Arylsulfatase

Arylsulfatase cleaves sulfate esters. Cerebroside sulfate is its natural substrate. The enzyme is also active toward p-nitrocatechol sulfate, which is the basis for this assay. 

The p-nitrocatechol formed was separated at 30°C with a reversed-phase Cjg column (4 millimeters X 125 mm) from EM Science. The mobile phase was prepared by mixing 13 g of monochloroacetic add, 4.99 g of NaOH, 0.177 g of sodium octylsulfate, 130 mL of acetonitrile, 10 mL of tetrahydrofu-ran, and approximately 620 of mL water. After the pH had been adjusted to 3.3 with approximately 220 mL of 8 M acetic acid, the mixture was diluted to 1 liter with water. The electrochemical detector was set at an applied potential of 0.750 V. 

The reaction mixture was prepared by mixing 800 fiL of substrate solution containing 0.01 M p-nitrocatechol sulfate (dipotassium salt), 1.71 M NaCl, and 0.5 M acetate buffer (pH 5.0) with 200 fiL of either leukocyte and platelet lysate or saliva. After incubation for an hour at 37°C, a 50 fiL aliquot was mixed with 100 fiL of bovine serum albumin (13 mg/mL) and 1800 fiL of cold 95% ethanol. After centrifugation to remove proteins, 20 /iL was injected into the HPLC system. The reaction was linear with up to 200 nL of lysate. 

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Purified by dissolving in the minimum vol of hot water (60°) and adding EtOH, with stirring, then left at 0° for Ih. Crystals were filtd off and recrystd from H2O until free of Cl and SO ions. Dried in a vac over P2O5 at room temperature. It is a specific substrate for arylsulfatases which hydrolyse it to p-acetylphenol [A,max 327nm (e 21700 M- cm )] [Milsom et al. Biochem J 128 331 1972]. 

In addition to the reactions of luciferin shown in Fig. 7.2.3, Trainor (1979) made an interesting discovery that arylsulfatase from Pattela vulgata (Sigma) converts luciferin into a violet compound (treatment conditions pH 5.05, 37°C, 4 hr). The compound showed absorption peaks at 344 nm and 560 nm (Fig. 7.2.7), and the peaks shifted reversibly to 347 nm and 547 nm in acid, and to 364 nm and 720 nm in an alkaline solution. The violet compound was also obtained from the pink compound by treatment with arylsulfatase, or from luciferin by heating in 50% trifluoroacetic acid at 100°C for 1 hr, followed by an addition of oxygenated water. These results, together with the... 

Fig. 7.2.7 Absorption spectrum of the violet compound produced by the treatment of Odontosyllis luciferin with arylsulfatase, in 50 mM acetate buffer, pH 5.05. From Trainor, 1979.

Klotz and associates120 found that PEI-D-Im-type models catalyzed hydrolyses of 4-nitrocatechol sulfate by a two step mechanism resembling that of a natural enzyme. The polymer was reported to accelerate the rate by a factor of 1012 arm-pared to imidazole itself, and of 102 compared to a type-11A arylsulfatase enzyme. 

MIM 253200) Sly (MIM 253220) MPS VII sulfatase (arylsulfatase B) P-Glucuronidase Dermatan sulfate, heparan sulfate, chondroitin... 

Arylsulfatase from Pmudamonas aarugmasa Sulfatase from Rhodopimllula baMce ... 

Familial demyelinative/dysmyelinative and axonal neuropathies may also be caused by impaired lysosomal lipid metabolism. Metachromatic leukodystrophy (sulfatide lipidosis) results from mutations of the arylsulfatase A gene, which encodes a lysosomal enzyme required for sulfatide turnover. Myelin is affected in both CNS and PNS, though dysfunction is restricted to the PNS in some patients, and the onset of symptoms can occur at any time between infancy and adulthood. Bone marrow transplantation can slow disease progression and improve nerve conduction velocities [57]. (See in Ch. 41.)... 

Metachromatic leukodystrophy is due to a defect in arylsulfatase A (ASA). There are three major forms late infantile, juvenile and adult. The overall incidence is 1 40,000. In the late infantile and early juvenile forms, which comprise about 80% of patients, the initial symptoms involve the motor system, with falls, loss of ability to walk, flaccid paralysis, difficulty in swallowing, loss of speech, vision, seizures, decerebrate state and death 1-7 years after onset of symptoms. In the adolescent and... 

Multiple sulfatase deficiency (MSD). The clinical presentation of MSD includes features of MLD and the MPS, with the features that resemble MLD, such as gait disturbances, psychomotor retardation and polyneuropathy predominating. MPS-like features, such as facial coarsening, hepatosplenomegaly and joint stiffness, are present to a milder and variable extent. A reduction in the activity of all sulfatases is the key diagnostic abnormality. Von Figura and associates have demonstrated that catalytic activity of all sulfatases requires a post-translational modification in which a cysteine residue (cysteine 69 in arylsulfatase A) is oxidized to an aldehyde. It is this modification that is defective in MSD [5]. 

EL. De Hostos, J. Schilling, AR. Grossman (1989) Structure and expression of the gene encoding the periplasmatic arylsulfatase of Chlamydomonas reinhardtii. Mol. Gen. Genet., 218 229-239... 

J. P. Davies, D. P. Weeks, A. R Grossman (1992) Expression of the arylsulfatase gene from the C2-tubulin promoter in Chlamydomonas reinhardtii. Nttcl. Acids Res., 20 2959-2965... 

T. Lien, O. Schreiner (1975) Purification of a derepressible arylsulfatase from Chlamydomonas reinhardtii. Biochim. Biophys. Acta, 384 168-179... 

The protocol consisted of preconditioning with methanol (1 mL) followed by water (1 mL). Urine samples (3 mL) were deconjugated by treatment with /3-glucuronidase and arylsulfatase (10 jt/L and 200 fig/fiL) in 0.1M sodium acetate (pH 5.5) and then loaded onto conditioned cartridges. After washing with water (1 mL) and methanoksodium acetate (3 mL, 4 6, pH 5.5), the PAH metabolites were eluted with dichloromethane (3 mL). The eluate was spiked with dodecane (used... 

Enzyme activity (urease, amidase, dehydrogenase, pl-glucosidase, phosphatase, arylsulfatase, fluorescein diacetate hydrolysis) Laboratory incubation Indicates potential microbial activity and nutrient cycling reactions determined in nonstandard laboratory with specialized equipment highly spatially and temporally variable dependent upon organic inputs Dick et al. (1996) Parham et aL (2002)... 

Some sulfatases (sulfuric ester hydrolases, EC 3.1.6) play a pharmacological role in the hydrolysis of sulfuric acid ester metabolites and the few such esters that are used as prodrugs. Arylsulfatase (sulfatase, arylsulfohydrolase,... 

EC 3.1.6.1) is a lysosomal enzyme that hydrolyzes sulfuric acid ester bonds. The enzyme exists in two forms, arylsulfatases A and B, that differ in substrate specificity and in sensitivity toward inhibitors [142][143]. Human tissues contain more arylsulfatase A than arylsulfatase B. The natural substrates of these enzymes are complex lipids such as cerebroside 3-sulfate, and gly-cosaminoglycans such as chondroitin 4-sulfate and derman sulfate [144], Deficiencies of these enzymes are associated with a number of lysosomal disorders. 

A number of enzymes known as sulfuric ester hydrolases (EC 3.1.6) are able to hydrolyze sulfuric acid esters. They comprise arylsulfatase (sulfatase, EC 3.1.6.1), steryl-sulfatase (steroid sulfatase, steryl-sulfate sulfohydrolase, arylsulfatase C, EC 3.1.6.2), choline-sulfatase (choline-sulfate sulfohydrolase, EC 3.1.6.6), and monomethyl-sulfatase (EC 3.1.6.16). Whereas mono-methyl-sulfatase is highly specific and does not act on higher homologues, arylsulfatase has a broad substrate specificity and is of particular significance in the hydrolysis of sulfate conjugates of phenols, be they endogenous compounds, drugs, or their metabolites [167-169],... 

Studies on the fate of dopamine 4-sulfate (9.88) will serve to illustrate the reaction of deconjugation mentioned above. With this substrate, arylsulfatase activity in the dog was found to be highest in the kidney and liver, low in the intestine and heart, and almost nil in the brain and skeletal muscle [171]. Since this conjugate exists in high amounts in the plasma of humans, monkeys and dogs, the possibility was raised that it might be looked upon as a precursor or reservoir of free dopamine. 

Arylsulfatase [EC 3.1.6.1 ], also known simply as sulfatase, catalyzes the hydrolysis of a phenol sulfate, thereby producing a phenol and sulfate. This enzyme classification represents a collection of enzymes with rather similar specificities. (1) Steryl-sulfatase [EC3.1.6.2],also referred to as arylsulfatase C and steroid sulfatase, catalyzes the hydrolysis of 3-j8-hydroxyandrost-5-en-17-one 3-sulfate to 3-j8-hydroxyandrost-5-en-17-one and sulfate. The enzyme utilizes other steryl sulfates as substrates. (2) Cere-broside-sulfatase [EC 3.1.6.8], or arylsulfatase A, catalyzes the hydrolysis of a cerebroside 3-sulfate to yield a cerebroside and sulfate. The enzyme will also hydrolyze the galactose 3-sulfate bond present in a number of lipids. In addition, the enzyme will also hydrolyze ascorbate 2-sulfate and other phenol sulfates. 

This enzyme [EC 3.1.6.8], also known as arylsulfatase A, catalyzes the hydrolysis of a cerebroside 3-sulfate to produce a cerebroside and sulfate. The enzyme will also catalyze analogous reactions on the galactose 3-sulfate residues in a number of lipids as well as on ascorbate 2-sulfate and many phenol sulfates. 

Presently, FAB-MS spectra are routinely used to characterize synthetic tyrosine O-sulfate peptides.152,57,63-671 Since partial hydrolysis of the sulfate ester occurs in the gas phase, quantification of the tyrosine O-sulfate residue by mass spectrometry is not possible, but combined with one-peak assignment in HPLC, FAB-MS represents a powerful analytical tool. On the other hand, partial hydrolysis in the gas phase excludes the presence of sul-fonated species which should be perfectly stable. In early studies the presence of such species were excluded by quantitative recovery of tyrosine upon acid hydrolysis or upon hydrolysis with arylsulfatase.1361 Recently, even MALDI-TOF-MS spectra of CCK-peptides1441 and of conotoxins a-PnIA and a-PnlB 138 were reported which show that in the positive-ion mode the [M + H-S03]+ ions represent the base peaks, while in the negative-ion mode, [M-H]-ions consistently correspond to the base peaks. In the CCK peptides intramolecular salt bridging of the sulfate hemi-ester with proximal positive charges of arginine or lysine side chains was found to reduce the extent of hydrolysis in the gas phase significantly.144,1491... 

Conjugates of flavour compounds were also found in milk phenols can be liberated by /i-glucuronidase, arylsulfatase and acid phosphatase from their respective precursors [57]. 

Patients with multiple sulfatase deficiency also show low N-acetylglucosamine-6-sulfatase activity. Therefore, arylsulfatase A or another sulfatase should also be determined in case of low results for a single sulfatase, especially when jS-galactosidase activity is in the normal range for that sample. 

For each patient sample, 10 and 20 pi of leukocyte homogenate are transferred to individual wells on the microtiter plate. As two different incubation times are necessary, the same volumes of all samples are also pipetted into the lower part of the plate. Subsequently, 100 pi of substrate buffer is added to each well and the volume is adjusted to a total of 150 pi by adding sodium acetate buffer containing barium acetate. Arylsulfatase A will be inhibited in the presence of barium ions. The plate is briefly shaken, sealed with tape, and covered with aluminum wrapping. Incubation is carried out at 37°C for 30 min (upper half including the standard curve) and 90 min (lower half). The reaction is stopped with 200 pi stop solution and the absorbance is read at 490 nm. 

The activity is calculated using the standard curve and the result for the absorption (patient) because during the first 30 min of incubation some of the activity may be derived from arylsulfatase A instead of arylsulfatase B. Results are expressed in nmol/ mg protein-h. 

All enzymes are measured at acidic pH with artificial fluorogenic substrates, except for arylsulfatase A, which is measured with a chromogenic substrate. The fluorogenic substrates are derivatives of 4-methylumbelliferone or 6-hexadecanoylamino-... 

In case of a deficiency of arylsulfatase A, at least one other sulfatase should be measured to exclude multiple sulfatase deficiency (see Chap. 4.1 for the assays of arylsulfatase and other sulfatases). Sulfatide excretion in urine should be measured (see assay below) and/or mutation analysis should to performed to confirm the diagnosis, especially if the clinical symptoms are atypical and in order to exclude a pseudodeficiency of arylsulfatase A. The enzyme should always be measured in the parents to check for the presence of compound heterozygosity of a metachromatic leukodystrophy and a pseudodeficiency allele. This is very important for the interpretation of the results of arylsulfatase A assays, especially when performed in asymptomatic or presymptomatic siblings or in the context of a prenatal diagnosis. Sulfatide should also be measured in case a normal arylsulfatase A activity is found in a patient with symptoms characteristic of (juvenile) metachromatic leukodystrophy. Increased urinary sulfatide excretion is indicative of an activator protein/saposin deficiency (Fig. 4.4.1). 

Baum H, Dodgson KS, Spencer (1959) The assay of arylsulfatases A and in human urine. Clin Chim Acta 4 453-455... 

Lee-Vaupel M, Conzelmann E (1987) A simple chromogenic assay for arylsulfatase A. Clin Chim Acta 164 171-180... 

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