Carbohydrate specificity, metal content and molecular stability of a lectin from Sainfoin (Onobrychis viciifolia, Scop.)

Biochimica et Biophvsiea Acta, 787 (1984) 237-243

237

Elsevier

BBA 31904

CARBOHYDRATE SPECIFICITY, METAL CONTENT AND MOLECULAR STABILITY OF A LECTIN FROM SAINFOIN (ONOBR YCHIS VICIIFOLIA, SCOP.) ROZENN N. KOUCHALAKOS * and K E N N E T H D. HAPNER **

Chemistry Department and/Igricultural Experiment Station, Montana State University, Bozeman, M T 59717 (U.S.A.) (Received December 12th, 1983)

Key words." Lectin stability," Carbohydrate specifici(v," Metal content," (Sainfoin)

Sainfoin (Onobrychis viciifolia, Scop. var Eski) lectin has been characterized as a 'typical' leguminous lectin belonging to the D-ma.nnose (D-glucose) binding family. The dissociation constant, Kd, and the number of binding sites per monomer with D-glucose were 3.5 mM and 1.0, respectively, as determined by equilibrium dialysis. Affinity electrophoresis showed two binding species with K d values of 1.6 mM and 2.0 mM toward free D-glucose and K~ values of 0.8 mM and 3.3 mM toward immobilized D-glucose. Concentrations of several carbohydrates causing 50% inhibition of hemagglutination were in the range 0.4-3 raM. D-Mannose, methyl a-D-mannopyranoside, maltose and 3-O-methyI-D-glucopyranose were among the strongest inhibitors. Inhibition by D-mannose was 7-8-times more effective than by D-glucose. Metal analysis showed one Ca 2+, and lesser amounts of Mg 2+ and Mn 2+ per protein monomer. Native sainfoin lectin was not cleaved by trypsin and was stable to heating at 55°C. Heating in the presence of 10 mM EDTA rapidly destroyed hemagglutinating activity. Succinylation solubilized the protein but did not render it susceptible to trypsin. The succinylated and carboxymethylated protein was rapidly cleaved by trypsin, fort-exchange chromatography of the native protein on DEAE-cellulose separated three fractions that showed similar amino acid composition. The observed ionic heterogeneity is presumably due mainly to differences in protein amide content rather than isolectin forms with different electric charge.

Introduction Sainfoin lectin is a seed-derived glycoprotein composed of two identical 26 kDa non-covalently associated subunits [1,2]. The peptide chain contains 236 amino-acid residues, including one cysteine, and is devoid of methionine. Alanine and threonine, respectively, occupy the nitrogen and carboxyl positions and asparagine 118 is the single oligosaccharide attachment site [3]. The protein is isolated by affinity chromatography on o-man* Present address: Department of Biological Chemistry, California College of Medicine, University of California, lrvine, CA 92717, U.S.A. ** To whom correspondence should be addressed. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

nose-Sepharose and shows multiple bands on isoelectric focusing, three or four bands at pH 9.5 and a single band at pH 4.3 on polyacrylamide gel electrophoresis and a single band on SDS-polyacrylamide gel electrophoresis. The protein is homogeneous as judged by affinity chromatography profiles, ultracentrifugation and gel filtration. Sainfoin lectin shows agglutination specificity for trypsinized cat erythrocytes. Initial determinations of carbohydrate specificity indicated a binding preference for D-mannose and D-glucose and their a-glycosidic derivatives [1]. This paper presents further characterization of sainfoin lectin in terms of binding constants for D-glucose, comparative carbohydrate inhibition of hemagglutination, aspects of molecular stability,

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metal content and ionic heterogeneity. The work was done as part of an interdisciplinary program directed toward improvement of sainfoin as a forage legume [4] and in association with primary structural studies of sainfoin lectin [3]. Materials and Methods

Chemicals. Common chemicals were analytical grade. Carbohydrates and DPCC Trypsin (Type XI) were purchased from Sigma (St. Louis, MO). Radioactive D-[U-lac]glucose and iodo[1-14C]acetic acid were purchased from ICN (Irvine, CA). Sainfoin lectin preparation. Lectin was isolated from ground sainfoin seeds by affinity chromatography on D-mannose Sepharose with minor modifications of the previous description [1]. The concentration of D-glucose in the initial extraction step was increased to 0.1 M and the ammonium sulfate fractionation step was omitted. Filtration through cheese-cloth and centrifugation were used to remove insoluble debris and the lectin was absorbed to the affinity matrix in batch fashion. D-Glucose included in the extraction buffer was removed by dialysis prior to affinity absorption. Lectin concentrations were determined using E1'~,,1 c m = 14.3. 280 nm Equilibrium dialysis. Dialysis was carried out at 22°C in 0.5 ml cells of a rotating microvolume dialyzer. Solutions of D-[14C]glucose (0.5 ml) and lectin (1.58 mg/ml, 0.5 ml) in pH 7.0 buffer (10 mM sodium phosphate, 150 mM NaC1, 3.8 mM sodium azide) were placed in opposite compartments of the dialysis cells. Sugar concentrations and specific radioactivity ranged from 0.12-10 mM and (1.22-4.5). 105 cpm/p~mol respectively. Dialysis was continued to equilibrium (72 h) and radioactivity was determined in duplicate 50 t~l aliquots from each compartment. Binding data were analyzed according to Scatchard [5]. Affinity electrophoresis. Sainfoin lectin was electrophoresed at pH 4.5 on O-glycosyl polyacrylamide gel rods according to procedures described by Hofej~i et al. [6]. The a-allyl glycosides of D-glucose and D-galactose were synthesized from allyl alcohol and the monosaccharide according Ho~ej~i and Kocourek [7]. Each product migrated as a single spot on thin-layer chromatography using butanone/ ethanol / water, 75 : 8 : 3 (v/v) as

solvent. Solutions used for preparation of the gels were: A, 60 ml 1 M KOH, 21.5 ml glacial acetic acid, 5 ml N , N , N ' , N ' - t e t r a m e t h y l ethylenediamine, 13.5 ml H 2 0 (pH 4.5): B, 37.5 g acrylamide, 1 g bisacrylamide, H 2 0 to 100 ml: C, 0.35 g ammonium persulfate, H 2 0 to 100 ml. For polymerization of 7.5% gels, 1 ml A, 2 ml B and 4 ml C were combined with O-glycosyl copolymer, crystalline D-glucose and H 2 0 to 10 ml. The amount of copolymer and D-glucose was adjusted to give the desired final concentration of immobilized and free D-glucose, respectively. Reservoir buffer (pH 5.0) was 3.12 g p-alanine, 0.8 ml glacial acetic acid, H 2 0 to 1 liter. Sample size was 20/~g protein per gel. The gels were electrophoresed at 5 mA/gel, 10°C for 3.5 h. Gels were stained with Coomassie blue. Two identical affinity gels were run for each concentration of immobilized and free sugar. Control gels contained either no immobilized sugar or D-galactose as immobilized sugar. Data were analyzed and plotted according to Hofejgi et al. [6]. lsoelectricfocusing. Protein samples (50/~g) were focused in 7.5% polyacrylamide gel rods (5 × 75 ram) in a pH 3-10 gradient according to Wrigley [8]. Disc-gel electrophoresis. Polyacrylamide disc-gel electrophoresis with or without sodium dodecyl sulfate was performed as previously described [1]. Metal analysis. Metals were determined by atomic absorption using a Varian 1200 instrument. Lyophilized protein (20 mg) that had been exhaustively dialyzed in deionized water was digested in 5 ml hot concentrated nitric acid followed by dilution to 25 ml with H:O. Amino-acid analysis. Protein hydrolysis was performed in 6 M HC1 at I10°C for 20 h in sealed evacuated tubes. Hydrolyzates were analyzed on a Beckman 120C instrument according to Spackman et al. [9]. Succinylation. Solid succinic anhydride, 50-fold molar excess over the total protein lysine content, was added at 22°C at five 10 rain intervals to protein (1 m g / m l ) dissolved in buffer (10 mM Tris-HCl/100 mM NaCI/100 mM D-glucose) at pH 8.0. The pH was held constant by addition of 100 mM NaOH. Reagents were removed by dialysis in H 2 0 and the extent of lysine modification was determined by trinitrobenzene sulfonic acid

239

analysis [10].

AIkylation. The single cysteine residue of sainfoin lectin was alkylated with a 3-fold molar excess of iodo[~aC]acetic acid (spec. act. 7.47. l0 s c p m / m m o l ) after the method of Thomas et al. [11]. 1 mM EDTA was included in the reaction solution. Excess reagents were removed by dialysis in H20. Hemagglutination assay. Agglutination tests were done in microtiter plates by mixing serial 2-fold dilutions of lectin (25 ~1) and 25 /.tl 2.5% (v/v) suspension of trypsinized cat erythrocytes (Colorado Serum Co., Denver, CO) in phosphatebuffered saline (10 mM potassium phosphate/150 mM NaCI (pH 7.2)) [12]. Agglutination patterns were visually determined after 30 rain at 22°C. Titer value was the reciprocal of the highest dilution showing agglutiriation. Carbohydrate inhibition of hemagglutination was determined by performing the assay in phosphate-buffered saline which contained carbohydrate at 2-fold decreasing concentrations ranging from 50-0.1 mM. Lectin solutions were adjusted to titer 32 and all determinations were done in duplicate. The carbohydrate concentration which reduced the titer by one agglutination well was considered to cause 50% inhibition. DEAE-cellullose chromatography. Sainfoin lectin, 32 mg in 4 ml pH 8.3 buffer (5 mM sodium phosphate/100 mM sucrose), was chromatographed on a 2.6 × 95 cm column of DEAE-cellulose. Elution was performed at 22 m l / h with pH 7.9 buffer (10 mM sodium phosphate/100 mM sucrose/3 mM sodium azide). Effluent fractions (2 ml) were monitored for A2s0. Results

A second quantitative measurement of D-glucose binding to sainfoin lectin was determined by affinity electrophoresis. The experiments were performed at pH 4.5 in an attempt to simplify results, since the protein migrates as a single electrophoretic band at this pH [1]. Fig. 2 shows that sainfoin lectin, when electrophoresed on O-glycosyl polyacrylamide gels, separated into two bands that were retarded by the affinity gel. A third band (not shown) representing about 10% of the sample travelled identically with non-interacting controls, showing it to be non-binding (denatured) protein. The faster and slower of the two retarded bands respectively accounted for approx. 60% and 30% of the total load. Dissociation constants toward free and immobilized D-glucose, K a and K~ respectively were 1.6 ___0.2 mM and 0.8 + 0.2 mM for the slower component and 2.0 + 0.1 mM and 3.3 _+ 1.0 mM for the faster component. These values represent the mean + standard deviation from three experiments. The relatively large difference in affinity toward immobilized o-glucose presumably accounted for separation of the two protein bands on the affinity gel. Relative dissociation constants of several D-glucose- and D-mannose-related carbohydrates with sainfoin lectin were approximated through determination of the concentration which inhibited hemagglutination by 50%. Table I lists the inhibitory strengths of the carbohydrates examined. DMannose and a-glycosidic derivatives of o-man-

oo 2

Nature of carbohydrate binding The carbohydrate binding characteristics of sainfoin lectin were examined by equilibrium dialysis, affinity electrophoresis and comparative carbohydrate inhibition of hemagglutination. The results of equilibrium dialysis with o-glucose are shown in Fig. 1. The dissociation constant and the number of binding sites per protein monomer were calculated to be 3.5 mM and 1.0, respectively. The sainfoin lectin dimer therefore contained two Dglucose binding sites per 52 kDa.

2 BOUND

GLUCOSE x IOSM

Fig. l. Equilibrium dialysis binding of D-glucose to sainfoin lectin at pH 7.2, 22°C. The slope shown was calculated by regression analysis with correlation coefficient of 0.87. Binding constant and number of sites were calculated according to Scatchard [5].

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nose and D-glucose were the strongest inhibitors and were effective at 0.4 raM. D-Mannose was about 8-fold more effective than D-glucose; however, 3-O-methyl-D-glucose was equal to D-mannose in inhibitory strength. Formation of derivatives of D-glucose at the 2-position had little effect, whereas 6-deoxy-D-glucose and 2-position derivatives of o-mannose were much poorer inhibitors. The a-anomeric forms of the methyl- and pnitrophenyl-D-glucosides were better inhibitors than the corresponding p-anomer. Raffinose, 6-deoxy-D-glucose and EDTA were ineffective at 100 m M concentration. It has been demonstrated previously that B-linked disaccharides such as cellobiose and lactose are non-inhibitory [1].

2 ..~

2.

~2

2

4.

C x 103M

Fig. 2. Affinity electrophoresis of sainfoin lectin at pH 4.5. L and L 0 are the distances travelled in the presence and absence of immobilized D-glucose and C is concentration of free D-glucose. The faster component is represented by • and the slower by A.

Metal content Analysis of three different lectin preparations gave the following metal composition expressed as tool metal per mol protein monomer (mean +_ S.D.): Ca 2+, 1.11 + 0.06; Mn 2+, 0.43 + 0.16; Mg 2+ 0.25 + 0.21. Thus, the protein contained a stoichiometric amount of Ca 2+ and variably lesser amounts of Mn 2+ and Mg 2+ Carboxymethylation Sainfoin lectin which had been reacted with

TABLE I INHIBITION OF SAINFOIN LECTIN H E M A G G L U T I N A T 1 O N : C A R B O H Y D R A T E C O N C E N T R A T I O N S H O W I N G 50% INHIBITION Concentration (mM) 0.4

0.8 1.6 3.1 6.2 12.5 25 50 100 > 100 " " No inhibition at 100 mM.

Inhibitor D-mannose, methyl a-D-mannopyranoside, 3-0methyl-D-glucopyranose, sucrose, maltose, melizitose p-nitrophenyl a-D-glucopyranoside, J3-D-fructose turanose, trehalose methyl oL-D-glucopyranoside, palatinose D-glucose, 2-deoxy-D-glucopyranose, 2-deoxy2-amino-D-glucopyranose, L-sorbose 2-deoxy-2-acetamido-D-glucopyranose, p-nitrophenyl/9D-glucopyranoside methyl/3-D-glucopyranoside, 2-deoxy-2acetamido-D-mannopyranose 2-deoxy-2-amino-o-mannopyranose raffinose, 6-deoxy-D-glucopyranose, EDTA

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labeled iodoacetic acid showed incorporation of 0.9 tool ]4C per mol protein monomer. Amino acid analysis gave 0.9 residues S-carboxymethylcysteine per protein monomer.

a

b

Stability Conformational stability of sainfoin lectin was examined by exposure of different protein samples to trypsin, heat, EDTA and succinic anhydride. Resistance to trypsin was shown by the following experiment. Lectin (0.3 m g / m l phosphate-buffered saline) was incubated with 10% (w/v) of active trypsin for 3 h at 37°C. No change in hemagglutination titer occurred during that period and the treated protein gave one band at M r 26 000 on SDS-polyacrylamide gel electrophoresis, identical to the molecular weight previously determined for untreated protein [1]. Preincubation for 15 min at pH 2.5 or pH 11.0 did not sensitize the protein toward trypsin. An analogous experiment involving lectin that was 90% succinylated gave identical results. When the protein was both S-carboxymethylated and succinylated, it was extensively digested by trypsin at 37°C. Subsequent treatment with carboxypeptidase B [13] showed that 10 of 12 arginine residues were exposed by the trypsin treatment. The effect of EDTA and heat on lectin activity was examined. Lectin (0.3 m g / m l phosphatebuffered saline) was heated at 55 ° for 3 h. The hemagglutination titer remained unchanged at 32 for 2 h and decreased to 16 at 3 h. When 10 mM EDTA was included in the buffer, and the same heat treatment was applied, 75% of hemagglutination activity was lost after 15 min and zero activity remained after 1 h incubation, in contrast to the finding that 100 mM EDTA at 22°C did not inhibit hemagglutination (Table I).

Ion-exchange chromatography Fig. 3 shows the behavior of sainfoin lectin toward DEAE-cellulose chromatography. The protein was separated into three fractions, the largest of which (I) was subsequently separated into two subfractions at pH 8.3 (subfractionation not shown). Disc-gel electrophoresis at pH 9.5 of fractions I, II and III gave single bands with fractions II and III and a double band with fraction I. The individual electrophoretic bands corresponded to

E

I 2,~0

II 3;0 EFFLUENT

~

3;0 VOLUME,ml

III

),410

Fig. 3. Elution profile of the ion-exchange chromatography of sainfoin lectin (SL) on a column (2.6 × 95 cm) of DEAE-cellulose at pH 7.9. Insets a and b show polyacrylamide gel electrophoresis and isoelectric focusing patterns of the three DEAE-cellulose fractions and unfractionated lectin.

portions of the banding pattern observed in unfractionated lectin (Fig. 3, inset a). Isoelectric focusing of each of the three DEAE-cellulose fractions produced multiple bands (Fig. 3, inset b) which generally corresponded to portions of the multiple banding pattern observed in unfractionated lectin. Each of the three DEAE-cellulose fractions showed agglutination activity toward cat erythrocytes. Amino-acid analysis of each fraction gave essentially identical compositions (data not shown) which were indistinguishable from that previously published for sainfoin lectin purified only by affinity chromatography [1]. We feel that these indications of ionic heterogeneity in the protein are most consistent with molecular forms differing in amide content as opposed to isolectin molecular forms. Although determination of the amino-acid sequence for sainfoin lectin [3], supports this view in that no charge replacements were found, evidence for at least one site of heterogeneity was observed and the possibility of electrophoretically distinct variants occurring in these samples exists.

242

Discussion

These experiments show that sainfoin lectin is a stable phytoagglutinin with primary carbohydrate specificity directed toward D-mannose and D-glucose and their a-glycosidic derivatives. As such, it is a member of the class of leguminous D-mannose- (D-glucose-) binding proteins composed (among others) of concanavalin A, lentil, pea and faba bean lectins. The carbohydrate binding character, metal content and other physicochemical properties of sainfoin lectin described here and previously [1] are similar to those properties generally descriptive of the D-mannose (D-glucose) binding family of lectins [14]. The amino-acid sequence of sainfoin lectin also exhibits primary structural homology with other sequenced leguminous lectins [3]. The binding strength of D-glucose with sainfoin lectin as estimated by affinity electrophoresis, hemagglutination inhibition and equilibrium dialysis gave reasonably consistent dissociation constants in the range 1-4 mM. This good agreement of the D-glucose-binding constants served to support the less precise equilibrium dialysis data. Repeated dialysis experiments produced comparably scattered data, the probable result of experimental limitations due to the relatively weak binding constant and limited solubility of the protein. Sainfoin lectin contains one carbohydrate binding site per subunit which means the active molecular species is bivalent. Related D-mannose- (D-glucose-) binding lectins are also bivalent, except for concanavalin A which is tetravalent at neutral pH [15]. The hemagglutination studies showed that sainfoin lectin had greatest specificity for D-mannose, methyl a-D-mannopyranoside and a-glucosyl saccharides. D-Glucose was about 8-times less inhibitory than D-mannose; however, 3-O-methyl-D-glucose was equal in inhibitor strength to D-mannose. A similar stimulation of binding strength by 3-0methylation of D-glucose is seen with lentil, pea and faba bean lectins, but not with concanavalin A. The effect of methylation at position 3 on inhibition by carbohydrate is ascribed to a stabilizing hydrophobic binding site on the responsive lectins [16]. The 2-hydroxyl group of D-mannose was important for inhibitory activity, whereas the corre-

sponding D-glucose position could be modified with little effect. Two D-mannose derivatives, 2amino-2-deoxy- and 2-acetamino-2-deoxy-D-mannose, were 1/250 and 1/62 as effective as D-mannose, whereas several 2-position D-glucose derivatives were equally effective as D-glucose (Table !). Pea lectin is similar in this regard [17]. The lack of inhibition by 6-deoxy-D-glucose confirmed the importance of the 6-hydroxyl group for binding at the lectin site. A similar dependence has been observed with concanavalin A and lentil lectin [14]. Raffinose, a trisaccharide containing non-reducing terminal D-galactose, also failed to bind sainfoin lectin, a result consistent with earlier observations [1]. Sainfoin lectin contained 1 mol Ca 2÷ per protein monomer and variable but lesser amounts of Mg 2+ and Mn 2+, the sum of which approximated one atom per mol of protein monomer. These results, particularly when viewed relative to other lectins, suggest that the protein contains two metal-binding sites, one highly specific for Ca 2+ and a second, less specific, site. The observation that EDTA destabilized the protein to heat denaturation yet had no effect on hemagglutination at 22°C supports the presence of a stabilizing high-affinity metal-binding site. The dependence of concanavalin A subunit structure and carbohydrate binding on occupied metal binding sites is well known [18]. Only Ca 2+ is required for saccharide binding to concanavalin A at neutral pH [19], suggesting that occupancy of the second metal ion site in sainfoin lectin may, by analogy, not be required for hemagglutinating activity. Nonstoichiometric amounts of metals have been observed in lentil [20] and pea [21] lectins and they show different susceptibility to sequestration by EDTA. Sainfoin lectin was relatively stable to trypsin cleavage and to heat denaturation in the absence of EDTA. The trypsin resistance persisted after exposure to extreme pH and after extensive succinylatilon of the molecule. Carbohydrate binding capacity of the protein was also retained, as shown by retention on D-mannose-Sepharose (data not shown). SDS gel electrophoresis confirmed the molecular stability toward trypsin and the absence of any 'natural' peptide bond cleavages similar to those associated with concanavalin A [22]. This

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result is consistent with previous work that showed the sainfoin lectin subunit to consist of a single polypeptide chain [1]. Previous work had indicated several electrophoretic forms of sainfoin lectin [1] and the heterogenity observed here is consistent with variable protein amide content and consequent variations in molecular charge. The DEAE-cellulose fractions travelled as single distinct protein bands during electrophoresis at pH 9.5; however, they exhibited multiple isoelectric focusing bands and each had amino-acid composition indistinguishable from that of the parent sainfoin lectin purified by affinity chromatography. Apparently the DEAE-cellulose column separated the protein into groups of similar but not identically charged molecules. Heterogeneity due to amide differences would be suppressed at pH values near 4, which may account for the single electrophoretic band at that pH [1]. The two o-glucose-binding species seen at pH 4.5 in affinity electrophoresis were not interpreted as isolectin forms because they are not present in the absence of immobilized sugar and all other attempts to produce different binding forms by D-glucose gradient elution from affinity chromatography columns were unsuccessful. The two bands might possibly be related to monomer and dimer forms of the lectin at pH 4.5 and their potentially different binding affinity with immobilized D-glucose [6]. Finally, the determination of the amino-acid sequence sainfoin lectin confirmed the absence of charged isolectin forms [3]. A similar situation involving apparent isolectin molecular forms which share a common amino-acid sequence has been observed with the o-mannose- (D-glucose-) binding lectin from Vicia cracca [23]. Among the Dmannose- (o-glucose-) binding lectins, isolectin forms have been demonstrated for the pea [24] and lentil [25] proteins. Whether differential amide content of sainfoin lectin is a natural or artifactually induced characteristic is unknown.

Acknowledgements This research was additionally supported by a grant from the United States Department of Agri-

culture (616-15-76) and is published as paper number J-1448 from the Montana Agricultural Experiment Station.

References 1 Hapner, K.D. and Robbins, J.E. (1979) Biochim. Biophys. Acta 580, 186-197 2 Namen, A.E. and Hapner, K.D. (1979) Biochim. Biophys. Acta 580, 198-209 3 Kouchalakos, R.N., Bates, O.J., Bradshaw, R.A. and Hapner, K.D. (1984) Biochemistry 23, 1825-1830 4 Ditterline, R.L. and Cooper, C.S. (1975) Mont. Agric. Exp. Stn. Bull. 681, 1-23 5 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51,660-672 6 Hofej~i, V., Tichfi, M. and Kocourek, J. (1977) Biochim. Biophys. Acta 499, 290-300 7 Hofej~i, V. and Kocourek, J. (1974) Methods Enzymol. 34, 361-367 8 Wrigley, C.W. (1971) Methods Enzymol. 22, 559-564 9 Spackman, D.H., Stein, W.H. and Moore, S. (1958) Anal. Chem. 30, 1190-1206 10 Habeeb, A.F.S.A. (1966) Anal. Biochem. 14, 328-336 11 Thomas, K.A., Silverman, R.E., Jeng, I., Baglan, N.C. and Bradshaw, R.A. (1981) J. Biol. Chem. 256, 9147-9155 12 Lis, H. and Sharon, N. (1973) Methods Enzymol. 28, 360-365 13 Ambler, R.P. (1972) Methods Enzymol. 25, 143-154 14 Goldstein, l.J. and Hayes, C.E. (1978) Adv. Carbohydr. Chem. Biochem. 35, 127-340 15 Kalb, A.J. and Lustig, A. (1968) Biochim. Biophys. Acta 168, 366-367 16 Allen, A.K., Desai, N.N. and Neuberger, A. (1976) Biochem. J. 155, 127-135 17 Van Wauwe, J.P., Loonteins, F.G. and De Bruyne, C.K. (1975) Biochim. Biophys. Acta 379, 456-461 18 Kalb, A.J. and Levitzki, A. (1968) Biochem. J. 109, 669-672 19 Christie, D.J., Alter, G.M. and Magnuson, J.A. (1978) Biochemistry 17, 4425-4430 20 Paulova, M., Ticha, M. and Entlicher, G. (1971) Biochim. Biophys. Acta 252, 388-395 21 Entlicher, G., Kostir, J.V. and Kocourek, J. (1970) Biochim. Biophys. Acta 221,272-281 22 Wang, J.L., Cunningham, B.A. and Edelman, G.M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 1130-1134 23 Baumann, C.M., Strosberg, A.D. and Rudiger, H. (1982) Eur. J. Biochem. 122, 105-110 24 Trowbridge, I.S. (1974) J. Biol. Chem. 249, 6004-6012 25 Howard, I.K., Sage, H.J., Stein, M.D., Young, N.M., Leon, M.A. and Dyckes, D.F. (1971) J. Biol. Chem. 246, 1590-1595