency of placental steroid sulfatase (E.C.3.1.6.2). Upon routine pregnancy monitoring these patients were detected on the basis of low estriol excretion and failing induction of labor. In all three cases a male was delivered and subsequently the diagnosis of partial deficiency of placental steroid sulfatase was confirmed enzymatically in placenta homogenates. In one case, fibroblast cultures were established from skin explants of mother and son. In fibroblasts of the child, as in placental tissue, the activity of steroid sulfatase was only 34% of normal. Similar values were obtained for arylsulfatase C, though this enzyme is clearly separable from steroid sulfatase by electrophoresis. In cells of the mother, enzyme activities were unremarkable.     

Development of novel steroid sulfatase inhibitors; II. TZS-8478 potently inhibits the growth of breast tumors in postmenopausal breast cancer model rats

In postmenopausal breast cancer tissue, steroid sulfatase (STS) activity is high and much estrone sulfate also exists; these facts reveal that estrone sulfate may be involved in the growth of breast cancer as an estrogen source. Steroid sulfatase is an enzyme, which catalyzes hydrolysis from estrone sulfate to estrone, and the development of steroid sulfatase inhibitors is expected as novel therapeutic drugs for postmenopausal breast cancer. We have developed a novel compound 2',4'-dicyanobiphenyl-4-O-sulfamate (TZS-8478), which has potent steroid sulfatase-inhibitory activity and exhibits no estrogenicity in vitro and in vivo. To elucidate its usefulness as a therapeutic drug for postmenopausal breast cancer, we examined the breast cancer cell proliferation- and breast tumor growth-inhibitory activity of TZS-8478 in postmenopausal breast cancer model rats. TZS-8478 dose-dependently suppressed the estrone sulfate-stimulated proliferation of MCF-7 cells. Regarding nitrosomethylurea (NMU)-induced postmenopausal breast cancer models, furthermore, TZS-8478 (0.5 mg/kg per day) markedly inhibited the estrone sulfate-stimulated growth of breast tumors similarly to estrone sulfate-depletion. TZS-8478 completely inhibited steroid sulfatase activity in tumor, uterus and liver, and also markedly lowered plasma concentrations of estrone and estradiol. The above mentioned results suggested that TZS-8478 may be useful as a therapeutic drug for estrogen-dependent postmenopausal breast cancer.     

Sex steroid binding protein (SBP) receptors in estrogen sensitive tissues

Since the discovery of a specific membrane binding site for sex steroid binding protein (SBP) in human decidual endometrium and in hyperplastic prostate numerous speculations have been raised on the existence of an additional non-receptor-mediated system for steroid hormone action. In the present work SBP cell membrane binding was investigated in human estrogen target tissues other than those previously studied either in the absence of steroids or in the presence of varying amounts (10⁻¹⁰−10⁻⁶M) of estradiol, testosterone and dihydrotestosterone, respectively. Plasma membranes obtained by differential centrifugation from homogenized samples of pre-menopausal endometrium, endometrium adenocarcinoma, normal liver and post-menopausal breast showed a specific binding of highly purified [¹²⁵I]SBP: a major displacement of labeled SBP was elicited by radioinert SBP, while no significant displacement occurred when other human plasma proteins were used as cold competitors (molar excess ranging 500–10,000-fold). A specific, time-dependent binding of [¹²⁵I]SBP was also observed in MCF-7 and in Hep-G2 cell lines. The different patterns of specific binding, observed in membranes from different tissues when SBP was liganded with different sex steroid molecules, leads us to consider the tissue individuality of the receptor as a further entity in the membrane recognition system for SBP.     

New assay for steroid sulfatase (EC 3.1.6.2) and its application for studies of human placental and skin sulfatase

A new, simple, fast and highly practicable sulfatase assay and its application is described. Sterol sulfatase sulfohydrolase (EC 3.1.6.2) activity is determined by a two-phase scintillation technique separating the unreacted [4-14C]dehydroepiandrosterone sulfate from carbon-14-labeled products. The principle of the separation relies on the limited emulsifying capacity of the dioxane-based scintillation solution for water and the different partition of dehydroepiandrosterone sulfate and sulfate-free steroid products between the scintillation fluid and the aqueous phase as recently applied for determination of aromatase activity [1]. [7-3H]Dehydroepiandrosterone sulfate can also be used as a substrate for this assay. This test was applied to studies of microsomal sulfatase prepared from human term placenta and to the detection of sulfatase activity in human skin biopsies. Using placental microsomes, the Km of dehydroepiandrosterone sulfate was determined to be 5.0 X 10(7)M. Sulfatase activity in frozen scrotal skin was found to be 2-3 fold than with vaginal skin. Using an incubation time of 24h/skin sulfatase can be detected in biopsies as small as 2.5 mm2. The sulfatase assay can be applied for routine detection of human placental sulfatase deficiency and, furthermore, the application of this assay has to be demonstrated for the analysis of sulfatase activity in patients with congenital ichthyosis (X-chromosomal, recessive type).     

Immunohistochemical localization of sex hormone-binding globulin in normal and neoplastic breast tissue.

The distribution of sex hormone-binding globulin-like antigens (SHBG-LA) in normal and neoplastic human breast tissues was investigated by immunohistochemistry, employing a monospecific polyclonal antiserum against highly purified human SHBG and an avidin-biotin-peroxidase complex (ABC) method in formalin-fixed paraffin embedded tissue sections. In normal breast tissues the staining of SHBG-LA was present exclusively in the cytoplasm of epithelial cells of ductal and ductular types. Nuclei as well as stromal and lymphatic tissues remained unstained. While the staining was positive in all cases of intraductal carcinoma, only 4 out of 15 infiltrating carcinomas revealed SHBG-LA. The demonstration of a plasma sex steroid binding globulin in the cytoplasm of endocrine target cells is consistent with the hypothesis that steroid-binding globulins are able to enter target cells. The apparent loss of this specific cell function in infiltrating carcinomas may result from dedifferentiation and change of cell membrane properties occurring during the process of neoplastic progression.     

Steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities in mouse tissues

The metabolism of estrone sulfate and dehydroisoandrosterone sulfate to the free, unconjugated steroids, estrone and dehydroisoandrosterone, was demonstrated in more than thirty different tissues from male and female BALB/c mice. The activity of steroid sulfatase, when expressed per mg tissue, was greatest in both the pituitary gland and the adrenal glands. The pituitary gland, however, had the lowest capacity for hydrolysis of steroid sulfates while the liver had the greatest capacity. 17 beta-Hydroxysteroid oxidoreductase activity also was demonstrated in all mouse tissues by the formation of estradiol-17 beta when using estrone sulfate as the substrate. The highest apparent activity for 17 beta-hydroxysteroid oxidoreductase was found in lung tissue, and the greatest capacity to form estradiol-17 beta from estrone sulfate was found in liver, lungs, kidneys and testes. This study demonstrates that the majority of mouse tissues have steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities.     

Human placental steroid sulfatase: purification and properties

Steroid sulfatase is recovered quantitatively from the 105,000 g h supernatant of human placental microsomes extracted with Triton X-100. The solubilized enzyme has been purified using conventional techniques. Throughout the purification procedure, steroid sulfatase appears to be heterogeneous as evidenced by certain, but not all, criteria. Following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the final preparation exhibits a major component and varying amounts of two minor ones. Antibodies raised in rabbits with the heterogeneous immunogen give rise to a single precipitation line when the native enzyme is analyzed by double immunodiffusion or by immunoelectrophoresis. In addition, using aged preparations of microsomes and immunoaffinity techniques, steroid sulfatase activity was found to be associated with the fastest migrating minor component. This finding would suggest that the apparent heterogeneity of purified steroid sulfatase is linked to degradation processes occurring within the microsomal preparations. Steroid sulfatase has a Stokes radius of 56 A, a sedimentation coefficient of 4.85 +/- 0.15S (in Triton-containing buffers) and binds 1.3 g of Triton X-100-per g of protein. The molecular weight of the Triton-protein complex was calculated to be 166,000 in which the glycoprotein portion contribution is about 43% (72,000). In contrast, the apparent molecular weight of the major polypeptide determined on calibrated SDS-gels is 62,000. The purified enzyme exhibits two pH optima with cholesterol sulfate as substrate, an acidic one at pH 5.0 and a second one at pH 7.5. The Km values for cholesterol sulfate, dehydroandrosterone sulfate and p-nitrophenylsulfate were 5.26, 14 and 1,320 microM, respectively.     

Genetic heterogeneity of steroid sulfatase deficiency revealed with cDNA for human steroid sulfatase

Three cDNA clones with inserts of 1.2-1.6 kb that reacted both with antibodies and oligonucleotides specific for steroid sulfatase were isolated from a human placental library in lambda gt11. The 5'-end of one of the inserts, STS-3, was sequenced and colinearity with the amino acid sequence of 3 peptides of steroid sulfatase encompassing 64 amino acids was demonstrated. STS-3 hybridized with 2.5, 4.6 and 6.3 kb species in poly(A)+RNA and with 2.5, 4 and 9 kb fragments of EcoRI digested human DNA. The frequency of the EcoRI fragments in DNA from females was approximately twice that in DNA from males. DNA from two patients with steroid sulfatase deficiency and X-linked ichthyosis did not hybridize with STS-3. DNA from a third patient showed a normal hybridization pattern. It is concluded that steroid sulfatase deficiency is a genetically heterogenous disorder.