Effect of O-Sulphate Groups in Lactose and N-Acetylneuraminyl-Lactose on Their Enzymic Hydrolysis by Nasi MIAN, Caroline E

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

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-D- galactosidase (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 Bearing in mind the resistance of compounds the presence of unsubstituted carboxy groups on the towards neuraminidase action owing to the presence N-acetylneuraminic acid is of great importance of substituents on the N-acetylneuraminic acid mole- (Gottschalk, 1962; Yu & Ledeen, 1969). Bacterial cule and the inhibition of the enzyme activity in the and viral neuraminidases recognize substituents on presence of chemically sulphated sialoglycopeptides, the N-acetylneuraminic acid molecule. The resistance it was considered important to chemically sulphate to hydrolysis by neuraminidase of substances some simple low-molecular-weight substrates and to containing the substituted N-acetylneuraminic acid compare their enzymic hydrolysis reactions with the molecule have been a subject of great interest from unsubstituted compounds. To this end, two enzyme the point of view of specificity of enzyme action systems, neuraminidases (EC 3.2.1.18) from Clostri- (for review see Drzeniek, 1973). In addition, viral dium perfringens and rat liver lysosomes and lactase neuraminidases specifically hydrolyse the different (EC 3.2.1.23) from suckling rat intestine and Escher- types of a-ketosidic linkage between N-acetyl- ichia coli ,B-galactosidase were chosen and chemically neuraminic acid and adjacent carbohydrate mole- sulphated derivatives of N-acetylneuraminyl-lactose cules. The nature of this carbohydrate is of minor and lactose were prepared. In the present paper, we importance for the recognition by these enzymes, report a comparative kinetic study on the action of but large substituents on these carbohydrates make these enzymes on their sulphated and unsubstituted such compounds resistant towards bacterial and substrates and also discuss the biological role of viral neuraminidases owing to steric hindrance 0-sulphate ester groups. (Drzeniek, 1973). However, we have observed that the chemically sulphated sialoglycopeptides inhibited Experimental the hydrolysis of N-acetylneuraminic acid from N-acetylneuraminyl-lactose, bovine submaxillary- Chemical estimations gland mucin and from the native sialoglycopeptides The 2-thiobarbituric acid method of Warren (1959) by steric hindrance (Mian et al., 1979). was used for the estimation of free N-acetylneura- Vol. 181 388 N. MIAN, C. E. ANDERSON AND P. W. KENT minic acid. The resorcinol method of Svennerholm Authentic glucose, galactose and lactose were (1957) was used for the determination of total free eluted with water, whereas N-acetylneuraminic acid and bound N-acetylneuraminic acid. Lactose was was eluted with 0.3 M-formic acid. Galactose 6-0-sul- determined by the anthrone-reagent method des- phate, glucose 6-0-sulphate, glucose 3-0-sulphate cribed by Cook (1976). Glucose was determined by were eluted with 4.5 M-formic acid, whereas N-acetyl- glucose oxidase reaction by using glucose-test- neuraminic acid 0-sulphate was eluted with 5.OM- combination kit (Boehringer) based on the method formic acid. of Werner et al. (1970). Galactose was estimated by galactose dehydrogenase according to the method Chemical sulphation ofcarbohydrates described by Asp & Dahlqvist (1972). Sulphation of carbohydrates was carried out by The sulphate content of the samples after hydroly- using the pyridine-SO3 reagent (Aldrich Chemical sis in 25 % (v/v) formic acid for 24h at 100°C was Co., Gillingham, Dorset, U.K.) by a modification of determined by the method of Mende & Whitney the method of Lloyd (1960). Carbohydrates were (1978), which involved precipitation of the inorganic dissolved in anhydrous pyridine at 60°C, excess of sulphate on cellulose thin-layer plates with 133BaC12. the pyridine-SO3 was added and the reaction was maintained at 37°C for 12 or 36h with constant Paper chromatography shaking. The reaction mixture was then kept at room temperature for 3 h, cooled to 4°C and then an ice- Descending chromatography on Whatman no. 1 cold suspension of Ba(OH)2 was added with constant paper was performed with solvent system I (ethyl stirring over a period of 10min. After centrifugation acetate/pyridine/water, 10:5:6 by vol.), system II at 5000g at 4°C for 15 min, the sediment was discarded (n-butanol/n-propanol/0.1M-HCl, 1:2:1 by vol.), and solid CO2 was added to the supernatant liquid system III (ethyl acetate/pyridine/acetic acid/water, and centrifugation was repeated. The resulting 5 :5:1 :3 by vol.), and system IV (butan-l-ol/ethanol/ supernatant was concentrated by rotary evaporation water, 3 :1 :1 by vol.) containing 3 % (w/v) cetylpy- at 30°C with repeated additions of water to remove ridinium chloride. The chromatograms were de- excess pyridine and finally concentrated to 4ml. The veloped following a dip in ethanolic NaOH/AgNO3 sulphated products from unreacted carbohydrates (for reducing compounds) or with p-dimethylamino- were separated by repeated ion-exchange chroma- benzaldehyde spray (for N-acetylneuraminic acid). tography on Dowex 1 (X8; formate form). Carbo- hydrates present in the eluted fractions throughout were detected by using anthrone reagent for sulphated Ion-exchange chromatography or unchanged hexoses and resorcinol reagent for The authentic carbohydrate samples were run N-acetylneuraminic acid or its sulphated forms. individually on ion-exchange columns (Dowex 1; X8; Eluted fractions were freed from pyridine/formate by x 1 rotary evaporation at 30°C and concentrated to 1 ml. formate form; 200-400 mesh; 25cm cm) and These were converted salts eluted with a gradual continuous gradient of 0 to finally into potassium by 5.0M-formic acid. Similarly in every preliminary passage through Dowex 5OW (H+ form) followed by run, the sulphation reaction mixture was passed neutralization with KHCO3 (0.1 M). Samples were through an ion-exchange column and eluted with a then concentrated to dryness at room temperature gradual continuous gradient of 0 to 5.0M-pyridine/ in vacuo over P205. formate solution pH4.4 to assess approximately the gradient concentrations that eluted the carbohydrate Identification of 0-sulphate esters in sulphated test-positive materials. In the subsequent ion- substrates exchange chromatography, the columns after being Sulphated sugars used in the present work, i.e. loaded with the reaction mixture were washed with glucose 3-0-sulphate, glucose 6-0-sulphate and water and then eluted with a stepwise gradient galactose 6-0-sulphate (Lloyd, 1960) were all as ranging between minimum and maximum concen- potassium salts. Di- and tri-saccharide sulphates were trations of pyridine/formate required to elute the hydrolysed in 0.1 M-HCI for 90min at 100°C (Ryan carbohydrate material, as assessed from the pre- et al., 1965). Hydrolysis under these conditions liminary runs. This procedure minimized the time yielded monosaccharides and monosaccharide 0-sul- taken for ion-exchange chromatography and im- phates from di- or tri-saccharide derivatives. The proved the recovery of the added material from the hydrolysates werecooled in an ice bath and dried in the columns. The material recovered from these columns presence of NaOH pellets over silica gel in an evacu- was subjected to rechromatography on Dowex ated desiccator. The hydrolysis products were 1 (X8) and was eluted either by using a gradual examined in comparison with the reference authentic continuous gradient or by a narrow-range stepwise compounds by using analytical paper and ion- gradient. exchange chromatography. Samples were loaded on 1979 ENZYMIC DEGRADATION OF O-SULPHATED OLIGOSACCHARIDES 389

Dowex 1 (X8; formate form) columns (25cm x 1 cm) (a) and eluted with water followed by a linear gradient of 4.0 0 to 5.5M-formate. The eluted fractions were freed [I from pyridine/formate and concentrated by rotary evaporation. These were examined chemically, (b) enzymically and by analytical paper chromatography (c) 3.0 _ for the presence of glucose, galactose, N-acetyl- (d) neuraminic 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 6-0- @ 2.0 sulphate were not substrates for the enzyme. Galac- if tose dehydrogenase oxidized galactose 6-0-sulphate at halfthe rate ofthat ofgalactose. On the other hand, both anthrone and resorcinol reactions did not discriminate between unsubstituted and substituted hexoses and N-acetylneuraminic acid respectively. 1 .0 ,

Fractionation of structural isomers of N-acetyl- I , neuraminyl-lactose tb.cc The two structural isomers (2--*3) and (2-÷6) of a -1 I 1- N-acetylneuraminyl-lactose, commercially available o 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 as a mixture of the two (Sigma), were separated by Elution volume (litre) ion-exchange chromatography by the method of Fig. 1. Fractionation of lactose 6'-0-sulphate on Dowex Schneir & Rafelson (1966). About 70 % of the 1 (X8; formate form) material applied to the Dowex 1 (X2; acetate form) Pyridine-free concentrated reaction products (4ml) of column was recovered in a ratio of 5 :1 of (2->3)- and lactose obtained after 36h reaction with pyridine-SO3 (2-*6)-isomers. Descending chromatography on complex were applied to a column (2cm x 30cm) of Whatman no. 3 paper in solvent system III gave Dowex 1 (X8; formate form; 200-400 mesh). The discrete spots on development ofthe chromatograms. 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 Lactose 6'-0-sulphate [D-galactopyranosyl 6-0-sul- total hexoses by using anthrone reagent test. Peak I, phate-(fl1 -÷4)-0-D-glucopyranose] unchanged lactose; peak II, lactose 6'-0-sulphate. 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 for glucose 3-0-sulphate, glucose 6-0-sulphate or from Dowex 1 (X8; formate form) yielded two glucose bis-sulphate described by Rees (1960) were fractions; unchanged lactose was eluted with water detected on paper chromatography in solvent and the sulphated derivative was located in 0.5M- system IV. Galactose 6-0-sulphate liberated by acid pyridine/formate eluate in a preparative chroma- hydrolysis of lactose 6'-0-sulphate was eluted with tographic run with stepwise gradient elution (Fig. 1). 4.5M-formate from Dowex 1 (X8; formate form) and Potassium lactose 6'-O-sulphate (0.0046mol, yield had an RF value of0.40 in solvent system IV on paper 46%) was a crystalline solid that moved as a single chromatography (Lloyd, 1962). No unchanged component in solvent systems I and IV on paper galactose or a disulphate derivative was detected on chromatography. On rechromatography on Dowex paper chromatography. Oxidation of lactose and 1 (X8), the compound was eluted as a single peak lactose 6'-0-sulphate (2mg each) was carried out in with 0.2M-pyridine/formate or by 2.7M-formate 10ml of0.015M-sodium metaperiodate and periodate when the column was eluted with a linear gradient of oxidation was measured spectrophotometrically 0-5M-formate. The compound had a carbohydrate/ (Guthrie, 1962a). Lactose 6'-0-sulphate and lactose sulphate ratio of 2: 1 and on hydrolysis in 0.1 M-HCI consumed 2.54 and 3.96mol of periodate/mol of yielded glucose and galactose 6-0-sulphate. Glucose carbohydrate respectively (72h terminal). The analy- was identified by paper and ion-exchange chroma- sis of formaldehyde produced in the periodate tography and by glucose oxidase reaction. No reaction, carried out by the method of Guthrie component with RF values similar to those obtained (1962b) by using the chromotropic acid reaction, Vol. 181 390 N. MIAN, C. E. ANDERSON AND P. W. KENT indicated that lactose 6'-O-sulphate and lactose pro- glucose, galactose 6-0-sulphate and N-acetylneura- duced 1.1 and 1.8mol of formaldehyde/mol of the minic acid, which were identical with the correspond- compound respectively. These values are in close ing authentic carbohydrates on paper and ion- agreement with the previously published data (Ryan exchange chromatography. No sulphated derivatives et al., 1965). Reaction of lactose with pyridine-S03 ofglucose and N-acetylneuraminic acid were detected Complex, whether for 12 or 36h, produced lactose by paper chromatography in solvent systems I, III 6'-O-sulphate only and no disulphate isomers were and IV or by ion-exchange chromatography. Simi- detected. larly, estimation of sulphate in these products showed the carbohydrate/sulphate ratio of galactose 6-0-sulphate to be 1 :1, whereas no sulphate was N-Acetylneuraminyl-(2--3)-lactose 6'-O-sulphate [N- detected in glucose and N-acetylneuraminic acid. acetylneuraminyl-(a2--33)-O-galactopyranosyl 6-0- The identity of glucose and galactose 6-0-sulphate sulphate-(fl -+4)-0-glucopyranose] was also checked enzymically. N-Acetylneuraminyl-(2-+3)-lactose (0.001 mol) was reacted with pyridine-SO3 (0.002mol) in N-Acetylneuraminyl sulphate-(2-÷3)-lactose 6'-O- anhydrous pyridine for 12h at 37°C. Preparative sulphate [N-acetylneuraminyl ?-O-sulphate-(a2->3)- ion-exchange chromatography of the finally 0-galactopyranosyl 6-0-sulphate-(fl1 -4)-O-gluco- processed reaction mixture yielded two fractions: pyranose] unchanged N-acetylneuraminyl-(2--3)-lactose, which was eluted by 0.01 M-pyridine/formate, pH4.4, and N-Acetylneuraminyl-(2-+3)-lactose (0.001 mol) the sulphated derivative (52% yield), which was was reacted with pyridine-SO3 complex (0.002mol) obtained in 0.5M-pyridine/formate eluate in a step- in anhydrous pyridine for 36h at 37°C. Preparative wise gradient elution (Fig. 2). The potassium salt of ion-exchange chromatography of the finally pro- N-acetylneuraminyl-(2---3)-lactose 6'-O-sulphate cessed reaction mixture yielded three fractions: moved as a single component on paper chroma- unchanged N-acetylneuraminyl-(2--3)-lactose, tography in solvent systems II and IV, and was which was eluted with 0.01 M-pyridine/formate, pH eluted by 0.22M-pyridine/formate during rechro- 4.4, and two sulphated fractions, which were both matography on Dowex 1 (X8; formate form) by using eluted by 0.5 M-pyridine/formate from Dowex 1 (X8; a gradual continuous gradient of 0 to 1.0M-pyridine/ formate form) column as two separate peaks (Fig. 3). formate. The compound had a carbohydrate/sulphate The major and the minor fractions, 60 and 10% in ratio of 3: 1 and on hydrolysis in 0.1 M-HCI yielded yield respectively, were subjected to rechro-

(d) (b) ( (c) A .7 0.7 0.6 0.6 A 0.5 0.5 c04 0.4 x 0.3 ~~~~~~~~~~.19~~~~~~~~~~~~~~~~~~~~~~~1 0.3 0.2 0.2 0.1 */ X*, 0.1 0 0.3-X04-0-X-0-X-.-X-6-.80. 1l. I I 0.3 0.4 0.5 0.6 0.7 0.8 0.9 l o 1.1.1 1.2 1.3 1.4 1.5 Elution volume (litre) Fig. 2. Fractionation ofN-acetylneuraminyl-(2--3)-lactose 6'-O-sulphate o0n Dowex 1 (X8; formateform) 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. 1979 ENZYMIC DEGRADATION OF O-SULPHATED OLIGOSACCHARrDES 391

(d) (b) (d) (c) 0.7 0.7 0.6 0.6 0.5 0.5 ° III 0.4 o oo 0.3 111 0.3 *T 0.2 0.2

0.1 *-I , 0.1

A I I IA 0 0 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 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 ofwater 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) N-Acetylneuraminyl sulphate-(2--6)-lactose [N-ace- columns by using a gradual continuous gradient of tylneuraminyl ?-O-sulphate-(a2-+6)-0-galactopy- to 1.0M-pyridine/formate, pH4.4. -The major ranosyl-(fl-1 -*4)-0-glucopyranose] fraction was eluted as a single peak between 0.28M- and 0.34M-pyridine/formate and the minor fraction N-Acetylneuraminyl-(2-.6)-lactose could not be was eluted between 0.18M- and 0.22M-pyridine/ sulphated during 12h reaction with pyridine-SO3 formate as a single peak. The minor sulphated complex. However, reaction ofthis isomer (0.001 mol) at component was found to be identical with N-acetyl- with pyridine-SO3 complex (0.002mol) for 36h neuraminyl-(2-÷3)-lactose 6'-0-sulphate by analyses 37°C yielded a monosulphate derivative (45 % yield) the of its acid-hydrolysis products and had a carbohy- and unchanged compound (Fig. 4). The sulphated drate/sulphate ratio of 3 :1. The major sulphated derivative moved as a single component on paper component had a carbohydrate/sulphate ratio of 3 :2 chromatography in solvent system III and was eluted and on acid hydrolysis yielded glucose, galactose by pyridine/formate gradient between the concen- 6-0-sulphate and N-acetylneuraminyl sulphate. Glu- trated 0.30M and 0.34M on rechromatography on cose and galactose 6-0-sulphate were identified by Dowex 1 (X8; formate form) by using a linear paper and ion-exchange chromatography and by gradient of 0 to 1.OM-pyridine/formate, pH4.4. The their enzymic reactions as described in the preceding compound had a carbohydrate/sulphate ratio of 3 :1 on and section. N-Acetylneuraminic acid 0-sulphate had a and acid hydrolysis yielded glucose, galactose N-acetylneuraminyl sulphate, which were identified carbohydrate/sulphate ratio of 1: 1 and was eluted as described in the preceding section. from Dowex 1 (X8; formate) column with 5.OM- formic acid, whereas the authentic N-acetylneuraminic acid could be eluted with 0.3 M-formic acid. On N-Acetylneuraminyl-(2--->-3)- and -(2-*6)-lactose sul- paper chromatography in solvent system III, the RF phate (mixed isomers) value ofN-acetylneuraminic acid 0-sulphate was 0.36 compared with that of 0.48 for the authentic N- Sulphation of a commercial preparation of acetylneuraminic acid. No sulphated derivative of N-acetylneuraminyl-(2--3)- and -(2-*6)-lactose glucose or bis-sulphate of galactose was detected by (0.001 mol) by using pyridine-SO3 complex paper chromatography in solvent systems I and IV. (0.002mol) for 12h at 37°C yielded a sulphated The major sulphated component was thus assigned to product and unchanged compound. The sulphated be a disulphate product, i.e. N-acetylneuraminyl product was purified on ion-exchange chromato- sulphate-(2->3)-lactose 6'-0-sulphate. graphy and analysed as described previously. The Vol. 181 392 N. MIAN, C. E. ANDERSON AND P. W. KENT

(b) (d) (c) 0.4 r (a) 0.4

0.3 - 0.3

o 00.2 0.2 X

0.11- 0.1

I xx O-Ae-0-0-0-0-0-0-0 0-0-0-0 -0--j-rw 0J i x-v-x-x-x-x-X-*.X--K-X-%- x- 0

I I 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 Elution volume (litre) Fig. 4. Fractionation ofN-acetylneuraminyl ?-O-sulphate-(a%2-*6)-lactose on Dowex 1 (X8; formateform) 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) 0.15M- 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 pro- the direct method the enzymic reaction was stopped duct was 3 :0.8, and it yielded glucose, galactose by adding 0.1 ml of periodate reagent and the 6-0-sulphate and N-acetylneuraminic acid, which complete analysis was carried out as described by were identified as described in the preceding section. Warren (1959). For the chromatographic method, Of the total galactose only 80% was sulphated. The the enzymic reaction was terminated by heating the commercially available mixture contained (2--3)- tubes at 100°C for 2min. N-Acetylneuraminic acid and (2-+6)-isomers in the ratio 5 :1, the galactose was separated from the remainder of the mixture by moiety of (2-.6)-isomers remains unsulphated ion-exchange chromatography on Dowex 1 (X8; because of its linkage with the N-acetylneuraminyl formate form) (Cook, 1976) before its estimation by residue at C-6. The chromatographically purified the method of Warren (1959). The blanks were N-acetylneuraminyl-(2--*6)-lactose remained unsul- prepared in a similar way, except that the enzyme phated after 12 h reaction with pyridine-SO3 complex was added at the end-point of the reaction period. as described above. All estimations were carried out in duplicate. There was no non-enzymic hydrolysis of N-acetylneura- Assay ofneuraminidase activity minyl-lactose or its sulphated derivatives under the above assay conditions. The enzyme activity is The substrate solutions were used in concen- expressed throughout as nmol of N-acetylneura- trations up to 0.01 M with respect to N-acetyl- minic acid released/h per mg of enzyme protein. neuraminyl-(2--3)- and -(2->6)-lactose or its isomers in 0.05M-sodium acetate/acetic acid buffers, pH4.2 Assay oflactase activity and 5, as required. The incubation mixture (0.2ml) containing 0.1ml of rat liver lysosomal fraction or Brush-border lactase was assayed in the presence appropriately diluted commercial preparation of of 0.2 mM-p-hydroxymercuribenzoate by the,method enzyme from C. perfringens suspended in 0.05M- of Asp & Dahlqvist (1972) with lactose as substrate. sodium acetate/acetic acid buffer, pH4.2 or 5.0, and The incubation mixture (0.2ml) contained 0.032M- 0.05 ml of substrate solution was incubated for 1 h at lactose and 0.05M-sodium maleate buffer, pH5.5. 37°C. The amount. of N-acetylneuraminic acid The incubations were carried out at 37°C for 1 h over released by enzymic action was measured either which period the rate of reaction was linear. The directly or after its chromatographic separation. In 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 , 0.1g 0.4 / 0.10 Cld 0 the presence or absence of 0.2 mM-p-hydroxymercuri- s- 0.10 0 ~~~~~~~~~~~~~~~~~(Ilc) benzoate at 37°C for 1 h and the reaction was ter- ~~~~~~~~/0 minated by adding 0.4M-NaOH/glycine buffer, 0.2 0.05 0 pH11.0. Blanks were prepared with the enzyme or (U the substrate alone. 0 0 0 p-Nitrophenol (Sigma) was used t 3.5 4.5 5.5 3.5 4.5 5.5 3.5 4.5 5.5 as standard. 4.0 5.0 6.0 4.0 5.0 6.0 4.0 5.0 6.0 Protein was determined by the method of Lowry pH et al. (1951) with bovine serum albumin (Sigma) as Fig. 5. Effect of pH on the hydrolysis of N-acetylneura- standard. minyl-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 Preparation and general properties of rat liver lyso- acetate/acetic acid buffers and at pH6.0 by using some-bound neuraminidase O.05M-Na2HPO4/NaH2PO4 buffer (Gomori, 1955). (I) Rat liver neuraminidase; (II) C. perfringens neura- Lysosome fraction from male Wistar rats weighing minidase. (a) N-Acetylneuraminyl-(a2--3)- and between 250 and 300g was prepared by the method -(a2-+6)-lactose (mixed isomers); (b), N-acetylneura- of Horvat & Touster'(1968) based on that of de Duve minyl-(a2-.3)-lactose; and (c) N-acetylneuraminyl- et al. (1955). Livers were minced and passed through a (a26)-lactose as substrates. The values plotted are tissue press. Homogenization and the isolation of averages of three experiments. subcellular particles by differential centrifugation were performed by the method of de Duve et al. (1955). Different subfractions were characterized by assayingthe marker-enzyme activities (de Duve e#*l., 1955; Horvat & Touster, 1968). Neuraminidase and -(2-*6)-lactose and its isomers by both enzyme arylsulphatase, a typical lysosomal marker (Viala & preparations was linear with time for up to 1 h. Gianetto, 1955), showed highest relative total and specific activity in the same fraction sedimenting Preparation and general properties of suckling-rat between the mitochondria and microsomal fraction, brush-border lactase which were characterized by the presence of their respective enzyme markers (Horvat & Touster, Brush-border fraction from intestines of 12-14- 1968). The lysosome fraction contained 51 % of the day-old rats was prepared by the method of Schmitz total neuraminidase activity present in homogenates et al. (1973). A 1 % mucosal homogenate was made and the specific activity of the enzyme was 64 times in 0.05M-mannitol/0.002M-Tris, pH7.1, at 4°C. The higher than that of the original homogenate. C. homogenate was filtered through nylon mesh (40,um perfringens neuraminidase was obtained from Boeh- pore size). Solid CaCl2 was added to a final concen- ringer. tration of 0.01 M with constant stirring. After 10min pH-Activity curves of the two neuraminidases with occasional mixing by inversion the suspension tested by using N-acetylneuraminyl-(2--3)- and was centrifuged at 2000g for 10min to yield a heavy -(2->6)-lactose and its fractionated isomers as whitish pellet, which was resuspended in 0.05 M-man- substrates are given in Fig. 5. The pH optima in the nitol/0.002M-Tris, pH 7.1. It was then centrifuged at case of rat liver lysosome-bound enzyme was 4.2 for 20000g for 15min to yield a small brownish pellet these substrates. On the other hand, pH optimum for containing brush-border fraction. The final pellet the hydrolysis of the two isomers of N-acetylneura- contained 68 % of the total lactase activity present in minyl-lactose by C. perfringens neuraminidase were the homogenate and the specific activity of the en- 4.5 and 5.0, as reported for influenza-virus neura- zyme in the brush-border fraction was 76 times minidase previously (Schneir & Rafelson, 1966). The higher than the corresponding activity of the original rate of hydrolysis of N-acetylneuraminyl-(2-*3)- and homogenate. Vol. 181 394 N. MIAN, C. E. ANDERSON AND P. W. KENT

Lactase and fi-D-galactosidase activities of the lysosomes and C. perfringens under optimal con- brush-border fraction were measured by using ditions of assay are given in Table 1. These values are lactose and p-nitrophenyl f,-D-galactoside as sub- in close agreement with those reported previously for strates in the presence of 0.2mM-p-hydroxymercuri- these substrates, where neuraminidase from an benzoate to inhibit acid IJ-galactosidase and hetero-fl- influenza virus strain was used (Schneir & Rafelson, D-galactosidase as described by Asp & Dahlqvist 1966). The specificity of neuraminidase towards (1972). Fig. 6 shows the pH-activity curves of lactase and p-hydroxymercuribenzoate-stable p-nitrophenyl fi-D-galactosidase. Lactase activity of the brush- border fraction was not inhibited by sodium galac- tonate, whereas galactono-1,4-lactone was a com- petitive inhibitor, the K, values measured by using Dixon plots (Dixon & Webb, 1964a) being 0. 0.75+0.16mM and 1.19±0.18mM (mean±s.D.) with cd respect to lactose andp-nitrophenyl fl-galactopyranoside as substrates. .E 0 Kinetic experiments and analysis ofthe data 0

Kinetic experiments were performed by using C- various concentrations of substrates and the initial velocities were measured. The Kin, Vinax. and K, C.) values were calculated from Lineweaver-Burk plots as described by Dixon & Webb (1964a). In mixed- substrate experiments sulphated and unsubstituted 3.0 4.0 5.0 6.0 7.0 8.0 substrates were mixed in equimolar and different pH proportions and the initial velocities of the joint Fig. 6. Effect ofpH on lactase and p-hydroxymercuriben- reactions were measured. The significance in the zoate-stable p-nitrophenyl fi-D-galactosidase activities difference between two sets of values was determined Assays of enzyme activities were carried out at pH by using Student's t test. 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 Results and Discussion (Gomori, 1955). Lactase activity (o) was measured * the presence of 0.2mM-p-hydroxymercuribenzoate Kinetics of hydrolysis of N-acetylneuraminyl-lactose, with 32mM-lactose as a substrate. p-Hydroxymer- its isomers and their sulphated derivatives by neura- curibenzoate-stable p-nitrophenyl fi-D-galactosidase minidases activity (D) was estimated with 20mM-p-nitrophenyl fi-D-galactopyranoside as substrate in the presence of The kinetic values of hydrolysis of N-acetylneura- 0.2mM-p-hydroxymercuribenzoate. The results are minyl-(2--3)- and -(2-+6)-lactose and its two expressed as percentages of the optimal activities and structural isomers by neuraminidases from rat liver are averages of three experiments.

Table 1. Kinetic valuesfor the hydrolysis ofN-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 ... N-Acetylneura- N-Acetylneuraminyl- N-Acetylneuraminyl- Enzyme preparation minyl-(2-.3)- and (2-*6)-lactose (2-*3)-lactose (2-*6)-lactose Rat liver neuraminidase pH 4.2 4.2 4.2 K. (mM) 1.78+0.12 (5) 0.24+ 0.02 (3) 1.10+0.16 (3) Vmax. (nmol/h per mg of pro- 133+ 16 (5) 720+14(3) 45± 8 (3) tein) C. perfringens neuraminidase pH 5.0 5.0 4.5 Km (mM) 2.39 +0.04 (5) 0.45 + 0.06 (3) 1.80+0.13 (3) Vm,x. (nmol/h per mg of pro- 212± 15 (5) 580± 70 (3) 90± 10 (3) tein) 1979 ENZYMIC DEGRADATION OF O-SULPHATED OLIGOSACCHARIDES 395 different types of linkage between N-acetylneura- (P<0.005) lower, whereas their Vmax. values were minic acid and the lactosyl moiety of the compound, significantly (P<0.005) higher compared with the i.e. (2->3)-, (2-*4)- and (2->6) isomers has been corresponding values (Table l) of the unsulphated tested previously (for review see Drzeniek, 1973). compounds. However, on the other hand, both Km Previously reported data show that C. perfringens and Vmax. values of N-acetylneuraminyl sulphate- (Cassidy et al., 1965), Vibrio cholerae (Drzeniek, (2-*3)-lactose 6'-O-sulphate and of N-acetylneura- 1967; Drzeniek & Gauhe, 1970) and liver neuramini- minyl sulphate-(2-->6)-lactose showed a significant dases (Horvat & Touster, 1968) hydrolysed N- (P< 0.005) decrease compared with the corresponding acetylneuraminyl-(2--6)-lactose half as fast as values ofthe unsulphated compound (Tables I and 2). N-acetylneuraminyl-(2--3)-lactose. It is clear that the O-sulphate ester substitution(s) N-Acetylneuraminyl-lactose 6'-0-sulphate, a influenced the kinetic constants of the reactions. If naturally occurring compound in rat mammary the Ki,, as assumed in the present paper, is the equili- gland (Ryan et al., 1965; Choi & Carubelli, 1968), brium constant for reaction forming the enzyme- has been reported to be hydrolysed at a faster rate substrate complex, it then appears that the presence of than N-acetylneuraminyl-lactose by soluble and 0-sulphate ester in galactosyl or N-acetylneuraminyl lysosome-bound neuraminidase from rat liver moieties or in both increases the affinity of the (Tulsiani & Carubelli, 1970), by C.perfringens enzyme sulphated substrates for the enzyme molecules, as (Tulsiani & Carubelli, 1970) by soluble neuraminidase indicated by a significant decrease in their Km values. from rat mammary gland (Carubelli et al., 1962) and On the other hand, if Vinax. is a measure of the by rat brain neuraminidase (Carubelli, 1968). No velocity constant of the breakdown of the enzyme- detailed study appears to have been carried out substrate complex, it then appears that not only the previously on the comparative kinetics of hydrolysis presence of 0-sulphate ester, but also its distribution of the sulphated and unsubstituted N-acetylneura- among different carbohydrate moieties of the sub- minyl-lactose by neuraminidase. strate influence the velocity of their reactions. The The pH-activity curves of the hydrolysis of sul- presence of 0-sulphate ester in N-acetylneuraminyl phated derivatives of N-acetylneuraminyl-lactose moiety appears to have an effect on both the for- and its isomners by rat liver and C. perfringens neura- mation and breakdown of the enzyme-substrate minidases were similar in profiles shown for un- complex. Thus 0-sulphate ester substitution in N- sulphated substrates (Fig. 5), thus indicating that the acetylneuraminyl moiety as in N-acetylneuraminyl presence of 0-sulphate ester in these substrates did O-sulphate-(2--3)-lactose 6'-0-sulphate increases the -not change the pH optima of their reaction. Km value by 2-fold, but decreases the Vmax. value by The data (Table 2), however, indicated that the Km more than 4.5-fold compared with the values for values ofN-acetylneuraminyl-(2-*3)- and -(2-*6)-lac- N-acetylneuraminyl-(2->3)-lactose 6'-O-sulphate tose 6'-O-sulphate and ofN-acetylneuraminyl-(2-*3)- (Table 2). Further comparison would indicate that lactose 6'-O-sulphate for neuraminidases from rat both Km and Vmax. values for N-acetylneuraminyl liver lysosomes and C. perfringens were significantly O-sulphate-(2->3)-lactose 6'-O-sulphate were de-

Table 2. Kinetic valuesfor the hydrolysis ofsulphated derivatives ofN-acetylneuraminyl lactose and its structural isomers by neuraminidases Values are means + S.E.M. (for the number of experiments shown in parentheses). Enzyme characteristics N-Acetyl- N-Acetyl- N-Acetyl- neuraminyl N-Acetyl- Sulphated sulphate ... neuraminyl- neuraminyl- ?O-sulphate- neuraminyl (2-->3)- and -(2-+6)- (2-+3)-lactose (2-.3)-lactose ?-O-sulphate- Enzyme preparation lactose 6'-O-sulphate 6'-O-sulphate 6'-O-sulphate (2-*6)-lactose Rat liver neuraminidase pH 4.2 4.2 4.2 4.2 Km (mM) 0.72+0.12 (3) 0.07 + 0.02 (5) 0.16+0.02 (5) 0.75 + 0.21 (5) Vmax. (nmol/h per mg of pro- 240+40 (3) 1160+180 (5) 250± 110 (5) 20±10 (5) tein) C. perfringens neuraminidase pH 5.0 5.0 n.d. n.d. Km (mM) 1.05±16(3) 0.13+ 0.03 (3) n.d. n.d. V.ax. (nmol/h per mg of pro- 360+50 (3) 970±160 (3) n.d. n.d. tein) Vol. 181 396 N. MIAN, C. E. ANDERSON AND P. W. KENT creased by about 1.5 and 3 times respectively com- observed values of the joint reaction (Va+b) are pared with the values for the corresponding unsul- closely similar to those calculated by using eqn. (1) or phated compound (Tables 1 and 2). A similar pattern eqn. (2), and less than the sum of Va+ Vb, thus showing a decrease in both Km and Vmax. for N-acetyl- suggesting a competition between the sulphated and neuraminyl O-sulphate-(2--6)-lactose in comparison unsulphated substrates for the same active site(s) on with those obtained for the corresponding unsul- the enzyme molecule. phated compound could be observed (Tables 1 and 2). The result presented above is noteworthy, since it These results suggest that 0-sulphate ester substitu- has been assumed previously that for a compound tion in the galactosyl moiety of the substrate in- to be cleaved by neuraminidase the presence of non- fluences the reaction by lowering the Km and increas- substituted carboxy groups in N-acetylneuraminic ing the Vmax., whereas such substitution in the N- acid is of great importance. Thus the methyl esters acetylneuraminyl moiety of the substrate influences of bovine submaxillary-gland glycoprotein (Gotts- the reaction by lowering both Km and Vmax. chalk, 1962) and the methyl ester of N-acetylneura- Bearing in mind the presence of a bulky anionic minyl-(2-*3)-lactose (Yu & Ledeen, 1969) were not substituent, 0-sulphate ester, in the N-acetylneura- hydrolysed by V. cholerae neuraminidase. N-Acetyl- minyl moiety of the substrate and its subsequent neuraminic acid was liberated after hydrolysis of the effect on the reaction constants the effect of the sul- ester group by dilute alkali. The replacement of the phated derivatives on the hydrolysis of the unsul- acetyl group at the nitrogen atom of N-acetylneura- phated compounds and vice-versa was studied. The minic acid by a large substituent such as a butyryl, experiments were carried out to measure Vmax. values benzoyl or benzyloxycarbonyl group in synthetic in joint reactions when N-acetylneuraminyl-lactose a-ketosides caused a complete resistance of these and its sulphated derivatives were present in equi- compounds towards V. cholerae or viral neuramini- molar concentrations in the reaction mixture. The dase (Meindl & Tuppy, 1966; Faillard et al., 1969). possibility of competition between the two corres- Similarly N-acetylneuraminic acid substituted at C(4) ponding substrates for the same active site(s) on the by an acetyl group was not released by V. cholerae enzyme were tested by using the relationship (Dixon and C. perfringens neuraminidases as shown by the & Webb, 1964b): resistance of horse submaxillary-gland glycoprotein Ka VaV-a+b containing N-acetyl-4-0-acetylneuraminic acid and N-acetyl-4,7-, -4,8- or -4,9-di-0-acetylneuraminic Kb Va+b-Vb (1) acid (Schauer & Faillard, 1968). On the other hand, where Ka, Va, Kb and Vb are the Km and Vmax. values neuraminic acid acetylated at C(7) and C(8) was of substrates a and b and V.+b is the maximum liberated by V. cholerae and C. perfringens neura- velocity observed with an equimolar mixture of minidases. Thus N-acetyl-7-0-acetylneuraminic acid substrates a and b. A corollary of this theoretical and N-acetyl-8-0-acetylneuraminic acid present in rate law for two substrates competing for a single bovine submaxillary-gland glycoproteins were com- active site is that the total rate of reaction (Va+b) will pletely released by C. perfringens neuraminidase be less than the sum of rates of reactions measured (Schauer & Faillard, 1968). The above findings separately (Va + Vb) when the two substrates are suggest that the resistance to enzymic hydrolysis present in equimolar concentrations (Dixon & Webb, depended on the nature and position of the sub- 1964b). Eqn. (1) was further extended (eqn. 2) to stituent in the N-acetylneuraminic acid molecule. examine the maximum velocity data, when the two Although the position of an 0-sulphate substituent substrates were present in different proportions in the in the N-acetylneuraminyl moiety of sulphated reaction mixture as follows: derivatives of N-acetylneuraminyl-lactose was not Ka VaV-a+b investigated, owing to the lack of material, it could = ~~~~~~~2) be speculated that C(7), C(8) or C(g) atoms might have aKb Va+b-Vb been 0-sulphated, since 0-acetylation at these Eqn. (2) has been previously used by Verpoorte (1972) carbon atoms in this molecule in bovine submaxillary- and by us (Mian et al., 1978; Pope et al., 1978) for gland glycoproteins also did not interfere during analysing the possibility of competition between the their hydrolysis by neuraminidase (Schauer & two substrates for the same binding site(s). a denotes Faillard, 1968). The subject has been reviewed more the ratio of concentrations of substrate a and b and recently by Schauer et al. (1974). should not be confused with the term used originally However, the kinetic data on the reactions between by Dixon & Webb (1964b) to designate the relative enzyme and sulphated substrates would suggest that concentration of substrate A to its Michaelis con- the presence of 0-sulphate ester, an anionic sub- stant, i.e. a = [AIIKA. The Km and Vmax. values for stituent, in the N-acetylneuraminyl moiety increased each substrate alone and Va+b of the mixed reaction its affinity for the active site of the enzyme, but de- were calculated from the initial-velocity measure- creased the rate of dissociation of enzyme-substrate ments (Table 3). It is clear from the data that the complex, whereas the presence of such an anionic 1979 ENZYMIC DEGRADATION OF O-SULPHATED OLIGOSACCHARIDES 397

Table 3. Calculated and observed maximum velocities in thejoint reaction ofN-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 Calculated according Ka Va Kb Vb [a]/[b] V. + Vb Observed to eqn. (1) or eqn. (2) N-Acetylneuraminyl-(;2--3)- N-Acetylneuraminyl-(2--3) and -(2-.6)-lactose and -(2-*6)-lactose 6'-O- sulphate 1.84 140 0.75 250 1.0 390 210 218 0.5 240 231 N-Acetylneuraminyl-(,2--3)- N-Acetylneuraminyl-(2-+3)- lactose lactose 6'-O-sulphate 0.22 710 0.08 1220 1.0 1930 1075 1984 0.5 1150 1142 N-Acetylneuraminyl-(:2-+3)- N-Acetylneuraminyl ?-O- lactose sulphate-(2---*3)-lactose 6'-O-sulphate 0.21 735 0.175 280 1.0 1015 480 487 0.5 410 414 N-Acetylneuraminyl-(,2-+6)- N-Acetylneuraminyl ?-O- lactose sulphate-(2->6)-lactose 1.18 40 0.64 25 1.0 65 36 30 0.5 30 28 N-Acetylneuraminyl-(:2-+3)- N-Acetylneuraminyl ?-O- lactose 6'-O-sulphaLte sulphate-(2-+3)-lactose 6'-O-sulphate 0.075 1150 0.18 260 1.0 1410 860 888 0.5 760 745 substituent on the adjacent galactose increased the in any other fractions. The ion-exchange chromato- affinity of the substrate for the enzyme (low Ki) graphic data also showed that lactose and N-acetyl- without interfering with the release of product and neuraminic acid 0-sulphate, the enzymically hydro- the increase in the Vmax. could be due to a rapid lysed products of N-acetylneuraminyl sulphate- turnover of the enzyme-substrate complex. (2-*6)-lactose, were eluted with water and 5.0M- formic acid respectively in a linear gradient elution from a Dowex 1 (X8; formate form) column. The Further characterization ofsulphated substrates based identity of all these products hydrolysed by neura- on chromatographic analysis of their enzymically minidase from their corresponding substrates was hydrolysedproducts further confirmed either directly or after their frac- Fractionation of neuraminidase-hydrolysed pro- tionation on the ion-exchange chromatographic ducts of N-acetylneuraminyl-(2--3)-lactose 6'-O- columns on paper chromatography against authentic sulphate on Dowex 1 (X8; formate form) columns reference carbohydrates. indicated that N-acetylneuraminic acid (tested by resorcinol reagent) was eluted by 0.3M-formic acid Kinetics of hydrolysis of lactose and lactose sulphate and lactose 6'-O-sulphate (tested by anthrone reagent) The Km and V..,, values of hydrolysis oflactose by was eluted by 2.7M-formic acid comparedwithlactose, brush-border lactase, at optimal pH5.5, were 25mM cleaved from the unsulphated substrates, which was and 0.188mmol/h per mg of enzyme protein respec- eluted with water under similar chromatographic tively. With the use of p-nitrophenyl ,B-D-galacto- conditions. Similarly, fractionation ofthe enzymically pyranoside as substrate at pH6.0 and in the presence hydrolysed products ofN-acetylneuraminyl sulphate- of 0.2mM-p-hydroxymercuribenzoate the corres- (2-*3)-lactose 6'-O-sulphate demonstrated that lac- ponding Km and Vmax. obtained for the brush-border tose 6'-O-sulphate and N-acetylneuraminic acid 0- fraction enzyme were 22.8mM and 0.054mmol/h per sulphate were eluted by 2.7M- and 5.0M-formic acid mg of enzyme protein respectively. respectively by using a linear gradient and no other Experiments with lactose 6'-O-sulphate indicated resorcinol- or anthrone-positive material was detected that it neither behaved as a substrate for the brush- Vol. 181 398 N. MIAN, C. E. ANDERSON AND P. W. KENT border lactase nor did it affect the hydrolysis of their corresponding unsulphated substrates. A lactose when tested as an inhibitor or as an activator. corollary of the present findings is that the 0-sul- Similar results were obtained when lactose 6'-O- phation of the substrate molecules may have some sulphate was tested against E. coli fi-D-galactosidase significance in terms of enzymic degradation of as a substrate or as an inhibitor with respect to glycoproteins, but a unified hypothesis on the role p-nitrophenyl fi-D-galactopyranoside as a substrate. of 0-sulphate esters cannot yet be formulated. No change in the behaviour of lactose 6'-O-sulphate towards rat intestinal lactase or E. coili f-D-galac- We thank Mrs. S. Jobling for typing the manuscript tosidase was observed by altering the pH between and Mrs. B. Chorley for general assistance. We are grate- 4.0 and 8.0 ofthe reaction mixture. ful to Crinos Biological Research Laboratories, Como, These results conclude that the presence of 0-sul- Italy, for financial support and Smith, Kline & French phate ester on C-6 of the galactosyl moiety of lactose for provision of apparatus. 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 References inhibitor of the enzyme activity with respect to Asp, N. G. & Dahlqvist, A. (1972) Anal. Biochem. 47, lactose and p-nitrophenyl fl-D-galactopyranoside as 527-538 substrate. Carubelli, P., Trucco, R. E. & Caputto, R. (1962) Biochim. Biophys. Acta 60, 196-197 Concluding Remarks Carubelli, R. (1968) Nature (London) 219, 955-956 Cassidy, J. T., Jourdian, G. W. & Roseman, S. (1965) J. The difference in the reaction behaviour of neura- Biol. Chem. 240, 3501-3506 minidase and lactase/fl-D-Salactosidase with regard Choi, H. U. & Carubelli, R. (1968) Biochemistry 7, 4423- to 0-sulphate ester substitution in their respective 4430 substrates is noteworthy. It would appear that Cook, G. M. W. (1976) in Biochemical Analysis of Mem- branes (Maddy, A. H., ed.), pp. 283-351, Chapman and intestinal brush-border lactase and E. coli fl-D- Hall, London galactosidase possess a high degree ofstereospecificity de Duve, C., Pressmann, B. C., Gianetto, R., Wattiaux, for the galactosyl moiety of their substrates, whereas R. & Appelmans, F. (1955) Biochem. J. 60, 604-617 neuraminidases from rat liver and C. perfringens Dixon, M. & Webb, E. C. (1964a) Enzymes, 2nd edn., show a lesser degree ofspecificity as these enzymes do pp. 54-90, Longmans, London not recognize the 0-sulphate ester substitution (the Dixon, M. & Webb, E. C. (1964b) Enzymes, 2nd edn., present study) or 0-acetyl groups at C(7), C(8) or C(s) pp. 86-87, Longmans, London of the N-acetylneuraminic acid molecule (Schauer & Drzeniek, R. (1967) Biochem. Biophys. Res. Commun. Faillard, 1968). 26, 631-638 During the course of this investigation, similar Drzeniek, R. (1973) Histochem. J. 5, 271-290 Drzeniek, R. & Gauhe, A. (1970) Biochem. Biophys. Res. preliminary observations were also made on glucose Commun. 38, 651-656 oxidase (EC 1.1.3.4) and galactose dehydrogenase Faillard, H., Do Amaral, C. F. & Blohm, M. (1969) (EC 1.1.1.48), which were used for routine measure- Hoppe-Seyler's Z. Physiol. Chem. 350, 798-802 ments of glucose and galactose respectively. Glucose Gomori, G. (1955) Methods Enzymol. 1, 138-146 6-0-sulphate was neither oxidized by glucose Gottschalk, A. (1962) Perspect. Biol. Med. 5, 327-337 oxidase nor did its presence interfere with the oxi- Guthrie, R. D. (1962a) Methods Carbohydr. Chem. 1, dation of the unsubstituted glucose to 3-D-glucono- 440-441 lactone by the enzyme. On the other hand, galactose Guthrie, R. D. (1962b) Methods Carbohydr. Chem. 6-0-sulphate was oxidized half as fast as unsub- 1, 441-444 Horvat, A. & Touster, 0. (1968) J. Biol. Chem. 243, stituted galactose by galactose dehydrogenase. 4380-4390 One would assume that such anomalies in the Lloyd, A. G. (1960) Biochem. J. 75, 478-482 behaviour of the individual enzymes from both Lloyd, A. G. (1962) Biochem. J. 83, 455-460 hydrolase and oxidoreductase systems could be due Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, to differences in the structural specializations of their R. J. (1951) J. Biol. Chem. 193, 265-275 active sites, which confer on them not only chemical Meindl, P. & Tuppy, H. (1966) Monatsh. Chem. 97, specificity but also stereospecificity. From the 1628-1647 evidence given in the present paper, it is proposed Mende, T. J. & Whitney, P. L. (1978) Anal. Biochem. 84, that the 0-sulphation of the cleavable moiety of the 570-573 Mian, N., Herries, D. G. & Batte, E. A. (1978) Biochim. substrates or of the adjacent carbohydrate residue Biophys. Acta 523, 454-468 may either render them completely unsuitable as Mian, N., Anderson, C. E. & Kent, P. W. (1979) Biochem. substrates or may alter the kinetics of their reaction J. 181, 377-385 with the enzymes, but it has so far shown no inhibi- Pope, A. J., Mian, N. & Herries, D. G. (1978) FEBSLett. tory effect on the enzyme reactions with respect to 93, 174-176 1979 ENZYMIC DEGRADATION OF O-SULPHATED OLIGOSACCHARIDES 399

Rees, D. A. (1960) Nature (Lonidon) 185, 309-3 10 Svennerholm, L. (1957) Biochimn. Biophys. Acta 24, 604- Ryan, L. C., Carubelli, R., Caputto, R. & Trucco, R. E. 611 (1965) Biochimn. Biophys. Acta 101, 252-258 Tulsiani, D. R. P. & Carubelli, R. (1970) J. Biol. Chenm. Schauer, R. & Faillard, H. (1968) H-oppe-Seyler's Z. 245, 1821-1827 Physiol. Chem. 349, 961-968 \'epoorte, J. A. (1972) J. Biol. Chein. 247, 4787-4793 Schauer, R., Buscher, H. P. & Casals-Stenzel, J. (1974) Viala, R. & Gianetto, R. (1955) Can. J. Biochent. Physiol. Biochem. Soc. Symp. 40, 87-116 33, 839-844 Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B. K., Warren, L. (1959) J. Biol. Chenm. 234, 1971-1975 Cerda, J. J. & Crane, R. K. (1973) Biochim. Biophys. Werner, W., Rey, H. G. & Wielinger, H. (1970) Z. Anal. Acta 323, 98-112 Cheini. 252, 224-228 Schneir, M. L. & Rafelson, M. E. (1966) Biochinm. Biophys. Yu, R. K. & Ledeen, R. (1969)J. Biol. Chemii. 244, 1306- Acta 130, 1-11 1313

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