hymecromone and 4-methylumbelliferyl-sulfate

LC-MS/MS has been proposed in various areas such as Therapeutic Drug Monitoring (TDM), Human Biomonitoring (HBM), disease diagnosis, clinical toxicology and doping control to identify and quantify chemical parents and their metabolites in biological matrices. To determine the total content of a xenobiotic (unconjugated+conjugated forms), an enzymatic hydrolysis step is required. Most studies in the literature have not controlled the effectiveness of the deconjugation process because no method has been described for that purpose. Therefore the aim of this study was to develop and validate a deconjugation probe using a LC-MS/MS method. In order to estimate deconjugation using β-glucuronidase and/or sulfatase, 4-methyl-umbelliferone (MU) and its conjugates were used as markers. Glucuronidase/sulfatase was added to plasma or urine spiked with 4-methylumbelliferyl-β-d-glucuronide (MUG) and 4-methylumbelliferyl sulfate (MUS) and umbelliferone, which was used as the internal standard. After incubation at 37°C during 90min, MU appears as a result of the deconjugation of MUG and MUS. The concentrations of the 3 markers were determined using LC-MS/MS. Trueness and precision of the LC-MS/MS method were determined by quality control analysis at three different levels of concentration covering the whole range of calibration. In both matrices, the analytical method allows quantification of the different compounds, with good linearity, trueness and precision and negligible matrix effects. The method was applied with success to deconjugation assay using active glucuronidase/sulfatase in plasma and urine. The probe developed in this study allows to ensure that enzymatic preparation is working properly in the frame of a quality system.

Topics: Chromatography, Liquid; Drug Monitoring; Glucuronides; Humans; Hymecromone; Limit of Detection; Linear Models; Reproducibility of Results; Sulfates; Tandem Mass Spectrometry

A continuous and real-time fluorometric assay for monoamine-preferring phenol sulfotransferase (SULT1A3) was developed. The methodology was based on the coupling of SULT1A1 to regenerate 3'-phosphoadenosine-5'-phosphosulfate (PAPS) using 4-methylumbelliferyl sulfate (MUS) as a sulfuryl group donor. The fluorophore product (4-methylumbelliferone, MU) was continuously produced and monitored when SULT1A3 catalyzed dopamine sulfation with PAPS. The optimal conditions of this turnover reaction and substrate inhibition of SULT1A3 were also determined. This coupled-enzyme assay allows the continuous measurement of initial reaction velocity and the sensitivity is comparable to that of end-point radioactive isotope assay.

Topics: Animals; Arylsulfotransferase; Enzyme Assays; Humans; Hymecromone; Phosphoadenosine Phosphosulfate; Rats; Spectrometry, Fluorescence; Sulfotransferases

Topics: Fluorescent Dyes; Humans; Hymecromone; Kinetics; Sulfatases

Topics: Arylsulfatases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glucosamine; HEK293 Cells; Heparitin Sulfate; Humans; Hymecromone; Kinetics; Molecular Structure; Substrate Specificity; Sulfatases; Sulfotransferases; Time Factors

Breast cancer resistance protein (Bcrp/Abcg2) is a member of the ATP-binding cassette transporter family with the ability to transport a variety of sulfate conjugates. In the present study, the regional expression and activity of Bcrp and sulfotransferases (SULTs/Sults) were investigated in mouse intestine. Western blotting analysis revealed the highest expression of Bcrp in the ileum over the duodenum, jejunum, and colon. Functional analysis of Bcrp was performed in everted intestinal sacs using 4-methylumbelliferone (4MU). The mucosal secretion clearance of 4MU sulfate formed in the enterocytes was markedly reduced in the jejunum, ileum, and colon of Bcrp (-/-) mice in comparison with wild-type mice, whereas a slight and nonsignificant reduction was observed in the duodenum. The reduction in the mucosal secretion clearance was most marked in the ileum followed by the colon and jejunum. In addition, the mucosal secretion clearance of minoxidil sulfate, an active metabolite of minoxidil, was also significantly reduced in the intestine of Bcrp (-/-) mice. The sulfation activity of 4MU was higher in the colon than in the small intestine where glucuronidation activity was somewhat higher than the sulfation activity. Real-time polymerase chain reaction analysis showed that the expression of sulfotransferases, such as Sult1a1/2, Sult1b1, and Sult1d1, was also highest in the colon. These results suggest that Bcrp activity is higher in the mid to lower intestine and that the cooperation of Bcrp and SULT provides an important detoxification pathway, particularly in the colon.

Topics: Animals; Arylsulfotransferase; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Enterocytes; Glucuronides; Hymecromone; Intestinal Mucosa; Male; Mice; Mice, Transgenic; Microfilament Proteins; RNA, Messenger

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder related to a deficiency in the enzyme iduronate-2-sulfatase (IDS). Clinical trials of enzyme replacement therapy are in progress, but effective treatment will require screening assays to enable early detection and diagnosis of MPS II. Our study evaluated the diagnostic accuracy of IDS protein and enzyme activity measurements in dried blood spots and plasma.. We collected dried-blood-spot and plasma samples from unaffected control individuals and from MPS II patients. We measured IDS protein concentration with a 2-step time-delayed dissociation-enhanced lanthanide fluorescence immunoassay. To measure enzyme activity, we immobilized anti-IDS antibody on microtiter plates to capture the enzyme and measured its activity with the fluorogenic substrate 4-methylumbelliferyl sulfate.. Dried-blood-spot samples from MPS II patients showed an almost total absence of IDS activity (0-0.075 micromol x h(-1) x L(-1)) compared with control blood spots (0.5-4.7 micromol x h(-1) x L(-1)) and control plasma (0.17-8.1 micromol x h(-1) x L(-1)). A dried-blood-spot sample from only 1 of 12 MPS II patients had detectable concentrations of IDS protein (24.8 microg/L), but no IDS protein was detected in plasma from MPS II patients. Ranges for IDS protein in control samples were 25.8-153 microg/L for blood spots and 22.8-349.4 microg/L for plasma.. Measurement of the IDS protein concentration and enzyme activity (as measured by a simple fluorogenic assay with an immune capture technique) enables identification of the majority of MPS II patient samples from both dried blood spots and plasma samples.

Topics: Adolescent; Adult; Animals; Blood Specimen Collection; Calibration; Child; Child, Preschool; CHO Cells; Clinical Enzyme Tests; Cricetinae; Cricetulus; Fluorometry; Humans; Hymecromone; Iduronate Sulfatase; Immunoassay; Indicators and Reagents; Infant; Middle Aged; Mucopolysaccharidosis II; Plasma; Recombinant Proteins

The hepatic excretion of hydrophilic conjugates, end products of phase II metabolism, is not completely understood. In the present studies, transport mechanism(s) responsible for the biliary excretion of 4-methylumbelliferyl glucuronide (4MUG) and 4-methylumbelliferyl sulfate (4MUS) were studied. Isolated perfused livers (IPLs) from Mrp2-deficient (TR(-)) Wistar rats were used to examine the role of Mrp2 in the biliary excretion of 4MUG and 4MUS. After a 30-micromol dose of 4-methylumbelliferone, cumulative biliary excretion of 4MUG was extensive in wild-type rat IPLs (25 +/- 3 micromol) but was negligible in TR(-) livers (0.4 +/- 0.1 micromol); coadministration of the Bcrp and P-glycoprotein inhibitor GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] had no effect on 4MUG biliary excretion in wild-type rat IPLs. In contrast, biliary excretion of 4MUS was partially maintained in Mrp2-deficient rat IPLs. Recovery of 4MUS in bile was approximately 50 to 60% lower in both control TR(-) (149 +/- 8 nmol) and wild-type IPLs with GF120918 coadministration (176 +/- 30 nmol) relative to wild-type control livers (378 +/- 37 nmol) and was nearly abolished in TR(-) IPLs in the presence of GF120918 (13 +/- 8 nmol). These changes were the result of decreased rate constants governing 4MUG and 4MUS biliary excretion. In vitro assays and perfused livers from Bcrp and P-glycoprotein gene-knockout mice indicated that 4MUS did not interact with P-glycoprotein but was transported by Bcrp in a GF120918-sensitive manner. In the rat liver, Mrp2 mediates the biliary excretion of 4MUG, whereas both Mrp2 and Bcrp contribute almost equally to the transport of 4MUS into bile.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Bile; Cell Line; Dogs; Hymecromone; Male; Rats; Rats, Wistar

A sensitive fluorometric assay was developed for alcohol sulfotransferase (AST). This was the first continuous fluorometric assay reported for AST. It used 3'-phosphoadenosine 5'-phosphosulfate regenerated from 3-phosphoadenosine 5'-phosphate by a recombinant phenol sulfotransferase (PST) using 4-methylumbelliferyl sulfate as the sulfuryl group donor. The recombinant PST did not use the alcohol substrate under the designed condition, and the sensitivity for AST activity was found to be comparable to that of radioactive assay as reported in the literature. The change of fluorescence intensity of 4-methylumbelliferone corresponded directly to the amount of active AST and was sensitive enough to measure nanogram or picomole amounts of the enzyme activity. This fluorometric assay was used to determine the activities of AST as purified form and in crude extracts of pig liver, rat liver, and Escherichia coli. Some properties of human dehydroepiandrosterone sulfotransferase were determined by this method and were found to be comparable to published data. Under similar assay conditions, the contaminated activities of arylsulfatase in crude extracts were also determined. This method not only is useful for the routine and detailed kinetic study of this important class of enzymes but also has the potential for the development of a high-throughput procedure using microplate reader.

Topics: Adenosine Diphosphate; Animals; Escherichia coli; Humans; Hymecromone; Kinetics; Liver; Phosphoadenosine Phosphosulfate; Rats; Recombinant Proteins; Sulfotransferases; Sulfur Radioisotopes; Swine

Renal ischaemia is associated with accumulation of fatty acids (FA) and mobilisation of arachidonic acid (AA). Given the capacity of UDP-glucuronosyltransferase (UGT) isoforms to metabolise both drugs and FA, we hypothesised that FA would inhibit renal drug glucuronidation. The effect of FA (C2:0-C20:5) on 4-methylumbelliferone (4-MU) glucuronidation was investigated using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7 as the enzyme sources. 4-MU glucuronidation exhibited Michaelis-Menten kinetics with HKCM (apparent K(m) (K(m)(app)) 20.3 microM), weak substrate inhibition with UGT1A9 (K(m)(app) 10.2 microM, K(si) 289.6 microM), and sigmoid kinetics with UGT2B7 (S(50)(app)440.6 microM) Similarly, biphasic UDP-glucuronic acid (UDPGA) kinetics were observed with HKCM (S(50) 354.3 microM) and UGT1A9 (S(50) 88.2 microM). In contrast, the Michaelis-Menten kinetics for UDPGA observed with UGT2B7 (K(m)(app) 493.2 microM) suggested that kinetic interactions with UGTs were specific to the xenobiotic substrate and the co-substrate (UDPGA). FA (C16:1-C20:5) significantly inhibited (25-93%) HKCM, UGT1A9 or UGT2B7 catalysed 4-MU glucuronidation. Although linoleic acid (LA) and AA were both competitive inhibitors of 4-MU glucuronidation by HKCM (K(i)(app) 6.34 and 0.15 microM, respectively), only LA was a competitive inhibitor of UGT1A9 (K(i)(app) 4.06 microM). In contrast, inhibition of UGT1A9 by AA exhibited atypical kinetics. These data indicate that LA and AA are potent inhibitors of 4-MU glucuronidation catalysed by human kidney UGTs and recombinant UGT1A9 and UGT2B7. It is conceivable therefore that during periods of renal ischaemia FA may impair renal drug glucuronidation thus compromising the protective capacity of the kidney against drug-induced nephrotoxicity.

Topics: Adult; Aged; Aged, 80 and over; Enzyme Inhibitors; Fatty Acids, Unsaturated; Glucuronosyltransferase; Humans; Hymecromone; Kidney; Kinetics; Male; Microsomes; Middle Aged; Recombinant Proteins; UDP-Glucuronosyltransferase 1A9; Uridine Diphosphate Glucuronic Acid

1. To elucidate the mechanisms involved in the sinusoidal efflux of sulfate and glucuronide metabolites of 4-methylumbelliferone (4MU), isolated rat liver perfusion studies were performed under several conditions. 2. The effect of sodium azide on the hepatic handling of both conjugates was examined. The net sinusoidal efflux clearance (CL(eff)) based on the unbound concentration in the liver did not change for 4MU glucuronide (4MUG) or significantly increase for 4MU sulfate (4MUS), suggesting that the sinusoidal efflux of both conjugates is not mediated by the transport systems dependent on adenosine triphosphate. 3. Under Cl(-)-depleted conditions, the CL(eff) of 4MUG significantly decreased, but the saturation of its sinusoidal efflux rather than the transport system dependent on Cl(-) might be involved because the hepatic concentration of 4MUG was extensively higher than that of the control study due to the extremely attenuated biliary excretion. The CL(eff) of 4MUS also significantly decreased, but its hepatic concentration was not different from that in the control study, suggesting that the transport system using Cl(-) is involved in the sinusoidal efflux of 4MUS. 4. The effect of glutathione was examined. CL(eff) of 4MUG was not affected by the additional glutathione, but CL(eff) of 4MUS decreased significantly, suggesting that some transport system sensitive to glutathione is involved in the sinusoidal efflux of 4MUS, but not of 4MUG. 5. Transporters such as Oatp1, Oatp2 and/or Npt1 might be involved in the sinusoidal efflux of 4MUS, but 4MUG is secreted from the sinusoidal membrane via the systems that are totally different from those for 4MUS.

Topics: Animals; Biological Transport, Active; Chlorides; Glucuronides; Glutathione; Hymecromone; In Vitro Techniques; Liver; Male; Models, Biological; Rats; Rats, Wistar; Sodium Azide; Sulfates; Xenobiotics

The venom of Naja nigricolis was found to contain a high level of the enzyme aryl sulfatase. The enzyme was isolated from the venom of N. nigriclois and purified to electrophoretic homogeneity by gel chromatography on Sephadex G-100, DEAE-cellulose, and phenyl-sepharose columns. The enzyme was optimally active at pH 5 and 40 degrees C. Arrhenius plot for the determination of the activation energy (E(a)) gave the value 25 kJ/mol with a half-life (t(1/2)) of 5 min at 50 degrees C. It was highly activated by Fe(2+) and Ca(2+) and inhibited by Co(2+) and Mn(2+). The enzyme catalyzed the hydrolysis of the fluorescent compound methylumbelliferyl-sulfate (MU-SO(4)). Double reciprocal plots of initial velocity data, using MU-SO(4) as substrate, gave a K(M) value of 110 microM and V(max) of 225 micromol min(-1) x mg(-1). N. nigricolis Aryl sulphatase also hydrolyzed chondroitin-4-sulphate. It was inhibited competitively by N-acetyl glucosamine sulfate (GlcNAc-SO(4)), glucose-6-sulfate (Glc-6-SO(4)), and glucose 1-sulfate (Glc-1-SO(4)). Extrapolated inhibition binding constants (K(i)) gave the values of 3, 25, and 315 microM for GlcNAc-SO(4), Glc-6-SO(4), and Glc-1-SO(4) respectively.

Topics: Acetylglucosamine; Animals; Arylsulfatases; Cations, Divalent; Chondroitin Sulfates; Elapid Venoms; Glucose; Hydrogen-Ion Concentration; Hydrolysis; Hymecromone; Kinetics; Protein Binding; Substrate Specificity; Temperature; Thermodynamics

X-linked recessive ichthyosis (XRI) is a genetic disorder of keratinization with extracutaneous manifestations due to deficiency of steroid sulfatase (STS). Because STS plays an important role in androgen metabolism, and elevated levels of dehydroepiandrosterone sulfate have been reported in young men with andogenetic alopecia (AGA), the hypothesis was advanced that men with XRI do not show AGA or develop only mild forms of common baldness.. Patients with a diagnosis of XRI confirmed by analysis of the microsomal sulfatases in our clinic between 1984 and 1998, and in whom study of the case histories depicted the typical clinical presentation of XRI, were sent a questionnaire with the Hamilton-Norwood scale of patterns of hair loss in men, inquiring them to designate the condition of their scalp hair.. Of 15 questionnaires returned, 7 indicated Hamilton-Norwood I, 3 Hamilton-Norwood II, 3 Hamilton-Norwood III-vertex, 1 Hamilton-Norwood IV and 1 Hamilton-Norwood VII. The results of this survey do not support the hypothesis that XRI and AGA are mutually exclusive, in as much as advanced AGA was found among these men.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Alopecia; Dehydroepiandrosterone Sulfate; Genes, Recessive; Humans; Hymecromone; Ichthyosis, X-Linked; Male; Middle Aged; Severity of Illness Index; Surveys and Questionnaires

The human genome contains six arylsulfatase genes (ARSA-ARSF), of which four are clustered in a distal region of the short arm of the X chromosome (Xp22.3). They were probably generated by a series of evolutionary duplication events; their exon-intron boundaries are identical. Nevertheless, different transcript lengths and the absence of cross-hybridizations point to a specific function of each gene in human cell metabolism, and multiple transcripts suggest the coding of protein isoforms. We identified a novel protein isoform of the ARSD gene by isolation of a series of cDNA clones from a human testis cDNA library. The clones were only partially identical to another series of ARSD clones isolated earlier (now designated ARSDalpha clones). Their specific C-terminal region (1160 nt) encodes a novel ARSD peptide of 48 amino acids and was identified as part of intron 6 of the ARSD gene in Xp22.3. We therefore designate them ARSDbeta clones. Expression analyses of ARSDalpha and ARSDbeta by semiquantitative RT-PCR revealed the presence of both in multiple human tissues, although in different quantities. A physiologic substrate for arylsulfatase D proteins is not known. We therefore estimated their sulfatase activities in vitro with the aid of the 4-methylumbelliferyl sulfate (4-MUS) assay. Surprisingly, neither ARSD protein isoform demonstrated any sulfatase activity alone or in combination, although their catalytic peptide domain is strongly conserved in comparison with that of the other X-chromosomal arylsulfatase enzymes (ARSC, ARSE, ARSF), all of which are functionally active in the 4-MUS assay.

Topics: Amino Acid Sequence; Animals; Arylsulfatases; Base Sequence; Catalytic Domain; Cloning, Molecular; Conserved Sequence; COS Cells; Exons; Gene Expression Profiling; Gene Library; Humans; Hydrogen-Ion Concentration; Hymecromone; Introns; Isoenzymes; Male; Molecular Sequence Data; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Alignment; Testis; Transfection; X Chromosome

The hepatocellular entry of 4-methylumbelliferyl sulfate (4MUS) a highly ionized and highly bound anion capable of futile cycling, was examined in the single-pass albumin-free perfused rat liver preparation. Desulfation of 4MUS to 4-methylumbelliferone (4MU) was verified in vitro to be a low-affinity, high-capacity process (Km = 731 micromol/L; Vmax = 414 nmol min(-1) g(-1) liver). With 4MUS given to the perfused rat liver, sulfation of 4MU, the formed metabolite, was attenuated in the presence of 2,6-dichloro-4-nitrophenol (DCNP), a sulfation inhibitor, and when sulfate ion was substituted by chloride ion. 4MU sulfation, being a high-affinity system, was reduced most effectively at the lowest 4MUS concentration (15 micromol/L) used, evidenced by the increased (24%) net hepatic extraction ratio of 4MUS and reduced utilization (72%) of infused tracer 35SO4(2-) by 4MU for 4MU35S formation. Single-pass multiple indicator dilution (MID) studies were thus conducted under identical conditions (DCNP and absence of inorganic sulfate), with injection of [3H]4MUS and a set of noneliminated vascular and cellular reference indicators into the portal vein (prograde) or hepatic vein (retrograde), against varying background bulk concentrations of 4MUS (5 to 900 micromol/L). The steady-state removal rate of 4MUS and formation rates of 4MU and its glucuronide conjugate (4MUG) were not altered with perfusion flow direction, suggesting the presence of even or parallel distributions of 4MUS desulfation and 4MU glucuronidation activities. When the outflow dilution profile of [3H]4MUS was evaluated with the barrier-limited model of Goresky, a slight red cell carriage effect was found for 4MUS. The permeability surface area product for cellular entry for prograde showed a dramatic concentration-dependent decrease (from 0.13 to 0.01 mL sec(-1) g(-1), or 7.4 to 0.56 times the blood perfusate flow rate) and was resolved as saturable and nonsaturable components, while data for retrograde were more scattered, varying from 2.8 to 1 times the blood perfusate flow rate. Efflux (coefficient = 0.0096 +/- 0.0024 and 0.0088 +/- 0.0062 mL sec(-1) g(-1), respectively) was relatively insensitive to concentration and flow direction. The same was observed for the removal capacity for metabolism and excretion (sequestration coefficient: for prograde, 0.0056 +/- 0.0017 mL sec(-1) g(-1); for retrograde, 0.0056 +/- 0.003 mL sec(-1) g(-1)). The decrease in the apparent partition coefficient (ratio o

Topics: Animals; Drug Carriers; Hymecromone; Indicator Dilution Techniques; Liver; Male; Nitrophenols; Perfusion; Rats; Rats, Sprague-Dawley; Sulfates

An assay was developed, using two similar formats, to simultaneously measure both the lysosomal targeting receptor binding and enzyme activity of the recombinant human enzyme N-acetylgalactosamine-4-sulfatase. This assay also has potential application for all phosphorylated lysosomal enzymes that contain mannose-6-phosphate residues. The receptor was either purified from fetal bovine sera then adsorbed, or produced in situ by growing and fixing diploid human fibroblast-like cells, to a solid phase. The enzyme substrate was 4-methylumbelliferyl sulfate which fluoresces after cleavage of the sulfate moiety. Both the precursor and mature forms of the recombinant enzyme were used to demonstrate the specificity and usefulness of the assay. The assay is rapid and sensitive and has a wide dynamic range. Association between the receptor and the mannose-6-phosphate residues was abrogated in the presence of a competitive inhibitor, mannose 6-phosphate. However, partial activity was still measured when the mature enzyme was incubated in the presence of mannose 6-phosphate when using the fixed fibroblast format. This would indicate that the recombinant enzymes contain at least one terminal sugar moiety other than mannose 6-phosphate which can recognize receptors on the surface of human fibroblast-like cells. Other possible applications of this assay are also discussed.

Topics: Adsorption; Animals; Binding, Competitive; Cattle; Cell Line; Chondro-4-Sulfatase; Fibroblasts; Humans; Hymecromone; Kinetics; Receptor, IGF Type 2; Recombinant Proteins; Spectrometry, Fluorescence

The vascularly perfused rat intestine and liver preparations were used to examine the effect of flow (8 and 10 ml/min) on the sequential metabolism of 4-methylumbelliferone (4MU), which forms primarily the glucuronide conjugate (4MUG) in intestine and the sulfate conjugate (4MUS) in liver at low input concentrations of 4MU. In this system, a constant tracer concentration of [3H]4MU was delivered systemically at 8 or 10 ml/min to the perfused rat small intestine preparation; the portal venous outflow perfusate at 8 and 10 ml/min was collected at steady state, reoxygenated and in tum delivered to the perfused rat liver preparation from a second rat donor. The intestinal extraction ratio and formation of 4MUG were decreased from 0.57 +/- 0.07 to 0.49 +/- 0.06 and 42 +/- 5 to 36 +/- 4% input rate, respectively, upon increasing the flow rate from 8 to 10 ml/min (P < .05). These decreases were the result of the reduction in transit time with increasing flows. In contrast, hepatic 4MU conjugation was increased (from 40 +/- 7% to 48 +/- 6% input rate to intestine) upon increasing the flow rate from 8 and 10 ml/min (P < .05), attributed primarily to increased formation of the major metabolite, 4 MUS, in liver (from 35 +/- 9% to 39 +/- 9% input rate to intestine). The unusual observation on increased hepatic metabolite formation with increasing flow could be rationalized. With increased flow to the serially perfused organs, there was an increased supply of substrate to the liver, the posterior organ, because of a faster intestinal transit time. Decreased intestinal metabolism (formation of 4MUG) at increased flow was compensated by increased hepatic metabolism (formation of 4MUS), albeit attenuated because of a faster hepatic transit time. The proportions of total 4MU conjugates formed (4MUG + 4MUS) across the intestine and liver remained constant at both flow rates. Hence, a rather constant overall extraction ratio (0.98 +/- 0.004 and 0.97 +/- 0.005, P > .05) existed across the two organs. The results demonstrate that the intestine, the anterior organ, plays a regulatory role on substrate supply to the posterior organ, the liver. With an increase in flow, the contribution of the intestine will decrease, whereas the contribution of the liver will increase in the overall first-pass metabolism.

Topics: Animals; Hymecromone; Intestinal Mucosa; Liver; Male; Perfusion; Rats; Rats, Sprague-Dawley

The uptake of estrone sulfate (E1S; 1 to 400 microM), harmol sulfate (HS; 5 to 900 microM), and 4-methylumbelliferyl sulfate (4MUS; 5 to 1000 microM) was investigated in isolated rat hepatocytes in the presence or absence of inhibitors. Uptake of all of the sulfate conjugates was rapid and exhibited saturation kinetics, best characterized by saturable and nonsaturable (linear transmembrane clearance) transport systems. The KM's were: 16 +/- 6, 123 +/- 28, and 64 +/- 6 microM for E1S, HS, and 4MUS, respectively, with corresponding Vmax's of 0.85 +/- 0.56, 0.48 +/- 14, and 0.42 +/- 0.07 nmol/min/10(6) cells. The nonsaturable uptake clearances, which displayed concentration-independent uptake, were 3 +/- 2, 1 +/- 0.1, 0.5 +/- 0.1 microliter/min/10(6) cells, respectively. Uptake of E1S was inhibited by ouabain (1 mM) and replacement of sodium by choline, whereas HS was insensitive to the addition or substitution. Uptake of both E1S and HS was significantly reduced by metabolic inhibitors (antimycin A, 2.7 microM, rotenone, 30 microM, and KCN, 2 mM) and temperature reduction (from 37 to 27 degrees C). 4,4'-Diisothiocyanostilbene-2-2-'disulfonic acid (2 mM), an inhibitor of anion transport, reduced E1S and HS uptake; E1S uptake was also reduced by HS. HS uptake by both saturable and nonsaturable transport components was depressed by 4MUS (300 microM); the apparent KM was increased by 83% while the Vmax remained unaltered, and the nonsaturable component was decreased by 48%. The data strongly suggest that multiple pathways exist for the uptake of E1S, HS, and 4MUS. E1S uptake is sodium-dependent, requires energy, and is inhibited by anions such as 4,4'diisothiocyanostilbene-2-2'-disulfonic acid and other sulfate conjugates. HS uptake, while being energy dependent, is not sodium dependent, and is inhibited by 4MUS in a competitive fashion. At least one of these pathways is shared.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport; Carrier Proteins; Choline; Estrogens, Conjugated (USP); Estrone; Harmine; Hymecromone; In Vitro Techniques; Liver; Male; Ouabain; Rats; Rats, Sprague-Dawley

Topics: Drug Stability; Hymecromone; Solubility

4-Methylumbelliferyl sulfate (4MUS), a polar metabolite of 4-methylumbelliferone (4MU), is known to undergo desulfation and participate in futile cycling with 4MU. Unusual parabolic or increasing profiles of the steady-state extraction ratio (Ess) of 4MUS with respect to concentration in rat livers perfused with a red cell (20%)-albumin (1%) medium have been reported (Ratna et al., 1993). In order to study this unusual phenomenon, we examined the desulfation of 4MUS in the single-pass rat liver in the absence of albumin. We further employed a tubular-flow model to describe the present observations and data previously obtained on 4MUS in order to predict the effects of protein binding and enzymatic constants for conjugation/deconjugation on the hepatic processing of 4MUS and its metabolites. The net hepatic extraction ratio from albumin-free perfusate decreased from 0.465 to 0.326 when the 4MUS input concentration was increased from 122 to 908 microM; moreover, the unusual profiles previously observed for ESS with increasing concentration in albumin-containing perfusate were not apparent. The hepatic clearances and desulfation rates of 4MUS were essentially identical to those observed in the presence of albumin, when the latter were expressed in terms of unbound concentrations (unbound input and logarithmic average unbound concentration of the input and output blood). Initial modeling indicated that first nonlinear protein binding (dissociation constant KD of 93 microM) and then saturable desulfation (Km of 382 microM) were responsible for the unusual increasing and then decreasing trend of ESS with concentration in the presence of albumin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blood Proteins; Hymecromone; Kinetics; Liver; Male; Perfusion; Protein Binding; Rats; Rats, Sprague-Dawley; Serum Albumin

Futile cycling between 4-methylumbelliferone and its sulfate and glucuronide conjugates was examined in the single-pass perfused rat liver preparation. The steady-state hepatic extraction ratio of 4-methylumbelliferone was found to be high (0.97) at a low input concentration of 0.005 mumol/L (tracer), with a net 4-methylumbelliferyl sulfate/4-methylumbelliferyl glucuronide ratio of about 5:1; at 63 mumol/L the steady-state extraction ratio had remained constant despite a shift from net sulfation to net glucuronidation. At higher input 4-methylumbelliferone concentrations, saturation was evidenced by a decreased steady-state extraction ratio and reduced net sulfation and net glucuronidation. Because 4-methylumbelliferyl sulfate and 4-methylumbelliferyl glucuronide deconjugation would result in an intracellular accumulation of 4-methylumbelliferone, the phenomenon was monitored with a shift in tracer [3H]4-methylumbelliferone metabolism from sulfation to glucuronidation with increased intracellular 4-methylumbelliferone concentration. When 4-methylumbelliferyl sulfate (0 to 890 mumol/L) or 4-methylumbelliferyl glucuronide (0 to 460 mumol/L) was delivered simultaneously with tracer [3H]4-methylumbelliferone to the rat liver, notable desulfation of 4-methylumbelliferyl sulfate (18% to 38% rate in) but little deglucuronidation of 4-methylumbelliferyl glucuronide (1.2% to 2.1% rate in) was observed. With 4-methylumbelliferyl sulfate, 4-methylumbelliferone and 4-methylumbelliferyl glucuronide were readily found as metabolites, whereas with 4-methylumbelliferyl glucuronide, levels of the metabolites, 4-methylumbelliferone and 4-methylumbelliferyl sulfate, were much reduced. 4-Methylumbelliferyl sulfate and not 4-methylumbelliferyl glucuronide shifted tracer [3H]4-methylumbelliferone metabolism from [3H]4-methylumbelliferyl sulfate to [3H]4-methylumbelliferyl glucuronide formation in a concentration-dependent fashion. The steady-state extraction ratio for 4-methylumbelliferyl sulfate (0.1 to 0.3) was comparatively higher than that for 4-methylumbelliferyl glucuronide (0.05), and it was found to increase with concentration, an observation explained by the nonlinear protein binding of 4-methylumbelliferyl sulfate. Biliary excretion rates for 4-methylumbelliferone and 4-methylumbelliferyl sulfate were proportional to their input or net formation rates, regardless of whether 4-methylumbelliferone, 4-methylumbelliferyl glucuronide or 4-methylumbelliferyl sulfate was ad

Topics: Animals; Bile; Blood Proteins; Hymecromone; Liver; Male; Models, Biological; Perfusion; Protein Binding; Rats; Rats, Sprague-Dawley

We used perfused rat livers to investigate the role of endosomes versus lysosomes in the hydrolysis of endocytosed material. When perfusions were performed at 37 degrees C with 125I-asialoorosomucoid, 125I-galactosylated albumin, or 125I-mannosylated albumin, there was a 15-min lag before trichloroacetic acid-soluble degradation products were detected. Furthermore, no hydrolysis was detected at 16 degrees C, indicating that there was no significant prelysosomal degradation of these proteins. Since detection by this method depends on extensive hydrolysis, we subsequently used three small synthetic molecules from which fluorescent products are generated by a single cleavage. These were 4-methylumbelliferyl sulfate, 4-methylumbelliferyl phosphate, and 4-methylumbelliferyl-beta-D-glucosaminide, which are substrates for aryl sulfatase, acid phosphatase, and beta-hexosaminidase, respectively. Using the first two compounds, hydrolysis was detected after 3 min at 37 degrees C and still occurred, albeit to a reduced extent, at 16 and 4 degrees C. This indicates that aryl sulfatase and acid phosphatase are active prelysosomally. We found a different result with 4-methylumbelliferyl-beta-D-glucosaminide. At 37 degrees C, there was a greater than 15-min lag before hydrolysis products were measured; furthermore, hydrolysis ceased at 16 degrees C, indicating that beta-hexosaminidase is active lysosomally. Taken together, these findings show that there is selective activation and/or delivery of hydrolases along the endocytic pathway.

Topics: Acetylglucosamine; Animals; Asialoglycoproteins; Cell Compartmentation; Endocytosis; Endosomes; Glucosamine; Glycoproteins; Hydrolases; Hymecromone; Liver; Lysosomes; Male; Pinocytosis; Rats; Rats, Inbred Strains; Serum Albumin, Bovine

A method combining immune capture and enzyme detection by fluorochemistry has been developed for the diagnostic assay of N-acetylgalactosamine-4-sulphatase (4-sulphatase). The procedure uses a monoclonal antibody 4-S 4.1 to immunoadsorb 4-sulphatase specifically from complex protein samples containing other sulphatases, and 4-methylumbelliferyl sulphate to detect captured 4-sulphatase. The assay provides an accurate and simple method for the diagnosis of Maroteaux-Lamy syndrome (Mucopolysaccharidosis type VI).

Topics: Antibodies, Monoclonal; Chondro-4-Sulfatase; Humans; Hymecromone; Immunoenzyme Techniques; Immunosorbent Techniques; Mucopolysaccharidosis IV; Spectrometry, Fluorescence

A direct high-performance liquid chromatographic (HPLC) assay was developed for the separation and determination of 4-methylumbelliferone (4MU) and its glucuronide (MUG) and sulfate (MUS) conjugates in the cell-free perfusate ("plasma") from in situ perfused rat intestine-liver preparation. In addition, a procedure was developed to extract and determine 4MU in the whole blood perfusate. Perfusate plasma containing an internal standard (umbelliferone) was precipitated with methanol (1:4, v/v), and injected into a reversed-phase HPLC system with gradient elution. 4MU and the same internal standard were also extracted directly from the whole blood perfusate with ethyl acetate and injected into a reversed-phase HPLC system with isocratic elution. Inter- and intra-day precision studies (n = 5 for each) for both the plasma and whole blood procedures demonstrated relative standard deviation of less than 10% at all concentrations studied. The compounds were stable in either the plasma or blood extracts at room temperature for up to 72 h. The procedures were successfully used to analyze perfusate samples obtained from the single-pass in situ perfusion of rat intestine-liver system with either trace (0.95 nM) or 32.3 microM concentrations of 4MU. The intestine was responsible for the formation of most of the MUG formed by the intestine-liver preparation during steady-state perfusion with either input concentration of 4MU.

Topics: Animals; Chromatography, High Pressure Liquid; Drug Stability; Erythrocytes; Humans; Hymecromone; Intestines; Liver; Perfusion; Rats

The anticoagulant phenprocoumon is mainly metabolized in humans to hydroxylated metabolites and their glucuronides. A method is described for the determination of phenprocoumon, 4'-hydroxyphenprocoumon, 6-hydroxyphenprocoumon, 7-hydroxyphenprocoumon, and their glucuronide and sulphate conjugates in human urine. Reversed-phase high-performance liquid chromatography is performed after selective extraction with disposable quaternary amine columns of untreated, and beta-glucuronidase- or sulphatase-treated urine samples. Urinary excretion data are presented for total, glucuronidated, sulphated and free phenprocoumon, 4'-hydroxyphenprocoumon, 6-hydroxyphenprocoumon and 7-hydroxyphenprocoumon in twelve patients after an average daily dosage of 1.3-4.2 mg phenprocoumon.

Topics: Adult; Aged; Chromatography, High Pressure Liquid; Glucuronidase; Humans; Hydrolysis; Hymecromone; Indicators and Reagents; Male; Middle Aged; Phenprocoumon; Spectrophotometry, Ultraviolet; Sulfatases

Arylsulphatase C (ASC) activity in leukocytes and fibroblasts measured with 4-methylumbelliferylsulphate, is caused by at least two genetically different sulphatases. One of these is steroid sulphatase (STS). Depending on the substrate concentration, about 10-50% of the ASC activity in leukocytes can be attributed to sulphatases other than STS. Steroid sulphatase can be measured specifically with 4-methylumbelliferylsulphate as the fraction of total ASC activity which is inhibitable by dehydroepiandrosterone sulphate. Using this assay, the adjusted ASC activity in leukocytes from patients with X-linked ichthyosis was 2% of normal. Obligate heterozygotes showed reduced activity.

Topics: Arylsulfatases; Dehydroepiandrosterone; Female; Fluorometry; Genetic Linkage; Humans; Hymecromone; Ichthyosis; Kinetics; Leukocytes; Male; Steryl-Sulfatase; Sulfatases; X Chromosome

Recessive X-linked ichthyosis (RXLI) has its biochemical basis in a defect of the enzyme steroid sulfatase. Since several studies have reported a simultaneous deficiency of arylsulfatase C and steroid sulfatase it has been hypothesized that both enzymes are identical. In human hair follicles, however, hydrolytic activity for 4-methylumbelliferone sulfate, the substrate for arylsulfatase C, is found, while dehydroepiandrosterone sulfate is not hydrolyzed at all. These findings suggested the possible existence of two different enzymes. In the present paper structure-activity studies and molecular energy calculations are used for the demonstration that the remaining sulfatase activity in hair follicles of RXLI patients can be explained on the basis of the assumption that the enzyme has not lost its total function but has become less efficient.

Topics: Adolescent; Adult; Dehydroepiandrosterone; Dehydroepiandrosterone Sulfate; Equilenin; Estrone; Female; Genetic Linkage; Hair; Humans; Hymecromone; Ichthyosis; Male; Middle Aged; Substrate Specificity; Sulfatases; X Chromosome

Ascorbic acid-2-sulfatase was isolated from rat liver by a multistep procedure. DEAE Sephacel ion-exchange chromatography resolved crude ascorbic acid-2-sulfatase into cationic and anionic fractions. These fractions were purified 75- and 230-fold, respectively. The comparative biochemical properties suggest that arylsulfatase B is responsible for the cationic ascorbic acid-2-sulfatase activity, while arylsulfatase A appears to be responsible for the anionic ascorbic acid-2-sulfatase activity. Partially purified arylsulfatase A hydrolyzed ascorbic acid-2-sulfate at 4% the rate of p-nitrocatechol sulfate hydrolysis, while arylsulfatase B hydrolyzed ascorbic acid-2-sulfate at 0.6% the p-nitrocatechol sulfate rate.

Topics: Animals; Arylsulfatases; Ascorbic Acid; Catechols; Cerebroside-Sulfatase; Chondro-4-Sulfatase; Chromatography, Ion Exchange; Hymecromone; Liver; Rats; Rats, Inbred Strains; Substrate Specificity; Sulfatases

Metal precipitation techniques for ultrastructural demonstration of arylsulfatase C activity were studied in rat kidney. Possible substrates for the techniques were biochemically tested with regard to their velocity of enzymatic hydrolysis and their specificity for arylsulfatase C. Effects of buffers and capturing metals were also examined. The results of these biochemical studies were then verified histochemically. Incubation in a medium containing 1 mM 4-methylumbelliferyl sulfate, 1% barium chloride, 0.1 M imidazole-HCl buffer (pH 7.5), and 5% sucrose achieved identifiable results in adequately fixed kidney. Precipitation of barium sulfate was localized mainly in the endoplasmic reticulum and perinuclear cisterns of the epithelial cells in the descending portions of proximal tubules.

Topics: Animals; Arylsulfatases; Barium Sulfate; Buffers; Chemical Precipitation; Endoplasmic Reticulum; Epithelium; Histocytochemistry; Hydrogen-Ion Concentration; Hymecromone; Kidney; Kinetics; Male; Microscopy, Electron; Rats; Rats, Inbred Strains; Steryl-Sulfatase; Substrate Specificity; Sulfatases

4-Methylumbelliferyl sulfate was used to characterize sulfatase activity in periportal and pericentral regions of the liver lobule in the perfused rat liver. Following infusion of 1.5 mM of this organic sulfatester, free 4-methylumbelliferone and 4-methylumbelliferyl glucuronide were formed at rates of 13 and 9 mumoles/g/h, respectively, in livers from fasted, phenobarbital-treated rats. 5-Pregnen-3 beta-ol, 20-one sulfate inhibited hydrolysis and metabolite production completely, whereas perfusion with nitrogen-saturated perfusate or FCCP decreased total metabolite formation by only 30%. 4-Methylumbelliferone formed from the hydrolysis of 4-methylumbelliferyl sulfate was monitored with micro-light guides placed on periportal and pericentral areas of the liver lobule. Detection of the desulfated product was always greater in the downstream region, i.e., infusion of 4-methylumbelliferyl sulfate produced a higher fluorescence signal in pericentral areas when perfusion was in the anterograde direction, while periportal areas demonstrated higher activity during perfusion in the retrograde direction. Perfusion with nitrogen-saturated perfusate abolished these differences. Taken together, these data suggest that uptake of organic sulfateesters is partially energy dependent, follows the hepatic oxygen gradient inversely, and is a major rate determinant for sulfatase activity in the liver.

Topics: Animals; Female; Fiber Optic Technology; Hydrolysis; Hymecromone; In Vitro Techniques; Liver; Perfusion; Rats; Rats, Inbred Strains; Spectrometry, Fluorescence; Umbelliferones

Rodent and bovine arylsulfatase B hydrolyze 4-methylumbelliferyl sulfate (4MUS) 10- to 30-fold more efficiently than arylsulfatase A. Therefore, 4MUS grossly underestimates arylsulfatase A activity in the presence of excess arylsulfatase B.

Topics: Animals; Catechols; Cattle; Cerebroside-Sulfatase; Chondro-4-Sulfatase; Hydrolysis; Hymecromone; Isoenzymes; Kinetics; Liver; Mice; Rats; Rats, Inbred Strains; Sulfatases

The dose-dependent first-pass hepatic metabolism and pharmacokinetics of 4-methylumbelliferone (4-MU) were studied at four dose levels (17.6 micrograms-5.29 mg) in rats. 4-MU was given intravenously and intraportally to determine the availability of 4-MU. The availability increased from 0.18 to 1.31 when the dose was increased from 17.6 micrograms to 5.29 mg/rat. The total body plasma clearance of 4-MU was accounted for mostly by the hepatic conjugative metabolism. The contribution of renal clearance to total plasma clearance was 11-35%, depending on the dose. The marked dose-dependency of availability may thus be explained by the saturable conjugative metabolism of 4-MU.

Topics: Animals; Biological Availability; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Femoral Vein; Hymecromone; Injections, Intravenous; Kinetics; Liver; Male; Portal Vein; Protein Binding; Rats; Rats, Inbred Strains; Time Factors; Umbelliferones

The hydrolysis of 4-methylumbelliferyl sulfate by liver sulfatases to free fluorescent 4-methylumbelliferone and the subsequent formation of the glucuronide conjugate were studied in the isolated perfused rat liver. In livers from fed, phenobarbital-treated rats, 4-methylumbelliferyl sulfate (0.25-1.5 mM) was hydrolyzed rapidly to free 4-methylumbelliferone at maximal rates of about 5 mumol/g/hr. A major fraction of the free 4-methylumbelliferone formed was converted to the glucuronide at maximal rates around 20 mumol/g/hr. Similar rates of hydrolysis were observed in livers from fasted, phenobarbital-treated or normal rats, although the ratio of glucuronide to free product was decreased markedly by fasting. In liver homogenates, however, rates of organic sulfate hydrolysis exceeded those observed in the perfused liver by at least 2-fold, suggesting that 4-methylumbelliferyl sulfate content is an important determinant of rates of hydrolysis in the perfused liver. There was a good correlation (r = 0.91) between rates of product formation and fluorescence of 4-methylumbelliferone detected from the liver surface with fiber optic light guides. Fluorescence of 4-methylumbelliferone produced from hydrolysis of 4-methylumbelliferyl sulfate was also monitored with micro-light guides placed on periportal and pericentral areas of the liver lobule for the estimation of local rates of product formation. When perfusions were in the anterograde direction, desulfation of 4-methylumbelliferyl sulfate was about 50% higher in pericentral (28.8 +/- 9.3 mumol/g/hr) than in periportal (18.2 +/- 2.7 mumol/g/hr) areas. Furthermore, 4-methylumbelliferyl sulfate content determined in microdissected samples was 1.5- to 2-fold higher in pericentral than in periportal regions of the liver lobule but the activity of 4-methylumbelliferyl sulfate sulfatase was identical in both zones of the liver lobule. We conclude, therefore, that the local substrate content is an important determinant of rates of 4-methylumbelliferyl sulfate hydrolysis in sublobular zones of the liver.

Topics: Animals; Fasting; Female; Hydrolysis; Hymecromone; Liver; Oxygen Consumption; Perfusion; Phenobarbital; Rats; Sulfatases; Umbelliferones

The role of the liver in the conjugation of 4-methylumbelliferone (4MU), mainly glucuronidation, was investigated in the rat in vivo. The liver extracted 4MU almost completely (97%) during steady-state infusion, as measured by the difference between 4MU concentration in portal and hepatic venous blood. Previously, it was shown that the intestinal region extracts 40% of the 4MU of the incoming arterial blood. The liver and the gastrointestinal region are so efficient that their conjugation activity can account for total body clearance of 4MU (50-60 ml/min per kg). These results and other evidence on extrahepatic conjugation of phenolic substrates suggest that glucuronidation may be limited to the liver, (the kidney) and the gastrointestinal region, while sulfation may occur more widespread throughout the body. Protein binding studies showed the sulfate conjugate to be even more protein-bound than unconjugated 4MU, while 4MU glucuronide was much less bound to rat plasma proteins.

Topics: Animals; Blood Proteins; Hymecromone; Infusions, Parenteral; Injections, Intravenous; Intestinal Mucosa; Liver; Male; Protein Binding; Rats; Rats, Inbred Strains; Umbelliferones

SWR/J mice possess two- to threefold higher 4-methylumbelliferyl sulfate (4MUS), dehydroepiandrosterone sulfate (DHEAS) and estrone sulfate (E1S) sulfatase activities in liver and kidney extracts than do A/J mice. These interstrain activity differences are maintained throughout the 6- to 45-day postnatal period. Characteristics of the hepatic activities of SWR/J mice suggest that all three activities reside in the same enzyme. Biochemical properties of the SWR/J and A/J enzyme were not significantly different. Expression of hepatic enzyme activity is subject to regulation by an autosomal locus possessing two alleles with additive effects. Postnuclear E1S- and DHEAS-sulfatase activities are primarily microsomal. Although postnuclear hepatic 4MUS-sulfatase activity is predominantly microsomal, renal activity is primarily nonmicrosomal. Only that portion of 4MUS-sulfatase occurring in cell membranes appears capable of hydrolyzing E1S and DHEAS. The hepatic- and renal-specific subcellular distributions of 4MUS-sulfatase activity may reflect tissue differences in enzyme processing. Renal 4MUS-sulfatase activity is also controlled by an autosomal gene with two alleles having additive effects. Positive correlation between hepatic and renal 4MUS-sulfatase activities indicates that both activities are most likely influenced by the same gene.

Topics: Alleles; Animals; Arylsulfatases; Dehydroepiandrosterone; Dehydroepiandrosterone Sulfate; Estrone; Hymecromone; Kidney; Mice; Mice, Inbred Strains; Microsomes, Liver; Steryl-Sulfatase; Substrate Specificity; Sulfatases

Topics: Chromatography, High Pressure Liquid; Humans; Hymecromone; Kinetics; Umbelliferones

Rabbit liver aryl sulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) is a glycoprotein containing 4.6% carbohydrate in the form of 25 residues of mannose, seven residues of N-acetylglucosamine, and three residues of sialic acid per enzyme monomer of molecular weight 140 000. Each monomer consists of two equivalent polypeptide chains. The protein has a relatively high content of proline, glycine and leucine, and the amino acid composition of rabbit liver aryl sulfatase A is similar to that of other known liver sulfatases. Rabbit liver aryl sulfatase A catalyzes the hydrolysis of a wide variety of sulfate esters, although it appears possible that cerebroside sulfate is a physiological substrate for the enzyme because the Km is very low (0.06 mM). The turnover rate for hydrolysis of nitrocatechol sulfate or related synthetic substrates is much higher than the rate with most naturally occurring sulfate esters such as cereroside sulfate, steroid sulfates, L-tyrosine sulfate or glucose 6-sulfate. However, the turnover rate with ascorbate 2-sulfate is comparable to the rates measured using most synthetic substrates. These results are discussed in relationship to several previously described sulfatase enzymes which were claimed to have unique specificities.

Topics: Amino Acids; Animals; Ascorbic Acid; Carbohydrates; Catalysis; Catechols; Cerebroside-Sulfatase; Cerebrosides; Hydrolysis; Hymecromone; Kinetics; Liver; Nitrobenzenes; Rabbits; Substrate Specificity; Sulfatases; Sulfuric Acids; Tyrosine