Microsomal Sulfatase Research Articles

Human hepatic microsomal sulfatase catalyzes the hydrolysis of polychlorinated biphenyl sulfates: A potential mechanism for retention of hydroxylated PCBs

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that continue to be of concern due to their varied toxicities. Upon human exposure, many PCBs with lower numbers of chlorine atoms are metabolized to hydroxylated derivatives (OH-PCBs), and cytosolic sulfotransferases can subsequently catalyze the formation of PCB sulfates. Recent studies have indicated that PCB sulfates bind reversibly with a high affinity to human serum proteins, and that they are also taken up by cells and tissues. Since PCB sulfates might be hydrolyzed to the more toxic OH-PCBs, we have investigated the ability of human hepatic microsomal sulfatase to catalyze this reaction. Twelve congeners of PCB sulfates were substrates for the microsomal sulfatase with catalytic rates exceeding that of dehydroepiandrosterone sulfate as a comparison substrate for steroid sulfatase (STS). These results are consistent with an intracellular mechanism for sulfation and de-sulfation that may contribute to retention and increased time of exposure to OH-PCBs.

Effect of 2,3,7,8-tetrachlorodibenzo- p-dioxin administration and high-fat diet on the body weight and hepatic estrogen metabolism in female C3H/HeN mice

We studied the effect of administration of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) by i.p. injection once every 2 weeks in combination with a high-fat (HF) diet for 8 or 16 weeks on the body and organ weight changes as well as on the hepatic enzyme activity for estrogen metabolism in C3H/HeN female mice. Administration of TCDD at 100 μg/kg b.w. once every 2 weeks for 8 weeks increased the body weight by 46% in the HF diet-fed animals, but not in the regular diet-fed animals. This is the first observation suggesting that TCDD at a high dose (100 μg/kg b.w.), but not at lower doses (1 or 10 μg/kg b.w.), may have a strong obesity-inducing effect in C3H/HeN mice fed an HF diet. While TCDD increased liver weight and decreased thymus weight in animals, these effects were enhanced by feeding animals an HF diet. Metabolism studies showed that TCDD administration for 8 or 16 weeks increased the liver microsomal activity for the 2- and 4-hydroxylation of 17β-estradiol in animals fed a control diet, but surprisingly not in animals fed an HF diet. Treatment with TCDD dose-dependently increased the hepatic activity for the O-methylation of catechol estrogens in both control and HF diet-fed animals, and it also decreased the levels of liver microsomal sulfatase activity for hydrolysis of estrone-3-sulfate. TCDD did not significantly affect the hepatic enzyme activity for the glucuronidation or esterification of endogenous estrogens. It is suggested that enhanced metabolic inactivation of endogenous estrogens by hepatic estrogen-metabolizing enzymes in TCDD-treated, control diet-fed animals contributes importantly to the reduced incidence of estrogen-associated tumors in animals treated with TCDD.

Different Biochemical Properties of Nuclear and Microsomal Estrone-3-Sulfatases: Evidence for the Presence of a Nuclear Isozyme

In female rats,totalestrone-3-sulfatase activity per liver in the nuclear fraction is comparable to the total activity per liver in the microsomal fraction. The combined estrone-3-sulfatase activity in the other fractions (lysosomal, mitochondrial, and cytosolic fractions) is negligible and only accounts for <5% of the total nuclear or microsomal sulfatase activity. Nuclear and microsomal estrone-3-sulfatases have different pH optima (pH 8.0 and 7.2, respectively). The apparent Km values for the nuclear and microsomal estrone-3-sulfatases are 2.5 and 10.1 μM, respectively, suggesting that the nuclear sulfatase has a considerably higher affinity for estrone-3-sulfate than the microsomal sulfatase. Moreover, the nuclear estrone-3-sulfatase is more sensitive to inhibition by several steroids than the microsomal sulfatase. The results suggest that estrone-3-sulfatase in the nuclear fraction is a different isozyme than that in the microsomal fraction.

Arylsulfatases: Colorimetric and fluorometric assays

This chapter discusses the colorimetric and fluorometric assays of arylsulfatases. The assay of arylsulfatases is simple in principle, and any of the reaction products may be determined. The general method is simple and requires only that the reaction mixture, after incubation with the enzyme, be made alkaline so that the liberated phenol can be determined, as the phenoxide, either spectrophotometrically or fluorometrically. With crude enzyme preparations deproteinization with, for example, ethanol or trichloroacetic acid may be necessary but any acid treatment should be as mild and as brief as possible because all aryl sulfates are acid-labile. In some cases, the spectra of the phenol and its sulfate ester differ sufficiently at the pH of arylsulfatase activity for the formation of the former to be followed directly so that continuous spec trophotometric assays are possible. Unhydrolyzed substrate can be determined spectrophotometrically. The lysosomal sulfatases A and B have pH optima at about 5, and acetate buffers, or formate buffers at lower pH, are generally suitable for their assay. These enzymes are inhibited by phosphate. The microsomal sulfatase C of mammalian tissues, and the arylsulfatases of mollusks and of microorganisms, have pH optima in the neutral or slightly alkaline region so that imidazole or Tris buffers may be used in their assay. Arylsulfatases may be quite sensitive to changes in the salt or buffer concentration of the reaction mixture so that careful control of the assay conditions is essential. Sulfatases A are hysteretic enzymes that undergo a substrate-induced inactivation with a half-time of a few minutes. The chapter also discusses the substrates for the arylsulfatases.

Early diagnosis of recessive X-linked ichthyosis: elevation of cholesterol sulfate levels in placental sulfatase deficiency before the onset of skin symptoms.

Children and adults with recessive X-linked ichthyosis have elevated levels of cholesterol sulfate, a substrate of the deficient enzyme microsomal sulfatase, in blood, erythrocyte membrane, and ichthyotic scale. We investigated cholesterol sulfate levels in patients with microsomal sulfatase deficiency before birth in amniotic fluid and in maternal plasma and erythrocytes, in cord blood (plasma and erythrocytes), and in plasma in the first month of life before and after onset of skin symptoms. Levels in amniotic fluid and cord blood were elevated. The plasma levels in the first month, before as well as after onset of skin symptoms, were within the range found in older patients with manifest skin symptoms.

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 K m of dehydroepiandrosterone sulfate was determined to be 5.0 × 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 mm 2. 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).

Steroid sulfatase = aryl sulfatase C? Chromatographic and electrophoretic properties in extracts from placental microsomes and skin fibroblasts.

Although the deficiency of steroid sulfatase (STS) as well as aryl sulfatase C (ASC) activities in patients with X-linked recessive ichthyosis has been confirmed by several groups all over the world, the question whether STS = ASC is not yet completely answered. To obtain more information, Miranol H2M extracts from placental microsomes and cultured skin fibroblasts were subjected to gel permeation chromatography and polyacrylamide gel electrophoresis. STS (3H-dehydroepiandrosterone sulfate) and ASC (4-methylumbelliferone sulfate) activities were estimated in the eluted gel permeation chromatography fractions and within the same gel cylinders in half gel slices. None of these two methods allowed a separation of the two microsomal sulfatase activities. From these results and from different behavior of STS and ASC (not deficient in uncultured skin preparations of X-linked recessive ichthyosis, different kinetic properties between STS and ASC, etc.) we propose microsomal sulfatase activities to be assembled in an enzyme aggregate.

Rapid laboratory diagnostic of X-linked ichthyosis.

Steroid sulfatase and aryl sulfatase C activities were assayed simultaneously in peripheral blood leucocytes of 4 patients with X-linked ichthyosis (XLI) and 4 patients with autosomal-dominant ichthyosis; 11 healthy subjects served as controls. The deficiency of steroid sulfatase as well as of aryl sulfatase C found in leucocytes of patients with XLI was confirmed by the histochemical demonstration of aryl sulfatase C deficiency in skin sections. Although results are obtained earlier with the histochemical method than with the biochemical assay in leucocytes, several disadvantages brought us to the conclusion that the biochemical assay of microsomal sulfatase activity in leucocytes offers a fast and safe method for the identification of patients with XLI.

Sulfatase activity of keratinizing tissues in X-linked ichthyosis.

Arylsulfatases A and B were measured in the stratum corneum of four normal controls and two individuals with sex-linked ichthyosis. For arylsulfatase A, the mean delta optical density/hr/mg protein value was 1.6 for controls and 2.0 for patients, whereas for arylsulfatase B values of 1.5 for controls and 1.4 for patients were observed. Assay of arylsulfatase C in the callus of four normal controls showed a mean delta optical density/hr/100 mg callus of 0.63, whereas no or trace activity was detected in callus from four patients with x-linked ichthyosis. The assay of steroid sulfatase is best for studying microsomal sulfatase activity. Table 1 shows the activity of this enzyme in nails, callus, and hair bulbs from controls and patients with x-linked ichthyosis. No steroid sulfatase could be demonstrated in patients with x-linked ichthyosis. The values in normal controls and obligate heterozygotes are compared in Table 2. The mean value of the two groups is statistically different with P less than or equal to 0.05 using the Student t test.

The hydrolysis of estrone sulfate by rat kidney microsomal sulfatase

A survey of the distribution of enzyme activity for the hydrolysis of estrone sulfate in various organs of the rat showed the kidney to be the most active. The hydrolytic activity is located primarily in the microsomal fraction of the kidney but could not be solubilized. The pH profile of the enzyme activity, its thermal stability, its substrate specificity, and its kinetic properties suggest that this enzyme is distinct from previously described kidney sulfatases.