Biochem. J. (1979) 181, 387-399 Printed in Great Britain

387

Effect of O-Sulphate Groups in Lactose and N-Acetylneuraminyl-lactose on their Enzymic Hydrolysis By Nasi MIAN, Caroline E. ANDERSON and Paul W. KENT Glycoprotein Research Unit, Science Laboratories, University of Durham, Durham DH1 3LE, U.K. (Received 18 December 1978) 1. Lactose 6'-0-sulphate, N-acetylneuraminyl-(a2->3)-D-lactose 6'-0-sulphate, N-acetylneuraminyl ?-O-sulphate-(a2-+3)-D-lactose 6'-0-sulphate, N-acetylneuraminyl ?-O-sulphate-(a2-÷6)-D-lactose and N-acetylneuraminyl-(a2->3)- and -(a2->6)-lactose 6'-O-sulphate were prepared by chemical sulphation of lactose, N-acetylneuraminyl-lactose and its isomers by using pyridine-SO3 reagent. 2. Significant kinetic differences were observed in the enzymic hydrolysis of the sulphated derivatives compared with unsubstituted substrates. 3. In the case of reactions catalysed by rat liver lysosomal and Clostridium perfringens neuraminidases (EC 3.2.1.18), the presence of an 0-sulphate group in the N-acetylneuraminyl moiety affected the reaction by decreasing the Km and the Vmax. its presence in the galactosyl moiety affected the reaction by decreasing the Km and increasing the Vmax. and its presence in both N-acetylneuraminyl and galactosyl moieties decreased the Km and the Vmax. of the reaction. 4. Mixed-substrate reaction kinetic data indicated competition between the sulphated and unsubstituted substrates for the same active sites on the neuraminidase molecule. 5. Lactose 6'-O-sulphate neither behaved as a substrate nor acted as an inhibitor with respect to unsubstituted lactose and p-nitrophenyl fi-D-galactopyranoside when tested with lactase of suckling rat intestine and Escherichia coli fJ-Dgalactosidase (EC 3.2.1.23). 6. Preliminary investigation also indicated that, whereas glucose 6-0-sulphate and glucose 3-0-sulphate were neither substrate nor inhibitor of glucose oxidase (EC 1.1.3.4), galactose 6-0-sulphate was oxidized half as fast as unsubstituted galactose by galactose dehydrogenase (EC 1.1. 1.48). For a compound to be cleaved by neuraminidase the presence of unsubstituted carboxy groups on the N-acetylneuraminic acid is of great importance (Gottschalk, 1962; Yu & Ledeen, 1969). Bacterial and viral neuraminidases recognize substituents on the N-acetylneuraminic acid molecule. The resistance to hydrolysis by neuraminidase of substances containing the substituted N-acetylneuraminic acid molecule have been a subject of great interest from the point of view of specificity of enzyme action (for review see Drzeniek, 1973). In addition, viral neuraminidases specifically hydrolyse the different types of a-ketosidic linkage between N-acetylneuraminic acid and adjacent carbohydrate molecules. The nature of this carbohydrate is of minor importance for the recognition by these enzymes, but large substituents on these carbohydrates make such compounds resistant towards bacterial and viral neuraminidases owing to steric hindrance (Drzeniek, 1973). However, we have observed that the chemically sulphated sialoglycopeptides inhibited the hydrolysis of N-acetylneuraminic acid from N-acetylneuraminyl-lactose, bovine submaxillarygland mucin and from the native sialoglycopeptides by steric hindrance (Mian et al., 1979). Vol. 181

Bearing in mind the resistance of compounds towards neuraminidase action owing to the presence of substituents on the N-acetylneuraminic acid molecule and the inhibition of the enzyme activity in the presence of chemically sulphated sialoglycopeptides, it was considered important to chemically sulphate some simple low-molecular-weight substrates and to compare their enzymic hydrolysis reactions with the unsubstituted compounds. To this end, two enzyme systems, neuraminidases (EC 3.2.1.18) from Clostridium perfringens and rat liver lysosomes and lactase (EC 3.2.1.23) from suckling rat intestine and Escherichia coli ,B-galactosidase were chosen and chemically sulphated derivatives of N-acetylneuraminyl-lactose and lactose were prepared. In the present paper, we report a comparative kinetic study on the action of these enzymes on their sulphated and unsubstituted substrates and also discuss the biological role of 0-sulphate ester groups. Experimental Chemical estimations The 2-thiobarbituric acid method of Warren (1959) was used for the estimation of free N-acetylneura-

388

minic acid. The resorcinol method of Svennerholm (1957) was used for the determination of total free and bound N-acetylneuraminic acid. Lactose was determined by the anthrone-reagent method described by Cook (1976). Glucose was determined by glucose oxidase reaction by using glucose-testcombination kit (Boehringer) based on the method of Werner et al. (1970). Galactose was estimated by galactose dehydrogenase according to the method described by Asp & Dahlqvist (1972). The sulphate content of the samples after hydrolysis in 25 % (v/v) formic acid for 24h at 100°C was determined by the method of Mende & Whitney (1978), which involved precipitation of the inorganic sulphate on cellulose thin-layer plates with 133BaC12.

Paper chromatography Descending chromatography on Whatman no. 1 paper was performed with solvent system I (ethyl acetate/pyridine/water, 10:5:6 by vol.), system II (n-butanol/n-propanol/0.1M-HCl, 1:2:1 by vol.), system III (ethyl acetate/pyridine/acetic acid/water, 5 :5:1 :3 by vol.), and system IV (butan-l-ol/ethanol/ water, 3 :1 :1 by vol.) containing 3 % (w/v) cetylpyridinium chloride. The chromatograms were developed following a dip in ethanolic NaOH/AgNO3 (for reducing compounds) or with p-dimethylaminobenzaldehyde spray (for N-acetylneuraminic acid).

Ion-exchange chromatography The authentic carbohydrate samples were run individually on ion-exchange columns (Dowex 1; X8; formate form; 200-400 mesh; 25cm x 1 cm) and eluted with a gradual continuous gradient of 0 to 5.0M-formic acid. Similarly in every preliminary run, the sulphation reaction mixture was passed through an ion-exchange column and eluted with a gradual continuous gradient of 0 to 5.0M-pyridine/ formate solution pH4.4 to assess approximately the gradient concentrations that eluted the carbohydrate test-positive materials. In the subsequent ionexchange chromatography, the columns after being loaded with the reaction mixture were washed with water and then eluted with a stepwise gradient ranging between minimum and maximum concentrations of pyridine/formate required to elute the carbohydrate material, as assessed from the preliminary runs. This procedure minimized the time taken for ion-exchange chromatography and improved the recovery of the added material from the columns. The material recovered from these columns was subjected to rechromatography on Dowex 1 (X8) and was eluted either by using a gradual continuous gradient or by a narrow-range stepwise gradient.

N. MIAN, C. E. ANDERSON AND P. W. KENT Authentic glucose, galactose and lactose were eluted with water, whereas N-acetylneuraminic acid was eluted with 0.3 M-formic acid. Galactose 6-0-sulphate, glucose 6-0-sulphate, glucose 3-0-sulphate were eluted with 4.5 M-formic acid, whereas N-acetylneuraminic acid 0-sulphate was eluted with 5.OMformic acid.

Chemical sulphation of carbohydrates Sulphation of carbohydrates was carried out by using the pyridine-SO3 reagent (Aldrich Chemical Co., Gillingham, Dorset, U.K.) by a modification of the method of Lloyd (1960). Carbohydrates were dissolved in anhydrous pyridine at 60°C, excess of the pyridine-SO3 was added and the reaction was maintained at 37°C for 12 or 36h with constant shaking. The reaction mixture was then kept at room temperature for 3 h, cooled to 4°C and then an icecold suspension of Ba(OH)2 was added with constant stirring over a period of 10min. After centrifugation at 5000g at 4°C for 15 min, the sediment was discarded and solid CO2 was added to the supernatant liquid and centrifugation was repeated. The resulting supernatant was concentrated by rotary evaporation at 30°C with repeated additions of water to remove excess pyridine and finally concentrated to 4ml. The sulphated products from unreacted carbohydrates were separated by repeated ion-exchange chromatography on Dowex 1 (X8; formate form). Carbohydrates present in the eluted fractions throughout were detected by using anthrone reagent for sulphated or unchanged hexoses and resorcinol reagent for N-acetylneuraminic acid or its sulphated forms. Eluted fractions were freed from pyridine/formate by rotary evaporation at 30°C and concentrated to 1 ml. These were finally converted into potassium salts by passage through Dowex 5OW (H+ form) followed by neutralization with KHCO3 (0.1 M). Samples were then concentrated to dryness at room temperature in vacuo over P205.

Identification of 0-sulphate esters in sulphated substrates Sulphated sugars used in the present work, i.e. glucose 3-0-sulphate, glucose 6-0-sulphate and galactose 6-0-sulphate (Lloyd, 1960) were all as potassium salts. Di- and tri-saccharide sulphates were hydrolysed in 0.1 M-HCI for 90min at 100°C (Ryan et al., 1965). Hydrolysis under these conditions yielded monosaccharides and monosaccharide 0-sulphates from di- or tri-saccharide derivatives. The hydrolysates werecooled in an ice bath and dried in the presence of NaOH pellets over silica gel in an evacuated desiccator. The hydrolysis products were examined in comparison with the reference authentic compounds by using analytical paper and ionexchange chromatography. Samples were loaded on 1979

ENZYMIC DEGRADATION OF O-SULPHATED OLIGOSACCHARIDES Dowex 1 (X8; formate form) columns (25cm x 1 cm) and eluted with water followed by a linear gradient of 0 to 5.5 M-formate. The eluted fractions were freed from pyridine/formate and concentrated by rotary evaporation. These were examined chemically, enzymically and by analytical paper chromatography for the presence of glucose, galactose, N-acetylneuraminic acid and their sulphated derivatives. The molar ratios of sulphate to monosaccharides were calculated from their total sulphate contents. Glucose oxidase was specific for unsubstituted glucose as glucose 3-0-sulphate and glucose 6-0sulphate were not substrates for the enzyme. Galactose dehydrogenase oxidized galactose 6-0-sulphate at half the rate of that of galactose. On the other hand, both anthrone and resorcinol reactions did not discriminate between unsubstituted and substituted hexoses and N-acetylneuraminic acid respectively.

Fractionation of structural isomers of N-acetylneuraminyl-lactose The two structural isomers (2--*3) and (2-÷6) of N-acetylneuraminyl-lactose, commercially available as a mixture of the two (Sigma), were separated by ion-exchange chromatography by the method of Schneir & Rafelson (1966). About 70 % of the material applied to the Dowex 1 (X2; acetate form) column was recovered in a ratio of 5 :1 of (2->3)- and (2-*6)-isomers. Descending chromatography on Whatman no. 3 paper in solvent system III gave discrete spots on development of the chromatograms. Lactose 6'-0-sulphate [D-galactopyranosyl 6-0-sul-

phate-(fl1 -÷4)-0-D-glucopyranose] Lactose (0.01 mol) was reacted with pyridine-SO3 (0.02mol) in anhydrous pyridine for 12 or 36h at 37°C. Finally processed reaction mixtures were subjected to ion-exchange chromatography. Elution from Dowex 1 (X8; formate form) yielded two fractions; unchanged lactose was eluted with water and the sulphated derivative was located in 0.5Mpyridine/formate eluate in a preparative chromatographic run with stepwise gradient elution (Fig. 1). Potassium lactose 6'-O-sulphate (0.0046mol, yield 46%) was a crystalline solid that moved as a single component in solvent systems I and IV on paper chromatography. On rechromatography on Dowex 1 (X8), the compound was eluted as a single peak with 0.2M-pyridine/formate or by 2.7M-formate when the column was eluted with a linear gradient of 0-5M-formate. The compound had a carbohydrate/ sulphate ratio of 2: 1 and on hydrolysis in 0.1 M-HCI yielded glucose and galactose 6-0-sulphate. Glucose was identified by paper and ion-exchange chromatography and by glucose oxidase reaction. No component with RF values similar to those obtained Vol. 181

389

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Elution volume (litre) Fig. 1. Fractionation of lactose 6'-0-sulphate on Dowex 1 (X8; formate form) Pyridine-free concentrated reaction products (4ml) of lactose obtained after 36h reaction with pyridine-SO3 complex were applied to a column (2cm x 30cm) of Dowex 1 (X8; formate form; 200-400 mesh). The column was eluted stepwise with (a) water, (b) 0.01 M-, (c) 0.15M- and 0.5M-pyridine/formate solutions (d), pH4.4. Fractions (lOml) were collected and tested for total hexoses by using anthrone reagent test. Peak I, unchanged lactose; peak II, lactose 6'-0-sulphate.

for glucose 3-0-sulphate, glucose 6-0-sulphate or glucose bis-sulphate described by Rees (1960) were detected on paper chromatography in solvent system IV. Galactose 6-0-sulphate liberated by acid hydrolysis of lactose 6'-0-sulphate was eluted with 4.5 M-formate from Dowex 1 (X8; formate form) and had an RF value of 0.40 in solvent system IV on paper chromatography (Lloyd, 1962). No unchanged galactose or a disulphate derivative was detected on paper chromatography. Oxidation of lactose and lactose 6'-0-sulphate (2mg each) was carried out in 10ml of 0.015M-sodium metaperiodate and periodate oxidation was measured spectrophotometrically (Guthrie, 1962a). Lactose 6'-0-sulphate and lactose consumed 2.54 and 3.96mol of periodate/mol of carbohydrate respectively (72h terminal). The analysis of formaldehyde produced in the periodate reaction, carried out by the method of Guthrie (1962b) by using the chromotropic acid reaction,

N. MIAN, C. E. ANDERSON AND P. W. KENT

390

glucose, galactose 6-0-sulphate and N-acetylneuraminic acid, which were identical with the corresponding authentic carbohydrates on paper and ionexchange chromatography. No sulphated derivatives of glucose and N-acetylneuraminic acid were detected by paper chromatography in solvent systems I, III and IV or by ion-exchange chromatography. Similarly, estimation of sulphate in these products showed the carbohydrate/sulphate ratio of galactose 6-0-sulphate to be 1 :1, whereas no sulphate was detected in glucose and N-acetylneuraminic acid. The identity of glucose and galactose 6-0-sulphate was also checked enzymically.

indicated that lactose 6'-O-sulphate and lactose produced 1.1 and 1.8mol of formaldehyde/mol of the compound respectively. These values are in close agreement with the previously published data (Ryan et al., 1965). Reaction of lactose with pyridine-S03 Complex, whether for 12 or 36h, produced lactose 6'-O-sulphate only and no disulphate isomers were detected.

N-Acetylneuraminyl-(2--3)-lactose 6'-O-sulphate [N6-0-

acetylneuraminyl-(a2--33)-O-galactopyranosyl

sulphate-(fl -+4)-0-glucopyranose]

(0.001 mol) N-Acetylneuraminyl-(2-+3)-lactose was reacted with pyridine-SO3 (0.002mol) in anhydrous pyridine for 12h at 37°C. Preparative ion-exchange chromatography of the finally processed reaction mixture yielded two fractions: unchanged N-acetylneuraminyl-(2--3)-lactose, which was eluted by 0.01 M-pyridine/formate, pH4.4, and the sulphated derivative (52% yield), which was obtained in 0.5 M-pyridine/formate eluate in a stepwise gradient elution (Fig. 2). The potassium salt of

N-Acetylneuraminyl sulphate-(2-÷3)-lactose 6'-Osulphate [N-acetylneuraminyl ?-O-sulphate-(a2->3)0-galactopyranosyl 6-0-sulphate-(fl1 -4)-O-glucopyranose] N-Acetylneuraminyl-(2-+3)-lactose (0.001 mol) was reacted with pyridine-SO3 complex (0.002mol) in anhydrous pyridine for 36h at 37°C. Preparative ion-exchange chromatography of the finally processed reaction mixture yielded three fractions: N-acetylneuraminyl-(2--3)-lactose, unchanged which was eluted with 0.01 M-pyridine/formate, pH 4.4, and two sulphated fractions, which were both eluted by 0.5 M-pyridine/formate from Dowex 1 (X8; formate form) column as two separate peaks (Fig. 3). The major and the minor fractions, 60 and 10% in yield respectively, were subjected to rechro-

6'-O-sulphate N-acetylneuraminyl-(2---3)-lactose moved as a single component on paper chromatography in solvent systems II and IV, and was eluted by 0.22M-pyridine/formate during rechromatography on Dowex 1 (X8; formate form) by using a gradual continuous gradient of 0 to 1.0M-pyridine/ formate. The compound had a carbohydrate/sulphate ratio of 3: 1 and on hydrolysis in 0.1 M-HCI yielded

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1.5 1.3 1.4 1.1.1 1.2 lo Elution volume (litre) Fig. 2. Fractionation of N-acetylneuraminyl-(2--3)-lactose 6'-O-sulphate o0n Dowex 1 (X8; formate form) Pyridine-free concentrated reaction products (1 ml) of N-acetylneuraminyl-(2--3)-lactose after 12h reaction with pyridine-SO3 complex were applied to a column (2cm x 30cm) of Dowex I (X8; formate form; 200-400 mesh). The column was first washed with (a) 300ml of water and then eluted stepwise with (b) 0.01 M-, (c) 0.15M- and 0.5 M-pyridine/ formate solution (d), pH4.4. lOml Fractions were collected and tested for N-acetylneuraminic acid (x; A580) and total hexoses (M; A620) by using resorcinol and anthrone reagent test respectively. Fractions eluted with water gave negative tests with both reagents and are not shown. Peak I, unchanged N-acetylneuraminyl-(2->3)-lactose; peak II, N-acetylneuraminyl-(2->3)-lactose 6'-0-sulphate.

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ENZYMIC DEGRADATION OF O-SULPHATED OLIGOSACCHARrDES

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Elution volume (litre) Fig. 3. Fraction ofN-acetylneuraminyl ?-O-sulphate-(a2-÷3)-lactose 6'-O-sulphate on Dowex 1 (X8; formateforni) Pyridine-free concentrated reaction products (1 ml) of N-acetylneuraminyl-(2-.3)-lactose after 36h reaction with pyridine-S03 complex were applied to a column (2cm x 30cm) of Dowex 1 (X8; formate form; 200-400 mesh). The column was first washed with (a) 300ml of water and then eluted stepwise with (b) 0.01 M-, (c) 0.15 M- and 0.5 M-pyridine/ formate solution (d), pH4.4. Fractions (lOml) were collected and tested for N-acetylneuraminic acid (x; AS80) and total hexoses (0; A620) by using resorcinol and anthrone reagent tests respectively. Fractions eluted with water did not contain any carbohydrate and are not shown. Peak I, unchanged N-acetylneuraminyl-(a2-.3)-lactose; peak II, N-acetylneuraminyl-(L2--*3)-lactose 6'-0-sulphate; peak III, N-acetylneuraminyl ?-O-sulphate-(a2-+3)-lactose 6'-O-sulphate.

matography on Dowex 1 (X8; formate form) columns by using a gradual continuous gradient of to 1.0M-pyridine/formate, pH4.4. -The major fraction was eluted as a single peak between 0.28Mand 0.34M-pyridine/formate and the minor fraction was eluted between 0.18M- and 0.22M-pyridine/ formate as a single peak. The minor sulphated component was found to be identical with N-acetylneuraminyl-(2-÷3)-lactose 6'-0-sulphate by analyses of its acid-hydrolysis products and had a carbohydrate/sulphate ratio of 3 :1. The major sulphated component had a carbohydrate/sulphate ratio of 3 :2 and on acid hydrolysis yielded glucose, galactose 6-0-sulphate and N-acetylneuraminyl sulphate. Glucose and galactose 6-0-sulphate were identified by paper and ion-exchange chromatography and by their enzymic reactions as described in the preceding section. N-Acetylneuraminic acid 0-sulphate had a carbohydrate/sulphate ratio of 1: 1 and was eluted from Dowex 1 (X8; formate) column with 5.OMformic acid, whereas the authentic N-acetylneuraminic acid could be eluted with 0.3 M-formic acid. On paper chromatography in solvent system III, the RF value of N-acetylneuraminic acid 0-sulphate was 0.36

compared with that of 0.48 for the authentic Nacetylneuraminic acid. No sulphated derivative of glucose or bis-sulphate of galactose was detected by paper chromatography in solvent systems I and IV. The major sulphated component was thus assigned to be a disulphate product, i.e. N-acetylneuraminyl sulphate-(2->3)-lactose 6'-0-sulphate. Vol. 181

N-Acetylneuraminyl sulphate-(2--6)-lactose [N-acetylneuraminyl ?-O-sulphate-(a2-+6)-0-galactopyranosyl-(fl-1 -*4)-0-glucopyranose] N-Acetylneuraminyl-(2-.6)-lactose could not be sulphated during 12h reaction with pyridine-SO3 complex. However, reaction ofthis isomer (0.001 mol) with pyridine-SO3 complex (0.002mol) for 36h at 37°C yielded a monosulphate derivative (45 % yield) and the unchanged compound (Fig. 4). The sulphated derivative moved as a single component on paper chromatography in solvent system III and was eluted by pyridine/formate gradient between the concentrated 0.30M and 0.34M on rechromatography on Dowex 1 (X8; formate form) by using a linear gradient of 0 to 1.OM-pyridine/formate, pH4.4. The compound had a carbohydrate/sulphate ratio of 3 :1 and on acid hydrolysis yielded glucose, galactose and N-acetylneuraminyl sulphate, which were identified as described in the preceding section.

N-Acetylneuraminyl-(2--->-3)- and -(2-*6)-lactose sulphate (mixed isomers) Sulphation of a commercial preparation of N-acetylneuraminyl-(2--3)- and -(2-*6)-lactose (0.001 mol) by using pyridine-SO3 complex (0.002mol) for 12h at 37°C yielded a sulphated product and unchanged compound. The sulphated product was purified on ion-exchange chromatography and analysed as described previously. The

N. MIAN, C. E. ANDERSON AND P. W. KENT

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Elution volume (litre) Fig. 4. Fractionation of N-acetylneuraminyl ?-O-sulphate-(a%2-*6)-lactose on Dowex 1 (X8; formate form) Pyridine-free concentrated reaction products (1 ml) of N-acetylneuraminyl-(a2-*6)-lactose after 36h reaction with pyridine-SO3 complex were applied to a column (2cm x 30cm) of Dowex 1 (X8; formate form; 200-400 mesh). The column was eluted with (a) water, (b) 0.01 M-, (c) 0.15 M- and 0.5 M-pyridine/formate solution (d), pH 4.4. Fractions (10ml) were collected and tested for N-acetylneuraminic acid (x, A580) and total hexoses (0, A620) by using resorcinol and anthrone reagent tests respectively. Peak I, unchanged N-acetylneuraminyl-(a2-÷6)-lactose; peak 1I, N-acetylneuraminyl ?-O-sulphate-(a2->6)-lactose.

carbohydrate/sulphate ratio of the sulphated product was 3 :0.8, and it yielded glucose, galactose 6-0-sulphate and N-acetylneuraminic acid, which were identified as described in the preceding section. Of the total galactose only 80% was sulphated. The commercially available mixture contained (2--3)and (2-+6)-isomers in the ratio 5 :1, the galactose moiety of (2-.6)-isomers remains unsulphated because of its linkage with the N-acetylneuraminyl residue at C-6. The chromatographically purified N-acetylneuraminyl-(2--*6)-lactose remained unsulphated after 12 h reaction with pyridine-SO3 complex as described above. Assay of neuraminidase activity The substrate solutions were used in concentrations up to 0.01 M with respect to N-acetylneuraminyl-(2--3)- and -(2->6)-lactose or its isomers in 0.05M-sodium acetate/acetic acid buffers, pH4.2 and 5, as required. The incubation mixture (0.2ml) containing 0.1ml of rat liver lysosomal fraction or appropriately diluted commercial preparation of enzyme from C. perfringens suspended in 0.05Msodium acetate/acetic acid buffer, pH4.2 or 5.0, and 0.05 ml of substrate solution was incubated for 1 h at 37°C. The amount. of N-acetylneuraminic acid released by enzymic action was measured either directly or after its chromatographic separation. In

the direct method the enzymic reaction was stopped by adding 0.1 ml of periodate reagent and the complete analysis was carried out as described by Warren (1959). For the chromatographic method, the enzymic reaction was terminated by heating the tubes at 100°C for 2min. N-Acetylneuraminic acid was separated from the remainder of the mixture by ion-exchange chromatography on Dowex 1 (X8; formate form) (Cook, 1976) before its estimation by the method of Warren (1959). The blanks were prepared in a similar way, except that the enzyme was added at the end-point of the reaction period. All estimations were carried out in duplicate. There was no non-enzymic hydrolysis of N-acetylneuraminyl-lactose or its sulphated derivatives under the above assay conditions. The enzyme activity is expressed throughout as nmol of N-acetylneuraminic acid released/h per mg of enzyme protein.

Assay of lactase activity Brush-border lactase was assayed in the presence of 0.2 mM-p-hydroxymercuribenzoate by the,method of Asp & Dahlqvist (1972) with lactose as substrate. The incubation mixture (0.2ml) contained 0.032Mlactose and 0.05M-sodium maleate buffer, pH5.5. The incubations were carried out at 37°C for 1 h over which period the rate of reaction was linear. The reaction was terminated by thermal inactivation at 1979

ENZYMIC DEGRADATION OF O-SULPHATED OLIGOSACCHARIDES

393

100°C for 2min. Blanks were prepared in a similar way, but were heated immediately for 2 min at 100°C. Lactase activity was determined by measuring the amount of glucose or galactose produced by glucose oxidase or galactose dehydrogenase methods. Assay ofp-nitrophenyl f9-D-galactosidase fi-D-Galactosidase activity was measured by using p-nitrophenyl-fi-D-galactopyranoside as substrate in 0.05M-sodium citrate/phosphate buffer in the pH range 5-8.0. Incubations were carried out in the presence or absence of 0.2 mM-p-hydroxymercuribenzoate at 37°C for 1 h and the reaction was terminated by adding 0.4M-NaOH/glycine buffer, pH11.0. Blanks were prepared with the enzyme or the substrate alone. p-Nitrophenol (Sigma) was used as standard. Protein was determined by the method of Lowry et al. (1951) with bovine serum albumin (Sigma) as standard. Preparation and general properties of rat liver lysosome-bound neuraminidase Lysosome fraction from male Wistar rats weighing between 250 and 300g was prepared by the method of Horvat & Touster'(1968) based on that of de Duve et al. (1955). Livers were minced and passed through a tissue press. Homogenization and the isolation of subcellular particles by differential centrifugation were performed by the method of de Duve et al. (1955). Different subfractions were characterized by assaying the marker-enzyme activities (de Duve e#*l., 1955; Horvat & Touster, 1968). Neuraminidase and arylsulphatase, a typical lysosomal marker (Viala & Gianetto, 1955), showed highest relative total and specific activity in the same fraction sedimenting between the mitochondria and microsomal fraction, which were characterized by the presence of their respective enzyme markers (Horvat & Touster, 1968). The lysosome fraction contained 51 % of the total neuraminidase activity present in homogenates and the specific activity of the enzyme was 64 times higher than that of the original homogenate. C. perfringens neuraminidase was obtained from Boehringer. pH-Activity curves of the two neuraminidases tested by using N-acetylneuraminyl-(2--3)- and -(2->6)-lactose and its fractionated isomers as substrates are given in Fig. 5. The pH optima in the case of rat liver lysosome-bound enzyme was 4.2 for these substrates. On the other hand, pH optimum for the hydrolysis of the two isomers of N-acetylneuraminyl-lactose by C. perfringens neuraminidase were 4.5 and 5.0, as reported for influenza-virus neuraminidase previously (Schneir & Rafelson, 1966). The rate of hydrolysis of N-acetylneuraminyl-(2-*3)- and

Vol. 181

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(U t

0 0 0 3.5 4.5 5.5 3.5 4.5 5.5 3.5 4.5 5.5 4.0 5.0 6.0 4.0 5.0 6.0 4.0 5.0 6.0

pH Fig. 5. Effect of pH on the hydrolysis of N-acetylneuraminyl-lactose and its structural isomers by neuraminidases Assays of enzyme activity were carried out at pH values between 3.5 and 5.5 by using 0.05M-sodiurf acetate/acetic acid buffers and at pH6.0 by using O.05M-Na2HPO4/NaH2PO4 buffer (Gomori, 1955). (I) Rat liver neuraminidase; (II) C. perfringens neuraminidase. (a) N-Acetylneuraminyl-(a2--3)- and -(a2-+6)-lactose (mixed isomers); (b), N-acetylneuraminyl-(a2-.3)-lactose; and (c) N-acetylneuraminyl(a26)-lactose as substrates. The values plotted are averages of three experiments.

-(2-*6)-lactose and its isomers by both enzyme preparations was linear with time for up to 1 h. Preparation and general properties of suckling-rat brush-border lactase Brush-border fraction from intestines of 12-14day-old rats was prepared by the method of Schmitz et al. (1973). A 1 % mucosal homogenate was made in 0.05M-mannitol/0.002M-Tris, pH7.1, at 4°C. The homogenate was filtered through nylon mesh (40,um pore size). Solid CaCl2 was added to a final concentration of 0.01 M with constant stirring. After 10min with occasional mixing by inversion the suspension was centrifuged at 2000g for 10min to yield a heavy whitish pellet, which was resuspended in 0.05 M-mannitol/0.002M-Tris, pH 7.1. It was then centrifuged at 20000g for 15min to yield a small brownish pellet containing brush-border fraction. The final pellet contained 68 % of the total lactase activity present in the homogenate and the specific activity of the enzyme in the brush-border fraction was 76 times higher than the corresponding activity of the original homogenate.

0

N. MIAN, C. E. ANDERSON AND P. W. KENT

394 Lactase and fi-D-galactosidase activities of the brush-border fraction were measured by using lactose and p-nitrophenyl f,-D-galactoside as substrates in the presence of 0.2mM-p-hydroxymercuribenzoate to inhibit acid IJ-galactosidase and hetero-flD-galactosidase as described by Asp & Dahlqvist (1972). Fig. 6 shows the pH-activity curves of lactase and p-hydroxymercuribenzoate-stable p-nitrophenyl fi-D-galactosidase. Lactase activity of the brushborder fraction was not inhibited by sodium galactonate, whereas galactono-1,4-lactone was a competitive inhibitor, the K, values measured by using Dixon plots (Dixon & Webb, 1964a) being 0.75+0.16mM and 1.19±0.18mM (mean±s.D.) with respect to lactose and p-nitrophenyl fl-galactopyranoside as substrates.

Kinetic experiments and analysis of the data Kinetic experiments were performed by using various concentrations of substrates and the initial velocities were measured. The Kin, Vinax. and K, values were calculated from Lineweaver-Burk plots as described by Dixon & Webb (1964a). In mixedsubstrate experiments sulphated and unsubstituted substrates were mixed in equimolar and different proportions and the initial velocities of the joint reactions were measured. The significance in the difference between two sets of values was determined by using Student's t test.

lysosomes and C. perfringens under optimal conditions of assay are given in Table 1. These values are in close agreement with those reported previously for these substrates, where neuraminidase from an influenza virus strain was used (Schneir & Rafelson, 1966). The specificity of neuraminidase towards

0.

cd

.E 0 0 CC.)

3.0

4.0

5.0

6.0

7.0

8.0

pH Fig. 6. Effect ofpH on lactase and p-hydroxymercuriben-

Results and Discussion Kinetics of hydrolysis of N-acetylneuraminyl-lactose, its isomers and their sulphated derivatives by neuraminidases The kinetic values of hydrolysis of N-acetylneuraminyl-(2--3)- and -(2-+6)-lactose and its two structural isomers by neuraminidases from rat liver

zoate-stable p-nitrophenyl fi-D-galactosidase activities Assays of enzyme activities were carried out at pH values between 3 and 5.6 by using 0.05M-Na2HPO4/ citric acid buffers and at pH values between 5.8 and 8.0 by using 0.05 M-Na2HPO4/NaH2PO4 buffers (Gomori, 1955). Lactase activity (o) was measured

* the presence of 0.2mM-p-hydroxymercuribenzoate with 32mM-lactose as a substrate. p-Hydroxymer-

curibenzoate-stable p-nitrophenyl fi-D-galactosidase activity (D) was estimated with 20mM-p-nitrophenyl fi-D-galactopyranoside as substrate in the presence of 0.2mM-p-hydroxymercuribenzoate. The results are expressed as percentages of the optimal activities and are averages of three experiments.

Table 1. Kinetic valuesfor the hydrolysis of N-acetylneuraminyl-lactose and its structural isomers by neuranminidases Values are means + S.E.M. for the number of experiments shown in parentheses.

Enzyme characteristics

Substrate Enzyme preparation Rat liver neuraminidase pH K. (mM) Vmax. (nmol/h per mg of protein) C. perfringens neuraminidase pH Km (mM)

Vm,x. (nmol/h per mg of protein)

...

N-Acetylneura-

minyl-(2-.3)- and (2-*6)-lactose

N-Acetylneuraminyl(2-*3)-lactose

N-Acetylneuraminyl(2-*6)-lactose

4.2 1.78+0.12 (5) 133+ 16 (5)

4.2 0.24+ 0.02 (3) 720+14(3)

4.2 1.10+0.16 (3) 45± 8 (3)

5.0 2.39 +0.04 (5) 212± 15 (5)

5.0 0.45 + 0.06 (3) 580± 70 (3)

4.5 1.80+0.13 (3) 90± 10 (3)

1979

ENZYMIC DEGRADATION OF O-SULPHATED OLIGOSACCHARIDES

different types of linkage between N-acetylneuraminic acid and the lactosyl moiety of the compound, i.e. (2->3)-, (2-*4)- and (2->6) isomers has been tested previously (for review see Drzeniek, 1973). Previously reported data show that C. perfringens (Cassidy et al., 1965), Vibrio cholerae (Drzeniek, 1967; Drzeniek & Gauhe, 1970) and liver neuraminidases (Horvat & Touster, 1968) hydrolysed Nacetylneuraminyl-(2--6)-lactose half as fast as N-acetylneuraminyl-(2--3)-lactose. N-Acetylneuraminyl-lactose 6'-0-sulphate, a naturally occurring compound in rat mammary gland (Ryan et al., 1965; Choi & Carubelli, 1968), has been reported to be hydrolysed at a faster rate than N-acetylneuraminyl-lactose by soluble and lysosome-bound neuraminidase from rat liver (Tulsiani & Carubelli, 1970), by C. perfringens enzyme (Tulsiani & Carubelli, 1970) by soluble neuraminidase from rat mammary gland (Carubelli et al., 1962) and by rat brain neuraminidase (Carubelli, 1968). No detailed study appears to have been carried out previously on the comparative kinetics of hydrolysis of the sulphated and unsubstituted N-acetylneuraminyl-lactose by neuraminidase. The pH-activity curves of the hydrolysis of sulphated derivatives of N-acetylneuraminyl-lactose and its isomners by rat liver and C. perfringens neuraminidases were similar in profiles shown for unsulphated substrates (Fig. 5), thus indicating that the presence of 0-sulphate ester in these substrates did -not change the pH optima of their reaction. The data (Table 2), however, indicated that the Km values of N-acetylneuraminyl-(2-*3)- and -(2-*6)-lactose 6'-O-sulphate and of N-acetylneuraminyl-(2-*3)lactose 6'-O-sulphate for neuraminidases from rat liver lysosomes and C. perfringens were significantly

395

(P3)-lactose 6'-O-sulphate (Table 2). Further comparison would indicate that both Km and Vmax. values for N-acetylneuraminyl O-sulphate-(2->3)-lactose 6'-O-sulphate were de-

Table 2. Kinetic values for the hydrolysis of sulphated derivatives of N-acetylneuraminyl lactose and its structural isomers by neuraminidases Values are means + S.E.M. (for the number of experiments shown in parentheses). Enzyme characteristics

Sulphated sulphate

...

Enzyme preparation Rat liver neuraminidase pH Km (mM)

(nmol/h per mg of protein) C. perfringens neuraminidase pH Vmax.

Km (mM)

V.ax. (nmol/h per mg of protein) Vol. 181

N-Acetylneuraminyl(2-->3)- and -(2-+6)lactose 6'-O-sulphate

N-Acetylneuraminyl(2-+3)-lactose 6'-O-sulphate

N-Acetylneuraminyl ?O-sulphate(2-.3)-lactose 6'-O-sulphate

N-Acetylneuraminyl ?-O-sulphate(2-*6)-lactose

4.2 0.72+0.12 (3) 240+40 (3)

4.2 0.07 + 0.02 (5) 1160+180 (5)

4.2 0.16+0.02 (5) 250± 110 (5)

4.2 0.75 + 0.21 (5) 20±10 (5)

5.0 1.05±16(3) 360+50 (3)

5.0 0.13+ 0.03 (3) 970±160 (3)

n.d. n.d. n.d.

n.d. n.d. n.d.

396

N. MIAN, C. E. ANDERSON AND P. W. KENT

creased by about 1.5 and 3 times respectively compared with the values for the corresponding unsulphated compound (Tables 1 and 2). A similar pattern showing a decrease in both Km and Vmax. for N-acetylneuraminyl O-sulphate-(2--6)-lactose in comparison with those obtained for the corresponding unsulphated compound could be observed (Tables 1 and 2). These results suggest that 0-sulphate ester substitution in the galactosyl moiety of the substrate influences the reaction by lowering the Km and increasing the Vmax., whereas such substitution in the Nacetylneuraminyl moiety of the substrate influences the reaction by lowering both Km and Vmax. Bearing in mind the presence of a bulky anionic substituent, 0-sulphate ester, in the N-acetylneuraminyl moiety of the substrate and its subsequent effect on the reaction constants the effect of the sulphated derivatives on the hydrolysis of the unsulphated compounds and vice-versa was studied. The experiments were carried out to measure Vmax. values in joint reactions when N-acetylneuraminyl-lactose and its sulphated derivatives were present in equimolar concentrations in the reaction mixture. The possibility of competition between the two corresponding substrates for the same active site(s) on the enzyme were tested by using the relationship (Dixon & Webb, 1964b): Ka VaV-a+b (1) Kb Va+b-Vb where Ka, Va, Kb and Vb are the Km and Vmax. values of substrates a and b and V.+b is the maximum velocity observed with an equimolar mixture of substrates a and b. A corollary of this theoretical rate law for two substrates competing for a single active site is that the total rate of reaction (Va+b) will be less than the sum of rates of reactions measured separately (Va + Vb) when the two substrates are present in equimolar concentrations (Dixon & Webb, 1964b). Eqn. (1) was further extended (eqn. 2) to examine the maximum velocity data, when the two substrates were present in different proportions in the reaction mixture as follows: Ka VaV-a+b = ~~~~~~~2) aKb

Va+b-Vb

Eqn. (2) has been previously used by Verpoorte (1972) and by us (Mian et al., 1978; Pope et al., 1978) for analysing the possibility of competition between the two substrates for the same binding site(s). a denotes the ratio of concentrations of substrate a and b and should not be confused with the term used originally by Dixon & Webb (1964b) to designate the relative concentration of substrate A to its Michaelis constant, i.e. a = [AIIKA. The Km and Vmax. values for each substrate alone and Va+b of the mixed reaction were calculated from the initial-velocity measurements (Table 3). It is clear from the data that the

observed values of the joint reaction (Va+b) are closely similar to those calculated by using eqn. (1) or eqn. (2), and less than the sum of Va+ Vb, thus suggesting a competition between the sulphated and unsulphated substrates for the same active site(s) on the enzyme molecule. The result presented above is noteworthy, since it has been assumed previously that for a compound to be cleaved by neuraminidase the presence of nonsubstituted carboxy groups in N-acetylneuraminic acid is of great importance. Thus the methyl esters of bovine submaxillary-gland glycoprotein (Gottschalk, 1962) and the methyl ester of N-acetylneuraminyl-(2-*3)-lactose (Yu & Ledeen, 1969) were not hydrolysed by V. cholerae neuraminidase. N-Acetylneuraminic acid was liberated after hydrolysis of the ester group by dilute alkali. The replacement of the acetyl group at the nitrogen atom of N-acetylneuraminic acid by a large substituent such as a butyryl, benzoyl or benzyloxycarbonyl group in synthetic a-ketosides caused a complete resistance of these compounds towards V. cholerae or viral neuraminidase (Meindl & Tuppy, 1966; Faillard et al., 1969). Similarly N-acetylneuraminic acid substituted at C(4) by an acetyl group was not released by V. cholerae and C. perfringens neuraminidases as shown by the resistance of horse submaxillary-gland glycoprotein containing N-acetyl-4-0-acetylneuraminic acid and N-acetyl-4,7-, -4,8- or -4,9-di-0-acetylneuraminic acid (Schauer & Faillard, 1968). On the other hand, neuraminic acid acetylated at C(7) and C(8) was liberated by V. cholerae and C. perfringens neuraminidases. Thus N-acetyl-7-0-acetylneuraminic acid and N-acetyl-8-0-acetylneuraminic acid present in bovine submaxillary-gland glycoproteins were completely released by C. perfringens neuraminidase (Schauer & Faillard, 1968). The above findings suggest that the resistance to enzymic hydrolysis depended on the nature and position of the substituent in the N-acetylneuraminic acid molecule. Although the position of an 0-sulphate substituent in the N-acetylneuraminyl moiety of sulphated derivatives of N-acetylneuraminyl-lactose was not investigated, owing to the lack of material, it could be speculated that C(7), C(8) or C(g) atoms might have been 0-sulphated, since 0-acetylation at these carbon atoms in this molecule in bovine submaxillarygland glycoproteins also did not interfere during their hydrolysis by neuraminidase (Schauer & Faillard, 1968). The subject has been reviewed more recently by Schauer et al. (1974). However, the kinetic data on the reactions between enzyme and sulphated substrates would suggest that the presence of 0-sulphate ester, an anionic substituent, in the N-acetylneuraminyl moiety increased its affinity for the active site of the enzyme, but decreased the rate of dissociation of enzyme-substrate complex, whereas the presence of such an anionic

1979

397

ENZYMIC DEGRADATION OF O-SULPHATED OLIGOSACCHARIDES

Table 3. Calculated and observed maximum velocities in the joint reaction of N-acetylneuraminyl-lactose and its isomers and their sulphated derivatives Neuraminidase used was from rat liver lysosomes. The values given below are averages of two experiments. Symbols are described in the text. Va+b

Substrate a

Substrate b

Ka Va N-Acetylneuraminyl-(;2--3)and -(2-.6)-lactose

1.84

140

Kb

Vb

[a]/[b]

V. + Vb

Observed

Calculated according to eqn. (1) or eqn. (2)

1.0 0.5

390

210 240

218 231

1.0

1930

1075 1150

1984 1142

1.0 0.5

1015

480 410

487 414

1.0 0.5

65

36 30

30 28

1.0 0.5

1410

860 760

888 745

N-Acetylneuraminyl-(2--3) and -(2-*6)-lactose 6'-Osulphate 250 0.75

N-Acetylneuraminyl-(,2--3)- N-Acetylneuraminyl-(2-+3)lactose 6'-O-sulphate lactose 1220 0.22 710 0.08

0.5 N-Acetylneuraminyl-(:2-+3)- N-Acetylneuraminyl ?-Osulphate-(2---*3)-lactose lactose 6'-O-sulphate 280 0.21 735 0.175

N-Acetylneuraminyl-(,2-+6)- N-Acetylneuraminyl ?-Olactose sulphate-(2->6)-lactose 1.18 40 0.64 25 N-Acetylneuraminyl-(:2-+3)lactose 6'-O-sulphaLte

N-Acetylneuraminyl ?-O-

sulphate-(2-+3)-lactose 6'-O-sulphate

0.075

1150

0.18

260

substituent on the adjacent galactose increased the affinity of the substrate for the enzyme (low Ki) without interfering with the release of product and the increase in the Vmax. could be due to a rapid turnover of the enzyme-substrate complex. Further characterization of sulphated substrates based on chromatographic analysis of their enzymically

hydrolysedproducts Fractionation of neuraminidase-hydrolysed products of N-acetylneuraminyl-(2--3)-lactose 6'-Osulphate on Dowex 1 (X8; formate form) columns indicated that N-acetylneuraminic acid (tested by resorcinol reagent) was eluted by 0.3M-formic acid and lactose 6'-O-sulphate (tested by anthrone reagent) was eluted by 2.7 M-formic acid comparedwith lactose, cleaved from the unsulphated substrates, which was eluted with water under similar chromatographic conditions. Similarly, fractionation of the enzymically hydrolysed products of N-acetylneuraminyl sulphate(2-*3)-lactose 6'-O-sulphate demonstrated that lactose 6'-O-sulphate and N-acetylneuraminic acid 0sulphate were eluted by 2.7M- and 5.0M-formic acid respectively by using a linear gradient and no other resorcinol- or anthrone-positive material was detected Vol. 181

in any other fractions. The ion-exchange chromatographic data also showed that lactose and N-acetylneuraminic acid 0-sulphate, the enzymically hydrolysed products of N-acetylneuraminyl sulphate(2-*6)-lactose, were eluted with water and 5.0Mformic acid respectively in a linear gradient elution from a Dowex 1 (X8; formate form) column. The identity of all these products hydrolysed by neuraminidase from their corresponding substrates was further confirmed either directly or after their fractionation on the ion-exchange chromatographic columns on paper chromatography against authentic reference carbohydrates. Kinetics of hydrolysis of lactose and lactose sulphate The Km and V..,, values of hydrolysis of lactose by brush-border lactase, at optimal pH 5.5, were 25mM and 0.188mmol/h per mg of enzyme protein respectively. With the use of p-nitrophenyl ,B-D-galactopyranoside as substrate at pH 6.0 and in the presence of 0.2mM-p-hydroxymercuribenzoate the corresponding Km and Vmax. obtained for the brush-border fraction enzyme were 22.8mM and 0.054mmol/h per mg of enzyme protein respectively. Experiments with lactose 6'-O-sulphate indicated that it neither behaved as a substrate for the brush-

398 border lactase nor did it affect the hydrolysis of lactose when tested as an inhibitor or as an activator. Similar results were obtained when lactose 6'-Osulphate was tested against E. coli fi-D-galactosidase as a substrate or as an inhibitor with respect to p-nitrophenyl fi-D-galactopyranoside as a substrate. No change in the behaviour of lactose 6'-O-sulphate towards rat intestinal lactase or E. coili f-D-galactosidase was observed by altering the pH between 4.0 and 8.0 of the reaction mixture. These results conclude that the presence of 0-sulphate ester on C-6 of the galactosyl moiety of lactose not only rendered it unsuitable as a substrate for both intestinal brush-border lactase and for E. coli fi-D-galactosidase, but also made it unsuitable as an inhibitor of the enzyme activity with respect to lactose and p-nitrophenyl fl-D-galactopyranoside as substrate.

Concluding Remarks The difference in the reaction behaviour of neuraminidase and lactase/fl-D-Salactosidase with regard to 0-sulphate ester substitution in their respective substrates is noteworthy. It would appear that intestinal brush-border lactase and E. coli fl-Dgalactosidase possess a high degree of stereospecificity for the galactosyl moiety of their substrates, whereas neuraminidases from rat liver and C. perfringens show a lesser degree of specificity as these enzymes do not recognize the 0-sulphate ester substitution (the present study) or 0-acetyl groups at C(7), C(8) or C(s) of the N-acetylneuraminic acid molecule (Schauer & Faillard, 1968). During the course of this investigation, similar preliminary observations were also made on glucose oxidase (EC 1.1.3.4) and galactose dehydrogenase (EC 1.1.1.48), which were used for routine measurements of glucose and galactose respectively. Glucose 6-0-sulphate was neither oxidized by glucose oxidase nor did its presence interfere with the oxidation of the unsubstituted glucose to 3-D-gluconolactone by the enzyme. On the other hand, galactose 6-0-sulphate was oxidized half as fast as unsubstituted galactose by galactose dehydrogenase. One would assume that such anomalies in the behaviour of the individual enzymes from both hydrolase and oxidoreductase systems could be due to differences in the structural specializations of their active sites, which confer on them not only chemical specificity but also stereospecificity. From the evidence given in the present paper, it is proposed that the 0-sulphation of the cleavable moiety of the substrates or of the adjacent carbohydrate residue may either render them completely unsuitable as substrates or may alter the kinetics of their reaction with the enzymes, but it has so far shown no inhibitory effect on the enzyme reactions with respect to

N. MIAN, C. E. ANDERSON AND P. W. KENT

their corresponding unsulphated substrates. A corollary of the present findings is that the 0-sulphation of the substrate molecules may have some significance in terms of enzymic degradation of glycoproteins, but a unified hypothesis on the role of 0-sulphate esters cannot yet be formulated. We thank Mrs. S. Jobling for typing the manuscript and Mrs. B. Chorley for general assistance. We are grateful to Crinos Biological Research Laboratories, Como, Italy, for financial support and Smith, Kline & French for provision of apparatus.

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1, 441-444 Horvat, A. & Touster, 0. (1968) J. Biol. Chem. 243, 4380-4390 Lloyd, A. G. (1960) Biochem. J. 75, 478-482 Lloyd, A. G. (1962) Biochem. J. 83, 455-460 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Meindl, P. & Tuppy, H. (1966) Monatsh. Chem. 97, 1628-1647 Mende, T. J. & Whitney, P. L. (1978) Anal. Biochem. 84, 570-573 Mian, N., Herries, D. G. & Batte, E. A. (1978) Biochim. Biophys. Acta 523, 454-468 Mian, N., Anderson, C. E. & Kent, P. W. (1979) Biochem. J. 181, 377-385 Pope, A. J., Mian, N. & Herries, D. G. (1978) FEBSLett. 93, 174-176

1979

ENZYMIC DEGRADATION OF O-SULPHATED OLIGOSACCHARIDES Rees, D. A. (1960) Nature (Lonidon) 185, 309-3 10 Ryan, L. C., Carubelli, R., Caputto, R. & Trucco, R. E. (1965) Biochimn. Biophys. Acta 101, 252-258 Schauer, R. & Faillard, H. (1968) H-oppe-Seyler's Z. Physiol. Chem. 349, 961-968 Schauer, R., Buscher, H. P. & Casals-Stenzel, J. (1974) Biochem. Soc. Symp. 40, 87-116 Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B. K., Cerda, J. J. & Crane, R. K. (1973) Biochim. Biophys. Acta 323, 98-112 Schneir, M. L. & Rafelson, M. E. (1966) Biochinm. Biophys. Acta 130, 1-11

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Svennerholm, L. (1957) Biochimn. Biophys. Acta 24, 604611 Tulsiani, D. R. P. & Carubelli, R. (1970) J. Biol. Chenm. 245, 1821-1827 \'epoorte, J. A. (1972) J. Biol. Chein. 247, 4787-4793 Viala, R. & Gianetto, R. (1955) Can. J. Biochent. Physiol. 33, 839-844 Warren, L. (1959) J. Biol. Chenm. 234, 1971-1975 Werner, W., Rey, H. G. & Wielinger, H. (1970) Z. Anal. Cheini. 252, 224-228 Yu, R. K. & Ledeen, R. (1969)J. Biol. Chemii. 244, 13061313