Ascorbic acid-2-sulfate sulfhohydrolase activity of human arylsulfatase A.

Pure human arylsulfatase A (EC 3.1.6.1) was found to hydrolyze ascorbic acid 2-sulfate to ascorbic acid and inorganic sulfate at rates from 200 to 2000 mumol/mg per h depending on the method of assay. This rate was lower than that observed with the synthetic substrate 4-nitrocatechol sulfate, but higher than that seen with the physiological substrate cerebroside sulfate. Extracts of cultured fibroblasts from normal subjects were also shown to hydrolyze ascorbic acid 2-sulfate; extracts of fibroblasts from patients with metachromatic leukodystrophy, known to be deficient in arylsulfatase A, did not. Similarly, hydrolysis of ascorbic acid 2-sulfate was not observed when a partially purified preparation of human arylsulfatase B was tested under a variety of conditions. Thus, in the human, arylsulfatase A appears to be the major, if not the only, ascorbic acid-2-sulfate sulfohydrolase.     

Uridine diphospho-N-acetylgalactosamine-4-sulfate sulfohydrolase activity of human arylsulfatase B and its deficiency in the Maroteaux-Lamy syndrome.

The hydrolysis of UDP-N-acetylgalactosamine-4-sulfate by human arylsulfatase B has been demonstrated with an enzyme preparation purified 200-fold from placenta. No hydrolysis was observed with arylsulfatase A. UDP-N-acetylgalactosamine-4-sulfate is the first fully characterized physiological compound shown to be a substrate for arylsulfatase B, confirming that arylsulfatase B is an N-acetylgalactosamine-4-sulfate sulfohydrolase. Cultured fibroblasts derived from patients with Maroteaux-Lamy syndrome were deficient in UDP-N-acetylgalactosamine-4-sulfate sulfohydrolase to the same extent that they were deficient in arylsulfatase B.     

A simple radioimmunoassay for estriol 3-sulfate in pregnancy plasma without deconjugation

A highly specific anti-estriol 3-sulfate antiserum was treated with 50% ammonium sulfate, and the crude globulin fraction was coupled to CNBr-activated Sepharose-4B. Addition of 0.1M Tris-HC1 buffer (pH 8.3) containing 0.1M glutamine to the solution of antigen-antibody enabled assaying without solvent-extraction or chromatography to remove endogenous interference. Subsequently, a direct radioimmunoassay using [6,7-3H]-estriol 3-sulfate as a radioactive ligand without deconjugation has been established and applied to the determination of estriol 3-sulfate levels in pregnancy plasma. The increasing plasma levels of estriol 3-sulfate are correlated with estriol levels over the period of gestation. The mean values of sulfated estriol concentration (A) in late pregnancy plasma were approximately 7 times as high as unconjugated estriol (B), but individual ratios of A to B showed considerable variability.     

Purification and properties of arylsulfatase B from high- and low-activity mouse strains

Arylsulfatase B (arylsulfate sulfohydrolase; EC 3.1.6.1) activities in C57BL/6J, SWR/J, and A/J mouse liver approximate a 5:3:1 ratio. Each enzyme was purified to apparent homogeneity, and the properties of the three purified enzymes were compared. The purified enzyme behaved as a monomer with an apparent molecular weight of 50,000. The purified enzyme catalyzed the hydrolysis of p-nitrocatechol sulfate (pNCS), 4-methylumbelliferyl sulfate (4MUS), and chondroitin-4-sulfate (C4S) heptasaccharide. Purified SWR/J arylsulfatase B possessed a higher relative electrophoretic mobility at pH 4.0 than the A/J and C57BL/6J isozymes, and the SWR/J enzyme was more thermostable than either the C57BL/6J or the A/J enzyme. No differences were observed among the three enzymes with respect to their Michaelis constants for 4MUS and pNCS, isoelectric points, responses to inhibitors, pH optima, or electrophoretic mobilities at pH 8.3. The relative in vivo rates of synthesis of C57BL/6J, A/J, and SWR/J arylsulfatase B were comparable.     

Screening and Production of Arylsulfatases for Target Therapy with Etoposide 4′-Sulfate,an Antitumor Prodrug

Two arylsulfatase-producing streptomycetes that desulfated etoposide 4′-sulfate were isolated from soil samples. Taxonomical study identified one soil isolate as Streptomyces griseorubiginosus S980-14 (Es-1 arylsulfatase producer), while the other was considered new and tentatively designated Streptomyces sp. T109-3 (Es-2 arylsulfatase producer). Both strains produced extracellular arylsulfatase activities, provided that cultivation media were prepared with distilled water. Unlike the two known types of arylsulfatases, which had significant activity on p-nitrophenyl sulfate but none on etoposide 4′-sulfate, the crude streptomycete arylsulfatases efficiently desulfated etoposide 4′-sulfate and p-nitrophenyl sulfate, which supports the establishment of a new type of arylsulfatases.     

Catecholamine sulfates: end products or metabolic intermediates?

Dopamine-beta-hydroxylase catalyzes the beta-oxidation of dopamine to noradrenaline while phenylethanolamine-N-methyltransferase converts noradrenaline to adrenaline. Since catecholamine sulfates represent the predominant form of catecholamines in human tissues, we have studied the role of dopamine sulfate and noradrenaline sulfate as alternate substrates for dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase, respectively. Dopamine 3-sulfate, dopamine 4-sulfate and noradrenaline 3-sulfate were chemically synthesized and exhaustively purified by ion-exchange chromatography. Dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase were partially purified from human adrenals. Using tyramine as substrate, dopamine-beta-hydroxylase is slightly inhibited by dopamine 3-sulfate according to some irreversible or mixed mechanisms. When dopamine-beta-hydroxylase was incubated with dopamine 3-sulfate or dopamine 4-sulfate, we were not able to find any synthesis of either noradrenaline sulfate or free noradrenaline. Using phenylethanolamine as substrate, the enzymatic activity of phenylethanolamine-N-methyltransferase remains unchanged with addition of dopamine 3-sulfate, dopamine 4-sulfate or noradrenaline 3-sulfate. It was concluded that dopamine sulfate is not an alternate substrate for either dopamine-beta-hydroxylase or phenylethanolamine-N-methyltransferase nor is noradrenaline 3-sulfate an alternate substrate for phenylethanolamine-N-methyltransferase.     

Steroid sulfatase, arylsulfatases A and B, galactose-6-sulfatase, and iduronate sulfatase in mammary cells and effects of sulfated and non-sulfated estrogens on sulfatase activity

Sulfatase enzymes have important roles in metabolism of steroid hormones and of glycosaminoglycans (GAGs). The activity of five sulfatase enzymes, including steroid sulfatase (STS; arylsulfatase C), arylsulfatase A (ASA; cerebroside sulfatase), arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase), galactose-6-sulfatase (GALNS), and iduronate-2-sulfatase (IDS), was compared in six different mammary cell lines, including the malignant mammary cell lines MCF7, T47D, and HCC1937, the MCF10A cell line which is associated with fibrocystic disease, and in primary epithelial and myoepithelial cell lines established from reduction mammoplasty. The effects of estrogen hormones, including estrone, estradiol, estrone 3-sulfate, and estradiol sulfate on activity of these sulfatases were determined. The malignant cell lines MCF7 and T47D had markedly less activity of STS, ASB, ASA, and GAL6S, but not IDS. The primary myoepithelial cells had highest activity of STS and ASB, and the normal epithelial cells had highest activity of GALNS and ASA. Greater declines in sulfatase activity occurred in response to estrone and estradiol than sulfated estrogens. The study findings demonstrated marked variation in sulfatase activity and in effects of exogenous estrogens on sulfatase activity among the different mammary cell types.     

Method for analysis of dopamine sulfate isomers by high performance liquid chromatography

Conjugated dopamine occurs in the tissues and fluids of many species, and much of this is thought to occur as dopamine sulfate. This paper describes the development and use of a method utilizing reversed-phase paired-ion high performance liquid chromatography to separate and quantitate each of the two naturally occurring dopamine sulfate isomers. Use of the method permitted demonstration of dopamine-3-0-sulfate in human urine from drug-free control subjects. It was found that this compound accounted for 73.1 ± 27% of the total daily conjugated dopamine excretion in the four subjects studied.     

The hydration of proteoglycans of bovine cornea

The water sorptive and retentive capacities of three corneal proteoglycans with different keratan sulfate/chondroitin-4-sulfate compositions were investigated. The calcium salt of a predominantly keratan sulfate containing proteoglycan had hydration properties similar to that of calcium keratan sulfate. The proteoglycan containing predominantly calcium chondroitin-4-sulfate side chains sorbed water to a greater extent than pure calcium chondroitin-4-sulfate but its retentive power was somewhat less. The proteoglycan containing about twice as much keratan sulfate as chondroitin-4-sulfate, on a disaccharidic molar basis and had hydration properties which were closer to the behavior of chondroitin-4-sulfate than keratan sulfate. The results are discussed in terms of structure and polymer interaction in the proteoglycan matrices.     

The hydration of proteoglycans of bovine cornea.

The water sorptive and retentive capacities of three corneal proteoglycans with different keratan sulfate/chondroitin-4-sulfate compositions were investigated. The calcium salt of a predominantly keratan sulfate containing proteoglycan had hydration properties similar to that of calcium keratan sulfate. The proteoglycan containing predominantly calcium chondroitin-4-sulfate side chains sorbed water to a greater extent than pure calcium chondroitin-4-sulfate but its retentive power was somewhat less. The proteoglycan containing about twice as much keratan sulfate as chondroitin-4-sulfate, on a dissaccharidic molar basis and had hydration properties which were closer to the behavior of chondroitin-4-sulfate than keratan sulfate. The results are discussed in terms of structure and polymer interaction in the proteoglycan matrices.     

Comparative studies of rodent anionic arylsulfatases

Approximately 25 and 40%, respectively, of murine (Mus musculus) and rat (Rattus norvegicus) hepatic arylsulfatase (EC 3.1.6.1) activity eluted from DEAE-ion exchange resins under high salt conditions. This high salt fraction contained arylsulfatase A and an enzyme which was immunologically similar to arylsulfatase B. The latter enzyme was thermostable, resistant to inhibition by silver, completely inhibited by phosphate, displayed linear kinetics, and had a higher pH optimum than arylsulfatase A. Anionic arylsulfatase B also hydrolyzed chondroitin-4-SO4 heptasaccharide. Sephacryl S-300 gel filtration resolved anionic arylsulfatase B into 55 and 115 kd fractions. Rodent arylsulfatase A activity was grossly underestimated when 4-methyl-umbelliferyl sulfate was employed as substrate.     

The activity of arylsulfatase A and B on tyrosine O-sulfates.

L-Tyrosine O-sulfate was hydrolyzed by pure human arylsulfatase A (arylsufate sulfohydrolase, EC 3.1.6.1). The rate of hydrolysis was 1/20 of the rate with nitrocatechol sulfate, but was comparable to the rate with cerebroside sulfate. The reaction was optimal at pH 5.3--5.5 and displayed zero order kinetics with time and enzyme concentration. The Km was about 35 mM. The enzyme showed no stereospecificity and hydrolyzed D-tyrosine O-sulfate with Km and V similar to those for the L-isomer. Arylsulfatase B was less than 5% as effective as arylsulfatase A in catalyzing the hydrolysis of the tyrosine sulfates. The daily urinary excretion of tyrosine sulfate by a patient with metachromatic leukodystrophy (arylsulfatase A deficiency) was comparable to the excretion by control subjects. The biological relevance of the tyrosine sulfatase activity of arylsulfatase A remains uncertain.     

Determination of catecholamine sulfoconjugate isomers in normal human urine by use of high-performance liquid chromatography with a photochemical detector

A method for the determination of catecholamine sulfoconjugate isomers (CA-S) in urine was developed. The photo-induced fluorogenic reaction of CA-S with ethylenediamine reported previously was applied to the postcolumn labeling of HPLC for sensitive and selective detection. Special equipment for the reaction was made with a uv-irradiation lamp and a reaction coil of Teflon tubing inside a temperature-controlled reaction box. Lower determination limits of this system were 1 to 2 pmol. Urine samples pretreated with small ion-exchange resin columns were subjected to HPLC. Peaks corresponding to CA-S were identified quantitatively by two different separation methods. Thus, all six CA-S were first detected in the urine of normal individuals. The excretion rates of dopamine 3-sulfate, dopamine 4-sulfate, norepinephrine 3-sulfate, norepinephrine 4-sulfate, epinephrine 3-sulfate, and epinephrine 4-sulfate were 420 +/- 240, 98 +/- 55, 86 +/- 95, 15 +/- 14, 18 +/- 7, and 3 +/- 1 ng/min (+/- SD), respectively (n = 5).     

Extra-Lysosomal Localization of Arylsulfatase B in Human Colonic Epithelium

The enzyme arylsulfatase B (N-acetylgalactosamine-4-sulfatase; ARSB; ASB) removes 4-sulfate groups from the sulfated glycosaminoglycans (sGAG) chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Inborn deficiency of ARSB leads to the lysosomal storage disease mucopolysaccharidosis VI, characterized by accumulation of sGAG in vital organs, disruption of normal physiological processes, severe morbidity, and premature death. Recent published work demonstrated extra-lysosomal localization with nuclear and cell membrane ARSB observed in bronchial and colonic epithelial cells, cerebrovascular cells, and hepatic cells. In this report, the authors present ARSB immunostaining in a colonic microarray and show differences in distribution, intensity, and pattern of ARSB staining among normal colon, adenomas, and adenocarcinomas. Distinctive, intense luminal membrane staining was present in the normal epithelial cells but reduced in the malignancies and less in the grade 3 than in the grade 1 adenocarcinomas. In the normal cores, a distinctive pattern of intense cytoplasmic positivity at the luminal surface was followed by reduced staining deeper in the crypts. ARSB enzymatic activity was significantly greater in normal than in malignant tissue. These study findings affirm extra-lysosomal localization of ARSB and suggest that altered ARSB immunostaining and reduced activity may be useful indicators of malignant transformation in human colonic tissue.     

Diversity in the degree of sulfation and chain length of the glycosaminoglycan moiety of urinary trypsin inhibitor isomers

Five isomers with different electric charge were fractionated from human urinary trypsin inhibitor (UTI) by anion exchange HPLC. Intact low-sulfated chondroitin 4-sulfate chains from the isomers were analyzed by HPLC and mass spectrometry. Unsaturated disaccharide composition analysis of the chondroitin sulfate chain revealed that the five isomers differ in the numbers of 4-sulfated disaccharide units. Intriguingly, we detected the presence of multiple novel isomers with different numbers of non-sulfated disaccharide units even in the same charge isomer fraction. Our results demonstrate that UTI can vary in terms of both the degree of sulfation and the length of the low-sulfated chondroitin 4-sulfate chain.     

Identification of 5′-deoxypyridoxine-3-sulfate as the major urinary metabolite of 5′-deoxypyridoxine in rats with comments on the inhibition of arylsulfatase activity and manganese dioxide oxidation by neighboring groups

The major urinary metabolite of 5′-deoxypyridoxine in rats was shown to be identical to 5′-deoxypyridoxine-3-sulfate but different from 5′-deoxypyridoxine-4′-sulfate in its ultraviolet and infrared spectra, its migration in thin-layer chromatography, and its behavior in acid and base. Previous identification of the metabolite as 5′-deoxypyridoxine-4′-sulfate by other workers was based on its failure to be hydrolyzed by arylsulfatase and to be oxidized by manganese dioxide. We have now demonstrated that ortho-methyl groups inhibit arylsulfatase and that ortho-sulfate groups inhibit oxidation by manganese dioxide. Therefore, we conclude that under our conditions the major metabolite of 5′-deoxypyridoxine in the rat was the 3-sulfate.     

A study on heterogeneity in molecular species of shark cartilage chondroitin sulfate C. Fractionation of the polysaccharide on Sepharose CL-4B in the presence of high concentrations of ammonium sulfate

Shark cartilage chondroitin sulfate C was fractionated by chromatography on Sepharose CL-4B-2.5 to 1.5M ammonium sulfate in 10mM hydrochloric acid at 4 degrees. Both unit-disaccharide composition and molecular-size distribution clearly affected the fractionation. Comparison of this fractionation with the fractionation on Sepharose 6B gel in 0.2M sodium chloride revealed that the former is distinctly superior to the latter. The fractionation on Sepharose CL-4B in the presence of ammonium sulfate also showed that the chondroitin sulfate C molecules having a larger molecular size contain generally more chondroitin 6-sulfate units (as major constituent) and less chondroitin disulfate units (D type, as minor constituent) than those having a smaller molecular size).     

Lysosomal arylsulfatases A and B from horse blood leukocytes: purification and physico-chemical properties

Lysosomal arylsulfatases A and B (aryl-sulfate sulfohydrolases, EC 3.1.6.1) from horse leukocytes were purified about 680-fold and 70-fold, respectively, starting from a crude extract of the azurophil and specific granules of leukocytes, by affinity, ion exchange, and gel filtration chromatography. Purified arylsulfatase A displayed anomalous kinetics, a pH optimum at 5.2, an isoelectric point at 4.3, and a Km value for p-nitrocatechol sulfate (pNCS) of 0.37 mM. This enzyme was found to exist in two association states depending on pH: a high molecular weight form at pH 5.0 and a low molecular weight form at pH 7.5. Arylsulfatase B displayed normal kinetics, a pH optimum at 5.8, two isoelectric points at pH 8.6 and 8.9, and a Km value for pNCS of 3.38 mM. The thermostability of the two enzymes was different: arylsulfatase B was found to be more stable than arylsulfatase A. Arylsulfatase A was inhibited by sulfate, sulfite, silver, magnesium, manganese and calcium ions and arylsulfatase B by chloride, sulfate, sulfite and silver ions.     

Inhibition of Charonia lampas ascorbate-2-sulfate sulfohydrolase activity by adenosine 5'-diphosphate and related compounds.

1) ADP was a potent inhibitor of the ascorbic-2-sulfate sulfohydrolase activity of Charonia lampas liver. The inhibition was competitive with respect to ascorbate 2-sulfate. The Ki value was 5.9 muM. ADP did not inhibit arylsulfatase (EC 3.1.6.1) of the same organism. 2) Other nucleoside 5'-diphosphates and GTP showed similar inhibition of ascorbate-2-sulfate sulfohydrolase activity. 3) The effects of different nucleosides, nucleotides, and sugar phosphates on ascorbate-2-sulfate sulfohydrolase activity were investigated. Phosphate derivatives other than 3',5'-cyclic AMP were more or less inhibitory.     

Developmental profiles of arylsulfatases A and B in rat cerebral cortex and spinal cord.

Arylsulfatases A (EC 3.1.6.1) and B (EC 3.1.6.12) are lysosomal enzymes that can remove sulfate groups from sulfatides and sulfo-glycosaminoglycans, respectively. The activities of these enzymes in cerebral cortex and in spinal cord of developing rat pups were measured. The tissues were homogenized and the arylsulfatases A and B in the soluble fraction were separated from each other by anion exchange chromatography on DE-52 cellulose. Subsequently, the enzyme activities were assayed with p-nitrocatechol sulfate as substrate at 37 degrees C and pH 5.6. We observed a developmental profile of arylsulfatase A, similar to that previously reported for cerebroside sulfatase (EC 3.1.6.8; (Van der Pal et al. (1990) Biochim. Biophys. Acta 1043, 91-96]. The activity of arylsulfatase A increased gradually during development, whereas arylsulfatase B rose more steeply, peaked around day 15 and declined thereafter. As a consequence the ratio between B and A forms of arylsulfatase dropped from about 4 in 1-week-old pups to 2.2 (cortex) and 0.7 (cord) in 7-week-old rat pups.