Focal neurometabolic alterations in mice deficient for succinate semialdehyde dehydrogenase

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1-/-) deficient mice previously revealed elevated gamma-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified beta-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of beta-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.     

The metabolism of benzyl isothiocyanate and its cysteine conjugate.

1. The corresponding cysteine conjugate was formed when the GSH (reduced glutathione) or cysteinylglycine conjugates of benzyl isothiocyanate were incubated with rat liver or kidney homogenates. When the cysteine conjugate of benzyl isothiocyanate was similarly incubated in the presence of acetyl-CoA, the corresponding N-acetylcysteine conjugate (mercapturic acid) was formed. 2. The non-enzymic reaction of GSH with benzyl isothiocyanate was rapid and was catalysed by rat liver cytosol. 3. The mercapturic acid was excreted in the urine of rats dosed with benzyl isothiocyanate or its GSH, cysteinyl-glycine or cysteine conjugate, and was isolated as the dicyclohexylamine salt. 4. An oral dose of the cysteine conjugate of [14C]benzyl isothiocyanate was rapidly absorbed and excreted by rats and dogs. After 3 days, rats had excreted a mean of 92.4 and 5.6% of the dose in the urine and faeces respectively, and dogs had excreted a mean of 86.3 and 13.2% respectively. 5. After an oral dose of the cystein conjugate of [C]benzyl isothiocyanate, the major 14C-labelled metabolite in rat urine was the corresponding mercapturic acid (62% of the dose), whereas in dog urine it was hippuric acid (40% of the dose). 5. Mercapturic acid biosynthesis may be an important route of metabolism of certain isothiocyanates in some mammalian species.     

Genetic differences in alteration of albumin in the house mouse,Mus musculus

Two strains of mice were shown to differ in relative proportion of two major protein bands of liver under acid conditions of electrophoresis. Genetic control was autosomal and by a pair of dominant and recessive genes. The difference was observed only if liver homogenates were extracted by treating with dilute acid and alkali. The two protein bands were identified as albumin on the basis of electrophoretic migration, molecular weight, and immunological response. No differences were found between strains in relative amounts of comparable bands of plasma. However, in studies with extraction of mixtures, liver homogenates of one strain but not another were capable of converting a large proportion of albumin from several sources to a faster-migrating form. It was concluded that the strains differ in a factor in liver capable of causing a secondary modification of the albumin molecule.     

Biodegradation of 4-chlorobenzoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PA01 NC

A bacterial consortium capable of degrading chloroaromatic compounds was isolated from pulp and paper mill effluents by selective enrichment on 4-chlorobenzoic acid as sole source of carbon and energy. The four different bacterial isolates obtained from bacterial consortium were identified as Pseudomonas aeruginosa AY792969 (A), P. aeruginosa PA01 NC (B), Pseudomonas sp. ZZ5 DQ113452 (C) and Pseudomonas sp. AY762360 (D) based on their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. These bacterial isolates were found to be versatile in degrading a variety of chloroaromatic compounds including fluoro- and iodobenzoic acids. P. aeruginosa PA01 NC utilized 4-chlorobenzoic acid at 2 g/l as growth substrate. Biodegradation studies have revealed that this organism degraded 4-chlorobenzoic acid through 4-chlorocatechol which was further metabolized by ortho-cleavage pathway and the dechlorination occurred after the ring-cleavage.     

Cloning of the <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. FG1 dehalogenase genes and construction of hybrid pathways in <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strains

An Arthrobacter strain, able to utilize 4-chlorobenzoic acid as the sole carbon and energy source, was isolated and characterized. The first step of the catabolic pathway was found to proceed via a hydrolytic dehalogenation that leads to the formation of 4-hydroxybenzoic acid. The dehalogenase encoding genes (fcb) were sequenced and found highly homologous to and organized as those of other 4-chlorobenzoic acid degrading Arthrobacter strains. The fcb genes were cloned and successfully expressed in the heterologous host Pseudomonas putida PaW340 and P. putida KT2442 upper TOL, which acquired the ability to grow on 4-chlorobenzoic acid and 4-chlorotoluene, respectively. The cloned dehalogenase displayed a high specificity for para-substituted haloaromatics with affinity Cl > Br > I > F, in the order.     

Novel biotransformations of 4-chlorobiphenyl by a Pseudomonas sp.

A bacterium, tentatively identified as a representative of the genus Pseudomonas (strain MB86), was isolated from soil contaminated by wood-preservation chemicals by using 4-chlorobenzoate as an enrichment substrate. The pseudomonad was able to grow on 4-chlorobenzoic acid and 4-chlorobiphenyl as sole carbon and energy sources. Spent culture medium from 4-chlorobiphenyl-grown cells contained 4-chlorobenzoic acid, 4'-chloroacetophenone, 2-hydroxy,2-[4'-chlorophenyl] ethane, and 2-oxo,2-[4'-chlorophenyl] ethanol as metabolites. 4'-Chloroacetophenone was produced in large amounts, possibly as a dead-end metabolite.     

Novel biotransformations of 4-chlorobiphenyl by a Pseudomonas sp

A bacterium, tentatively identified as a representative of the genus Pseudomonas (strain MB86), was isolated from soil contaminated by wood-preservation chemicals by using 4-chlorobenzoate as an enrichment substrate. The pseudomonad was able to grow on 4-chlorobenzoic acid and 4-chlorobiphenyl as sole carbon and energy sources. Spent culture medium from 4-chlorobiphenyl-grown cells contained 4-chlorobenzoic acid, 4'-chloroacetophenone, 2-hydroxy,2-[4'-chlorophenyl] ethane, and 2-oxo,2-[4'-chlorophenyl] ethanol as metabolites. 4'-Chloroacetophenone was produced in large amounts, possibly as a dead-end metabolite.     

Metabolism of 4,4′-dichlorobiphenyl by white-rot fungi Phanerochaete chrysosporium and Phanerochaete sp. MZ142

Degradation experiment of model polychlorinated biphenyl (PCB) compound 4,4′-dichlorobiphenyl (4,4′-DCB) and its metabolites by the white-rot fungus Phanerochaete chrysosporium and newly isolated 4,4′-DCB-degrading white-rot fungus strain MZ142 was carried out. Although P. chrysosporium showed higher degradation of 4,4′-DCB in low-nitrogen (LN) medium than that in potato dextrose broth (PDB) medium, Phanerochaete sp. MZ142 showed higher degradation of 4,4′-DCB under PDB medium condition than that in LN medium. The metabolic pathway of 4,4′-DCB was elucidated by the identification of metabolites upon addition of 4,4′-DCB and its metabolic intermediates. 4,4′-DCB was initially metabolized to 2-hydroxy-4,4′-DCB and 3-hydroxy-4,4′-DCB by Phanerochaete sp. MZ142. On the other hand, P. chrysosporium transformed 4,4′-DCB to 3-hydroxy-4,4′-DCB and 4-hydroxy-3,4′-DCB produced via a National Institutes of Health shift of 4-chlorine. 3-Hydroxy-4,4′-DCB was transformed to 3-methoxy-4,4′-DCB; 4-chlorobenzoic acid; 4-chlorobenzaldehyde; and 4-chlorobenzyl alcohol in the culture with Phanerochaete sp. MZ142 or P. chrysosporium. LN medium condition was needed to form 4-chlorobenzoic acid, 4-chlorobenzaldehyde, and 4-chlorobenzyl alcohol from 3-hydroxy-4,4′-DCB, indicating the involvement of secondary metabolism. 2-Hydroxy-4,4′-DCB was not methylated. In this paper, we proved for the first time by characterization of intermediate that hydroxylation of PCB was a key step in the PCB degradation process by white-rot fungi.     

Metabolism of sialic acids from exogenously administered sialyllactose and mucin in mouse and rat

A mixture of N-acetyl-[4,5,6,7,8,9-14C]neuraminosyl-alpha (2-3(6]-galactosyl-beta (1-4-glucose[( 14C]sialyl-lactose) and N-acetylneuraminosyl-alpha (2-3(6]-galactosyl-beta(1-4)-glucit-1-[3H]ol(sialyl-[3H]lactitol) as well as porcine submandibular gland mucin labeled with N-acetyl- and N-glycoloyl-[9-(3)H]neuraminic acid were administered orally to mice. The distribution of the different isotopes was followed in blood, tissues and excretion products of the animals. One half of the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture given orally was excreted unchanged in the urine. The other half was hydrolysed by sialidase and partly metabolized further, followed by the excretion of 30% of the 14C-radioactivity as free N-acetyl-[4,5,6,7,8,9-14C]neuraminic acid and 60% of this radioactivity in the form of non-anionic compounds including expired 14CO2 within 24 h. The 14C-radioactivity derived from the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture which remained in the bodies of fasted mice after 24 h was less than 1%. In the case of well-fed mice, a higher amount of the sialic acid residues was metabolized. The bulk of radioactivity of the mucin was resorbed within 24 h. About 40% of the radioactivity administered was excreted by the urine within 48 h; 30% of this radioactivity represented sialic acid and 70% other anionic and non-anionic metabolic products. 60% of the radioactivity administered remained in the body, and bound 3H-labeled sialic acids were isolated from liver. Sialyl-alpha (2-3)-[3H]lactitol was injected intravenously into rats; the substance was rapidly excreted in the urine without decomposition. These studies show that part of the sialic acids bound to oligosaccharides and glycoproteins can be hydrolysed in intestine by sialidase and be resorbed. This is followed either by excretion as free sialic acid or by metabolization at variable degrees, which apparently depends on the compound fed and on the retention time in the digestive tract.     

Molecular cloning of the liver-specific rat F antigen

F antigen is a 43-kDa widely conserved liver protein that has been intensively used in studies of immunogenicity and tolerance; two murine allotypes have been identified. Immunization of specific responder inbred strains with liver homogenates from the opposite allotype leads to precipitating antibody and cell-mediated immunity against F. The antibodies produced are autoantibodies as they react equally well with self. We have identified a cDNA clone from rat liver that reacts with alloantisera to F. The fused polypeptide produced by the clone was shown to correspond to F by several experiments. First, alloantisera to F antigen reacted with the cloned fused polypeptide, but not control recombinant clones. Second, mice immunized with the fused polypeptide generate an antibody response that reacts specifically with the 43-kDa protein of mouse liver homogenates and with highly purified F antigen. Finally, both anti-F allosera and sera from mice immunized with the fused polypeptide react with the same 43-kDa liver protein on two-dimensional immunoblots. The nucleotide and deduced amino acid sequence of the clone are presented and the sequence was found to have a significant homology with L28, an Escherichia coli ribosomal protein. The availability of recombinant F antigen will allow definitive questions to be addressed with respect to epitopes and specifically the identification of the T cell epitope which allows for autoimmune responses.     

Preliminary studies on the metabolism of 3,2'-dimethyl-4-aminobiphenyl in the rat.

Distribution of 3,2-dimethyl-4-aminobiphenyl (DMAB) and its metabolites in vivo and the metabolism of DMAB by liver in vitro have been studied in the Wistar rat. DMAB-HCI purified by recrystallization and dissolved in ethanol was injected subcutaneously and extractions made from liver, feces, and urine. Similar technical procedures were used to study in vitro metabolism in rat liver homogenates. Two components were isolated from urine and liver having Rf values (thin-layer chromatography) of 0.13 and 0.59, respectively. Three additional metabolites were found in the hydrolyzed fecal fraction. Rechromatography of the major fecal component yielded 6 fluorescent compounds. Gas-liquid chromatography of the most highly fluorescent of these indicated at least 3 additional metabolites. The evidence presented indicates that the liver transforms DMAB to several metabolites which are rapidly transferred in conjugated form to the intestine via the bile. The urine does not appear to be the major excretory route. We have examined the purity of commercially available DMAB free base and DMAB-HCL and found an impurity that comprises approximately 10-24% of the total samle upon GLC analysis, depending upon the supplier. This contaminant was completely removed by recrystallization of the hydrochloride and the chemical identity of purified compound as DMAB confirmed. Recommendations are presented for the use of this purified compound in a biological system.     

4-Hydroxybenzoate:Polyprenyl transferase and the prenylation of 4-aminobenzoate in mammalian tissues

The effect of variously substituted derivatives of 4-hydroxybenzoic acid on 4-hydroxybenzoate:polyprenyltransferase activity in mitochondrial preparations derived from rat liver and brain has been investigated. Catecholamines such as dihyroxyphenylalanine and norepinephrine showed a minor inhibition of the activity of the enzyme in brain mitochondrial preparations, 4-aminobenzoic acid and 4-chlorobenzoic acid proved to be the most potent inhibitors of the reaction. Inhibition by 4-hydroxymercuribenzoate indicated that -SH groups were essential for activity. Studies using ¹⁴C-labeled compounds further revealed that 4-aminobenzoic acid was inhibitory by virtue of its ability to serve as an alternate substrate for prenylation. The product of the prenylation is identified as 3-polyprenyl-4-aminobenzoate based on chromatographic characteristics of the products formed in liver mitochondria and Escherichia coli, the retention of the carboxyl group of 4-[carboxyl-¹⁴C]aminobenzoate, the incorporation of isopentenyl pyrophosphate, the effect of bacitracin, and the retention of the amino group. 4-Chlorobenzoic acid was not prenylated. A survey of rat tissues shows that heart tissue contains maximum polyprenyltransferase activity when compared to liver, kidney, spleen and brain. The significance of the above results is discussed.     

Fxr(-/-) mice adapt to biliary obstruction by enhanced phase I detoxification and renal elimination of bile acids

Farnesoid X receptor knockout (Fxr(-/-)) mice cannot upregulate the bile salt export pump in bile acid loading or cholestatic conditions. To investigate whether Fxr(-/-) mice differ in bile acid detoxification compared with wild-type mice, we performed a comprehensive analysis of bile acids extracted from liver, bile, serum, and urine of naive and common bile duct-ligated wild-type and Fxr(-/-) mice using electrospray and gas chromatography mass spectrometry. In addition, hepatic and renal gene expression levels of Cyp2b10 and Cyp3a11, and protein expression levels of putative renal bile acid-transporting proteins, were investigated. We found significantly enhanced hepatic bile acid hydroxylation in Fxr(-/-) mice, in particular hydroxylations of cholic acid in the 1beta, 2beta, 4beta, 6alpha, 6beta, 22, or 23 position and a significantly enhanced excretion of these metabolites in urine. The gene expression level of Cyp3a11 was increased in the liver of Fxr(-/-) mice, whereas the protein expression levels of multidrug resistance-related protein 4 (Mrp4) were increased in kidneys of both genotypes during common bile duct ligation. In conclusion, Fxr(-/-) mice detoxify accumulating bile acids in the liver by enhanced hydroxylation reactions probably catalyzed by Cyp3a11. The metabolites formed were excreted into urine, most likely with the participation of Mrp4.     

Uptake and Translocation of Benthiocarb Herbicide by Plants

The uptake and translocation of ¹⁴C-benthiocarb labelled at benzyl methylene by rice plant, bamyardgrass, wild amaranth, smart weed and lambsquarters were investigated, ¹⁴C-Benthiocarb was absorbed through the roots and the radioactivity was translocated into whole plants. The rate of absorption and translocation varied by the kind of plants. The translocation was occurred not only from roots into leaves, but from a leaf into other leaves, and even into roots of some kinds of plant. The absorption and translocation was more easy in barnyard-grass than in rice plant. Benthiocarb was rapidly absorbed by seeds and accumulated mostly in the embryo. The uptake of benthiocarb by seedlings decreased with the order of mesocotyl (bamyardgrass only), coleoptyl, root and leaf. Benthiocarb was degraded rapidly in plants.     

Formation and degradation of dicarboxylic acids in relation to alterations in fatty acid oxidation in rats.

Dicarboxylic acids are excreted in urine when fatty acid oxidation is increased (ketosis) or inhibited (defects in beta-oxidation) and in Reye's syndrome. omega-Hydroxylation and omega-oxidation of C6-C12 fatty acids were measured by mass spectrometry in rat liver microsomes and homogenates, and beta-oxidation of the dicarboxylic acids in liver homogenates and isolated mitochondria and peroxisomes. Medium-chain fatty acids formed large amounts of medium-chain dicarboxylic acids, which were easily beta-oxidized both in vitro and in vivo, in contrast to the long-chain C16-dicarboxylic acid, which was toxic to starved rats. Increment of fatty acid oxidation in rats by starvation or diabetes increased C6:C10 dicarboxylic acid ratio in rats fed medium-chain triacylglycerols, and increased short-chain dicarboxylic acid excretion in urine in rats fed medium-chain dicarboxylic acids. Valproate, which inhibits fatty acid oxidation and may induce Reye like syndromes, caused the pattern of C6-C10-dicarboxylic aciduria seen in beta-oxidation defects, but only in starved rats. It is suggested, that the origin of urinary short-chain dicarboxylic acids is omega-oxidized medium-chain fatty acids, which after peroxisomal beta-oxidation accumulate as C6-C8-dicarboxylic acids. C10-C12-dicarboxylic acids were also metabolized in the mitochondria, but did not accumulate as C6-C8-dicarboxylic acids, indicating that beta-oxidation was completed beyond the level of adipyl CoA.     

Metabolism and renal clearance of 111In-labeled DOTA-conjugated antibody fragments

Radiometal-labeled antibody fragments are promising reagents for radioimmunotherapy due to their high tumor uptake and rapid pharmacokinetics, but their therapeutic potentials are limited by high uptake and retention in the kidney. Identification of metabolic products is a first step in designing rationale approaches to lower kidney uptake. Previous studies in rats have shown that 111In-labeled DTPA-conjugated antibody fragments (via lysine residues) were degraded to an DTPA-epsilon-amino-lysine derivative and retained in the lysosomal compartments of the liver and kidney [Rogers et al. (1995) Cancer Res. 55, 5714s-5720s]. To determine the metabolic profile of another widely used metal-chelate, [111In]DOTA conjugated to lysines in antibody fragments via active ester chemistry, we analyzed kidney homogenates from nude mice injected with an [111In]DOTA-Fab generated enzymatically from the anti-lymphoma intact antibody Rituxan. The major kidney metabolite was identified as [111In]DOTA-epsilon-amino-lysine by comparison to an authentic synthetic standard. This end product was also identified in the urine, along with relatively small amounts of [111In]DOTA-Fab. Since injection of [111In]DOTA-epsilon-amino-lysine into nude mice resulted in rapid clearance into the urine without kidney retention, it is likely that the renal retention observed was due to kidney uptake of [111In]DOTA-Fab, followed by lysosomal degradation to [111In]DOTA-epsilon-amino-lysine, which is only slowly cleared from this compartment. This observation is supported by autoradiographs of the kidney showing rapid localization of radioactivity into the distal regions of the kidney cortex. To extend this analysis to clinical trials, we have also analyzed urine taken from a patient injected with the intact antibody [111In]DOTA-cT84.66. In that example, we found that the major radioactive species was also [111In]DOTA-epsilon-amino-lysine.     

Degradation of 4,4'-dichlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl by the white rot fungus Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium has demonstrated abilities to degrade many xenobiotic chemicals. In this study, the degradation of three model polychlorinated biphenyl (PCB) congeners (4,4'-dichlorobiphenyl [DCB], 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl) by P. chrysosporium in liquid culture was examined. After 28 days of incubation, 14C partitioning analysis indicated extensive degradation of DCB, including 11% mineralization. In contrast, there was negligible mineralization of the tetrachloro- or hexachlorobiphenyl and little evidence for any significant metabolism. With all of the model PCBs, a large fraction of the 14C was determined to be biomass bound. Results from a time course study done with 4,4'-[14C]DCB to examine 14C partitioning dynamics indicated that the biomass-bound 14C was likely attributable to nonspecific adsorption of the PCBs to the fungal hyphae. In a subsequent isotope trapping experiment, 4-chlorobenzoic acid and 4-chlorobenzyl alcohol were identified as metabolites produced from 4,4'-[14C]DCB. To the best of our knowledge, this the first report describing intermediates formed by P. chrysosporium during PCB degradation. Results from these experiments suggested similarities between P. chrysosporium and bacterial systems in terms of effects of congener chlorination degree and pattern on PCB metabolism and intermediates characteristic of the PCB degradation process.     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The synthesis of dibenz[a,c]anthracene 10,11-oxide and its metabolism by rat liver preparations

The synthesis of dibenz[a,c]anthracene 10,11-oxide is described. The oxide was unstable and was rapidly decomposed with cold mineral acid into a mixture of 10- and 11- hydroxydibenz[a,c]anthracene. The oxide was converted by rat liver microsomal preparations and homogenates into a product that is probably 10,11-dihydro-10,11-dihydroxydibenz[a,c]anthracene and which was identical with the metabolite formed when dibenz[a,c]anthracene was metabolized by rat liver homogenates. The oxide did not react either chemically or enzymically with GSH. 10,11-Dihydrodibenz[a,c]anthracene and 10,11-dihydrodibenz[a,c]anthracene 12,13-oxide were both metabolized by rat liver preparations into trans-10,11,12,13-tetrahydro-10,11-dihydroxydibenz[a,c] anthracene and the oxide was converted chemically into this dihydroxy compound, and it reacted chemically but not enzymically with GSH. In the alkylation of 4-(p-nitrobenzyl)pyridine, the ;K-region' epoxide, dibenz[a,h]anthracene 5,6-oxide, was more active than either dibenz[a,c]anthracene 10,11-oxide or 10,11-dihydrobenz[a,c]anthracene 12,13-oxide.     

Human N-acetylgalactosamine-4-sulphate sulphatase. Purification, monoclonal antibody production and native and subunit Mr values.

Initial purification of N-acetylgalactosamine-4-sulphate sulphatase from human liver homogenates containing approx. 1 mg of enzyme in 26 g of soluble proteins was achieved by a six-column chromatography procedure and yielded approx. 40 micrograms of a single major protein species. Enzyme thus prepared was used to produce N-acetylgalactosamine-4-sulphate sulphatase-specific monoclonal antibodies. The use of a monoclonal antibody linked to a solid support facilitated the purification of approx. 0.5 mg of N-acetylgalactosamine-4-sulphate sulphatase from a similar liver homogenate. Moreover the enzyme isolated contained a single protein species, shown by SDS/polyacrylamide-gel electrophoresis to have an Mr of 57,000, which dissociated into subunits of Mr 43,000 and 13,000 in the presence of reducing agents. Essentially identical enzyme preparations were isolated from homogenates of human kidney and lung and from concentrated human urine. The native protein Mr of enzyme from human liver and kidney was assessed by gel-permeation chromatography to be 43,000 on Ultrogel AcA and Bio-Gel P-150. The liver N-acetylgalactosamine-4-sulphate sulphatase was shown to have pH optima of approx. 4 and 5.5 with the oligosaccharide substrate (GalNAc4S-GlcA-GalitolNAc4S) and fluorogenic substrate (methylumbelliferyl sulphate) respectively. Km values of 60 microM and 4 mM and Vmax. values of 2 and 20 mumol/min per mg were determined with the oligosaccharide and fluorogenic substrates respectively.