The use of streptavidin-biotinylglycans as a tool for characterization of oligosaccharide-binding specificity of lectin

ANALYTICAL

BIOCHEMISTRY

205,

17-82 (19%)

The Use of Streptavidin-Biotinylglycans as a Tool for Characterization of O ligosaccharide-Binding Specificity of Lectin’ Ming-Chuan

Shao

The Department

of Biochemistry,

Received January

23, 1992

Shanghai

Medical

University,

A new rapid and sensitive method for characterizing lectin specificity using streptavidin-biotinylglycans as a tool is presented. This assay is analogous to enzyme immunoassay and takes advantage of the strong, irreversible adsorption of streptavidin to the wells of the chambers of titer plates. A series of streptavidin-biotinylglycans was first coated on a microtiter plate, and then one of six lectins, concanavalin A, wheat germ agglutinin, Phaseolus vulgaris (red kidney bean) erythroagglutinin, Lens culinaris (lentil) agglutinin, Datura stramoniun agglutinin, or Sambucus nigra (elderberry bark) agglutinin coupled to horseradish peroxidase, was added. After incubation and thorough washing, only the lectin bound to a complementary glycan remained and could be detected and quantified by the peroxidase reaction. It was established that the lectins retained their oligosaccharide-binding specificities after coupling to the peroxidase, that the binding was inhibited by addition of the corresponding sugar inhibitors, and that the color intensity produced by the enzyme reaction is proportional to the amount of lectin-peroxidase bound to biotinylglycan complexed with streptavidin immobilized on the plate. As an example, it was found that the peroxidase-D. stramoniun agglutinin conjugate strongly bound biotinylglycans, GlcNAc,Man,-R, GalGlcNAc,Man,-R, and GlcNAc,-*Man,-R (R = GlcNAc,-[6-(biotinamido)hexanoyl]-Asn). As little as 10 pmollml of lectin was detected. W ith the growing availability of biotinylglycans, the method should represent a reliable and simple procedure for screening lectin-oligosaccharide recognition qualitatively and quantitatively. (? 1992 Academic Press, Inc.

There are several methods available for the characterization of lectin-oligosaccharide recognition. The most ’ Supported by a grant from the National People’s Republic of China. 0003.‘x97/92 $5.00 Copyright Q 1992 by Academic Press, All rights of reproduction in any form

Science Foundation

of

Shanghai

200032, People’s Republic of China

widely used method, hemagglutination, is semiquantitative and depends on the reproducible availability of a variety of erythrocytes. Some more accurate methods, such as affinity chromatography on Sepharose-bound lectin and affinity electrophoresis, require radiolabeled glycoproteins, glycopeptides, or oligosaccharides and are also quite time-consuming (l-6). Recently, neoglycoenzymes have been introduced to detect lectin-binding specificity by Gabius et al. (7), but some glycoenzymes are not easily available. Because of the lack of good testing systems, the oligosaccharide-binding specificity of some lectins has not been studied in detail. W e have developed a new reproducibly reliable, rapid, nonradioactive, and sensitive microassay for the characterization of lectin-oligosaccharide binding properties, using a series of streptavidin-biotinylglycans immobilized on a microtiter plate as probes. In this paper, the oligosaccharide-binding specificities of Con A,’ WGA, E-PHA, LCA, and SNA labeled with HRP were tested. It was found that the specificities of these lectins are consistent with those reported previously (l-6,8-11), and some new properties of DSA binding to glycans were found when the system was applied to this lectin. The data demonstrate that this new method represents a simple and reliable approach to the successful screening of the oligosaccharide-binding specificity of lectins. MATERIALS

AND

METHODS

Materials and reagents. Con A, WGA, LCA, DSA, SNA, streptavidin, BSA, ovalbumin, protease, ABTS,

* Abbreviations used: Con A, concanavalin A; WGA, wheat germ agglutinin; E-PHA, Phaseolus uulgaris erythroagglutinin; LCA, Lens culinaris (lentil) agglutinin; DSH, Datum stramoniun agglutinin; SNA, Sambucus nigra agglutinin; GlcNAc, N-acetylglucosamine; Man, mannose; Gal, galactose; Sia, sialic acid; HRP, horseradish peroxidase; ABTS, 2,2’-azinobis-(3-ethylbenzthiazolinesulfonic acid). 77

Inc. reserved.

78

MING-CHUAN

High Mannose

Hybrid

Type

Complex

Type

TVDe

MM’

(GnWM’ l-l’

M--R II’

A7 M’Gn*M*R M’

1 Gn

9 GlcNAc,.,Man,

I G-WC3

-R

10 Gal>GlcNAc2Man3

-R

FIG. 1. The structures of the 12 biotinylglycans used in this study which were tightly bound to streptavidin. The anomeric configuration and the linkage position are encoded as indicated. Unless otherwise noted, R = GlcNAc-[6-(biotinamido)hexanoyl]-Asn. The assignment of the specific structures to these glycan derivatives is based on previous extensive structural analyses of these compounds and should be correct (12-14).

and Sephadex G-25 were obtained from Sigma. Cation exchanger AG 50W (H+ form, 200-400 mesh) and BioGel P-4 (200-400 and -400 mesh) were from Bio-Rad. Sulfosuccinimidyl-6(biotinamido) hexanoate and Nhydroxysuccinimidobiotin were from Pierce Chemical Co. The 96-well microtiter plate was from Kontron. HRP (&,,,~A,,, = 3) was from Dong Feng Biochemical Reagent Factory, Shanghai Institute of Biochemistry, Academia Sinica. E-PHA was a gift from Dr. Sun Ce (Shanghai Institute of Biochemistry, Academia Sinica). All other reagents used in the study were commercially available and of special grade. of biotinylglycans. Man-R, Man,-R, Preparation GlcNAc,Man,-R, GlcNAc,Man,-R, GalGlcNAc,Man,R, and GlcNAc,_,Man,-R (R is either GlcNAc,-[6-(biotinamido)hexanoyl] -Asn or GlcNAc, -(biotinyl)-Asn) were prepared by biotinylation of their corresponding Asn-linked oligosaccharides derived from ovalbumin (12). GlcNAcMan-R, GlcNAcMan, -R, GlcNAc,Man, -R, Gal,GlcNAc,Man, -R, SiaGal,GlcNAc,Man, -R, and Sia,Gal,GlcNAc,Man,-R were biosynthesized with Man,-R as a primary substrate and with rat liver Golgi enzymes as catalysts by the procedure described by Shao et al. (12). All these compounds were further purified with a 1.2 X 200-cm Bio-Gel P-4 column (<400 mesh) (12). The purity of the products was more than 90% except for GlcNAc,_,Man,-R, which was a mixture of GlcNAc,Man,-R (30%) and GlcNAc,Man,-R (63%). Figure 1 shows the structures of all of the well-charac-

SHAO

terized glycans used (12-14). The concentrations of the biotinylglycans were microassayed by the procedure developed by Green (15) and modified by Shao et al. (12). Preparation of the lectins and streptavidin labeled covalently with HRP. Con A-HRP, WGA-HRP, EPHA-HRP, LCA-HRP, DSA-HRP, SNA-HRP, and streptavidin-HRP were prepared by the procedure developed by Wilson and Nakane (16) and modified by Guo and Guo (17). Briefly, 200 nmol HRP, dissolved in 0.75 ml of H,O, was reacted with 0.75 ml of 60 m M NaIO, (freshly prepared) at 4°C for 30 min, followed by addiglycol at room tion of 0.75 ml of 160 mM diethylene temperature for 30 min to terminate the oxidation. Fifty nanomoles of lectin subunits (the molar ratio of lectin:HRP was about 1:4), dissolved in 1.5 ml of H,O, was mixed with the above solution and dialyzed against 50 mM sodium carbonate buffer, pH 9.6, at 4°C overnight with gentle stirring. After the dialysis, 0.3 ml of 0.5% NaBH, was added to the dialyzed mixture and the reaction was allowed to proceed at 4°C for 2 h. An equal volume of saturated ammonium sulfate was added to precipitate the lectin-HRP conjugate, leaving unreacted HRP in the supernatant. The lectin-HRP was collected by centrifugation, desalted by dialysis against deionized water, and lyophilized for storage. The molar ratio of HRP to lectin in the conjugates prepared was 1.5-1.8. specificity of the The assay of oligosaccharide-binding lectins. The principle of the assay is shown in Fig. 2, and the standard procedure was as follows: streptavidin was dissolved in 50 mM sodium carbonate buffer, pH 9.6, containing 0.02% sodium azide (buffer A) at 30 pmol/ml (2 pg/ml) concentration. Each of the above 12 biotinylglycans were dissolved in buffer A at 150 pmol/

Polystyrene

surface

FIG. 2. The principle for determination of oligosaccharide-binding specificity of the lectins with the streptavidin-biotinylglycan system as a probe. The activity of HRP coupled covalently with lectin, which can specifically bind to streptavidin-biotinylglycans immobilized on a titerplate, was determined after interaction between the lectin and the glycan. S, substrate, ABTS; P, product, oxidized ABTS; and arm, aminohexanoyl group.

ASSAY

TO CHARACTERIZE

OLIGOSACCHARIDE

ml concentration. It should be noted that 1 mol of streptavidin can bind 4 mol of biotinylglycans; a molar ratio of streptavidin to biotinylglycan of 115 was used in order to ensure complete saturation of streptavidin by the biotinylglycans (13). A 50-~1 aliquot of streptavidin (0.1 pg, 1.5 pmol) was applied to each well of a microtiter plate, followed by the addition of 50 ~1 of a selected biotinylglycan (7.5 pmol) solution. The mixtures were incubated at 25°C overnight or for 3 h. The plate was rinsed three times with 50 mM sodium phosphate buffer, pH 7.4, containing 0.05% Tween 20 (buffer B). A 350-~1 aliquot of buffer A containing 1% bovine serum albumin was added to each well and the plate was incubated at 25°C for 1 h to coat any remaining sites on the plate. After washing three times with buffer B, 100 ~1 of lectin-HRP at about 40 pmol/ml concentration dissolved in 50 mM Tris buffer, pH 7.4, containing 150 mM NaCl, 1 mM MgCl,, 1 mM MnCl,, and 1 mM CaCl, was added and the plate was incubated at 25°C for 3 h. Finally, the plate was washed five times with buffer B, and then 100 ~1 of the enzyme substrate ABTS (0.3 mg/ml) dissolved in 50 mM citrate buffer, pH 5.0, containing 0.012% H,O, was added and incubated at 25°C for lo-30 min. The reaction was terminated by addition of 100 ~1 of 5% sodium dodecyl sulfate solution. The release of chromogen was measured by use of a microplate reader (EIA Reader, Kontron) at a wavelength of 405 nm. After the assay, the plate can be stored at -20°C for l-2 weeks without significant color loss. An alternative method, coating the wells with lectin and binding biotinylglycan and streptavidin-HRP, gave similar results, but generally much more lectin was required (the amounts varied for different lectins), and less HRP was bound in each positive well; the development of color generally required 2-3 h.

RESULTS

Some basic properties of the reaction of lectin-HRP conjugates with streptavidine-biotinylglycans are illustrated in Fig. 3. With a fixed amount of streptavidinebiotinylglycan in the well, the amount of lectin-HRP bound as a function of lectin-HRP added describes a typical saturation curve (Fig. 3A), in which the endpoint corresponds to the saturation of the available glycan sites. This conclusion is substantiated by the standard curve in Fig. 3B, showing that enzyme-catalyzed color production is linear with enzyme concentration well beyond the endpoint of the reaction, and that substrate exhaustion thus cannot be the reason for the saturation point. It is interesting to note that only about 1% of the added lectin-HRP is detected as bound lectin at any one concentration; it is not clear whether this means that only 1% of the lectin-HRP is active under the reaction conditions or whether it reflects some unique difference

BINDING

79

OF LECTIN

Con A-HRP added (pmol/well)

0

IO0

2OC

Con A-HRP added (fmol/well) FIG. 3. Some characteristics of the streptavidin-biotinylglycan and 1ectinHRP reaction. (A) Each well contained 3 pmol of streptavidin subunits saturated with Man,-R, and Con A-HRP was added in the amounts indicated and incubated for 3 h at 25°C. After incubation and washing according to the procedures described in the text, the lectin bound was determined by the standard peroxidase-catalyzed color production. The enzyme reaction was carried for 10 min. (Bl To assess the linear range of the color production, different amounts of free Con A-HRP were incubated with substrate ABTS under the same conditions as those used in A.

in the HRP-substrate reaction for the glycan-bound (Fig. 3A) and free lectin-HRP (Fig. 3B). It has been established that Con A binds to a series of glycan structures to various extents (2,3). Figure 4A shows a typical picture of binding of Con A-HRP to the 12 streptavidin-biotinylglycans coated on the plate. The level of Con A binding to the high mannose types of oligosaccharide Man-R and Man,-R and hybrid GlcNACMan,-R is highest. The binding intensity to the other three hybrids is decreased, consistent with previous findings that when GlcNAcMan,-R is bisected with a pl,4-linked N-acetylglucosamine residue to the P-linked mannose, the binding is weakened. The level of Con A binding to the complex type, GlcNAcMan-R, GlcNAc,Man,-R, is similar to that to GlcNAC,Man,-R. The compounds Gal,GlcNAc,Man,-R, SiaGal,GlcNAc,Man, -R, Sia,Gal,GlcNAc,Man, -R, and GlcNAc,_ ,Man,-R with a bisecting N-acetylglucosamine residue gave lower color with Con A, showing that the affinity for the lectin is very weak under these conditions (also see Table 1). As predicted, the addition of a-methylMan strongly inhibited the color formation (Fig. 4B).

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MING-CHUAN

I

2

3

4

5

6

SHAO

7

8

9

10

I2

11

(B), WGA-HRP (C), E-PHA-HRP (D), FIG. 4. A typical picture of bindings of Con A-HRP (row A), Con A-HRP plus o-methyl-Man LCA-HRP (E), DSA-HRP (F), and SNA-HRP (H) to the 12 streptavidin-biotinylglycans with an aminohexanoyl group and that of DSAHRP (G) to the 12 streptavidin-biotinylglycans without an amino hexanoyl group, coated to wells 1-12 of each column of a plate. The 12 streptavidin-biotinylglycans used are as follows: 1, Mane-R; 2, Man,&?; 3, GlcNAcMan,-R; 4, GlcNAc,Man,-R; 5, GlcNAc,Man,-R; 6, GalGlcNAc,Man,-R; 7, GlcNAcMan,-R; 8, GlcNAc,Man,-R; 9, GlcNAc,-,Man,-R; 10, Gal,GlcNAc,Man,-R; 11, SiaGal,GlcNAc,Man,-R; 12, Sia,Gal,GlcNAc,Man,-R (R = GlcNAc,-[6-(biotinamido)hexanoyl]-Asn or R = GlcNAc-biotinyl-Asn). The assays were carried out separately for each row according to the standard procedure described under Materials and Methods. The concentrations of all of the biotinylglycans used here were the same at 150 pmol/ml (equal to 7.5 pmol added for each well); only their structures varied. The inhibitor for Con A sugar binding, 100 mM a-methylmannoside dissolved in Con A-HRP solution, was added to each well of the plate (row B). The enzyme reactions were carried out for SNA for 10 min; for Con A, E-PHA, and LCA for 15 min; and for WGA and DSA for 30 min. Each well ofthe plate contained 400 ~1 of enzyme reaction solution from a combination of two experiments of a given lectin-glycan reaction in order to get better contrast for the photograph.

This documents the key role of the interaction between Con A-HRP and oligosaccharides in the assay. The binding specificity of WGA-Sepharose for different oligosaccharides is well-documented. Strong interaction requires the presence of the bisecting p1,4-Nacetylglucosamine residue linked to the P-linked mannose residue in the glycans (8). The results obtained from interaction between WGA-HRP and streptavidin-biotinylglycans are in agreement with this requirement. As shown in Fig. 4C and Table 1 the strongest binding intensity of WGA-HRP was observed with GlcNAc,Man,-R and was also strong with GlcNAc,Man,-R and GalGlcNAc,Man,-R, all of which are derived from ovalbumin and thus contain the bisecting N-acetylglucosamine residue. GlcNAcMan,-R and GlcNAc,_,Man,-R bound to WGA-HRP with lower affinity. The above binding could be significantly reduced by the addition of 1% N-acetylglucosamine, an inhibitor of WGA-glycan interaction (data not shown). In addition, no binding of WGA-HRP to sialylated compounds, SiaGal,GlcNAc,Man, -R and Sia,Gal,GlcNAc,Man, -R,

was found. The result is also consistent with previous observations that the presence of several sialyloligosaccharides on a peptide backbone is necessary for WGA binding to sialic acid residues. The binding specificity of E-PHA-HRP and LCAHRP to the 12 biotinylglycans was tested in the same way. Figures 4D and 4E show that none of the 12 oligosaccharides was found to interact with E-PHA-HRP and LCA-HRP. Because E-PHA and LCA specifically require the following sugar chain structures for their binding, respectively (9,10), the absence of any binding was expected and provides a useful negative control: E-PHA GtGn Y- GneM+Gn+GnMf I -Gn

LCA ;t -Gn Mf 4 -Gn

F-3 MeGntGn-

Using the method described here some new properties of DSA binding to glycans were found. The binding of

ASSAY

TO CHARACTERIZE

OLIGOSACCHARIDE TABLE

BINDING

81

OF LECTIN

1

The Summary of Binding Intensities of the Lectins Coupled with HRP to Streptavidin-Biotinylglycans” Binding intensity of lectin (O.D. at 405 nm)* Biotinylglycans High mannose Man,-R Man,-R Hybrid GlcNAcMan,-R GlcNAc,Man,-R GlcNAc,Man,-R GalGlcNAc,,Man,-R Complex GlcNAcMan,-R GlcNAc,Man,-R GlcNAc,-,Man,-R Gal,GlcNAc,Man,-R SiaGal,GlcNAc,Man,-R Sia,Gal,GlcNAc,Man,-R

Con A

WGA

DSA

SNA

(n = 6)

(n = 8)

(n = 6)

(n = 8)

0.68 f 0.07 0.55 AZ0.08

NBd NB

NB NB

NB NB

* i ii

0.54 0.46 0.34 0.31

k f f +

0.10 0.05 0.03 0.04

0.09 0.42 0.28 0.24

0.07 0.06 0.05 0.04

NB NB 0.24 +- 0.03 0.27 f 0.04

NB NB NB NB

0.47 0.39 0.09 0.20 0.16 0.16

f f -t * ? +

0.08 0.05 0.04 0.05 0.04 0.04

NB NB 0.04 -+ 0.02 NB NB NB

NB NB 0.17 t 0.03 NB NB NB

NB NB NB NB 0.40 k 0.06 0.37 -+ 0.07

a The binding of Con A-HRP + O-methyl-Man, E-PHA-HRP, and LCA-HRP to the 12 biotinylglycans with an amino hexanoyl group and the binding of DSA-HRP to the 12 biotinylglycans without an amino hexanoyl group were all negative and are not shown. b Values (minus blank, no biotinylglycan added to the well) are means i SE from the indicated number (n) of experiments under the same conditions. ’ R = GlcNAc,-[6-(biotinamido)hexanoyl]-Asn. d No binding (binding intensity, O.D. < 0.02).

DSA to oligosaccharides has been reported to require unsubstituted sugar chains consisting of a Gal Pl-4GlcNAc ol-G(Ga1 fll-4GlcNAc /31-2)Man group (19). Such structures are not present among the 12 biotinylglycans. But it was found that DSA-HRP did bind biotinylglycans, GlcNAc,Man,-R, GalGlcNAc,Man,R, and GlcNAc-,Man,-R (Fig. 4F and Table 1). SNA, a recently discovered plant lectin, recognizes the terminal Sia cu2-6Gal sequence (11). This conclusion was confirmed by this simple assay. It is clearly shown in Fig. 4H and Table 1 that in the 12 streptavidin-biotinylglycans immobilized on the plate only the 2 compounds containing terminal Sia (r2-6Gal sequence, SiaGal,GlcNAc,Man,-R and Sia,Gal,GlcNAc,Man,-R, reacted positively with SNA-HRP. Table 1 shows a summary of the values for lectin binding intensities by activities of HRP coupled with Con A, WGA, DSA, and SNA bound to the 12 streptavidin-biotinylglycans coated on the plate. Based on all the above data it can be concluded that the sugar-binding specificities of a given lectin can be conveniently screened both qualitatively and quantitatively by using the streptavidin-biotinylglycans as probes, and that the error of this method usually is less than +20%. In the above experiments all the biotinylglycans contained an aminohexanoyl group inserted between biotin and the glycan. When this group was removed from these compounds, it was found that Con A could still recognize the same glycan structures, but the binding

intensity was reduced to various extents from 5 to 90% for the different biotinylglycans (data not shown), and the binding of DSA-HRP to biotinylglycans, GlcNAc,-R Man,-R, GalGlcNAc,Man,-R, and GlcNAc,_,Man, disappeared (Fig. 4G). This suggests that the existence of the aminohexanoyl group in the complexes moves the glycan far away from the streptavidin surface and makes the glycan more flexible and accessible to the lectin (11-13). DISCUSSION

It has been well documented by others that lectins such as Con A, WGA, E-PHA, LCA, DSA, and SNA can be coupled to Sepharose and still retain their biological activity and remain capable of binding specific oligosaccharides (1). Similar conclusions can be drawn for the coupling of these lectins to HRP used in this work. The system introduced in this study compares favorably to the methods previously reported (2-7). It is quantitative under carefully defined conditions and within a relatively narrow range of reactant concentrations, since the binding intensity is expressed as the activity of HRP coupled covalently with lectin, rather than as a semiquantitative titer used in hemagglutination. The oligosaccharide structures displayed on natural glycoproteins are often heterogeneous, however. Here each oligosaccharide bound to streptavidin represents a single, well-characterized structure; thus the fine oligosaccharide-binding specificity differences be-

82

MING-CHUAN

tween lectins may be detected precisely and reproducibly. All the Asn-linked oligosaccharides are easily biotinylated and bound tightly to streptavidin (ll-13), allowing numerous streptavidin-biotinylglycans to be readily formed and used for the study of lectin recognition properties. The assay is very sensitive; as little as 10 pmol/ml lectin can be detected. Because of the extreme sensitivity of the assay, however, small methodological variations from run to run can yield changes in color yield from a given lectin-HRP. In particular, when the lectin activity in the conjugate decreases, high background and consequently poor signal-to-noise ratios occur. It should always be kept in mind that different lectinHRPs have different stabilities and that it is very important to carefully keep the lectin activity as high as possible. In addition, this system may be used to detect and characterize unknown oligosaccharide structures in a glycoprotein when the Asn-oligosaccharides are prepared by digestion of the glycoprotein by proteases, followed by biotinylation. It also can be used for the determination of the activity of some glycotransferases and glycosidases. These investigations are underway in this laboratory. ACKNOWLEDGMENTS I am grateful to Dr. Finn Wold for the help in preparing this manuscript. I am also grateful to Dr. Ke-Yi Wang and Dr. Hui-Li Chen for many good suggestions and discussions.

REFERENCES 1. Lis, H., and Sharon, H. (1986) Annu. Reu. Biochem. 55, 35-67. 2. Baenzinger, J. U., and Fiete, D. (1979) J. Biol. Chem. 254, 2400-

2407.

SHAO

3. Brewer, C. F., and Bhattacharyya,

L. (1986) J. Biol. Chem. 261, 7306-7311. 4. Cummings, R. D., and Kornfeld, S. (1982) J. Biol. Chem. 257, 11230-11234. 5. Lesniak, A. P., and Liu, E. H. (1984) Anal. Biochem. 142, 140-

147. 6. Horejsi, V. (1981) Anal. Biochem. 112, 1-8. 7. Gabius, S., Hellmann, K.-P., Hellmann, T., Brinck, U., and Gabius, H.-J. (1989) Anal. Biochem. 182, 447-451. 8. Yamamoto, K., Tsuji, T., Matsumoto, I., and Osawa, T. (1981) Biochemistry 20, 5894-5899. 9. Yamashita, K., Hotoi, I., and Kobata, A. (1983) J. Biol. Chem.

258,14753-14755. 10. Kornfeld, K., Reitman, Chem. 256,6633-6640.

M. L., and Kornfeld,

R. (1981) J. Biol.

11. Shibuya, N., Goldstein, J. I., Broekaert, W. F., Nsimba-Lubaki, M., Peeters, B., and Peumans, W. J. (1987) J. Biol. Chem. 262, 1596-1601. 12. Shao, M. C., Chen, L. M., and Wold, F. (1989) in Methods in Enzymology (Wilchek, M., and Bayer, E. A., Eds.), Vol. 184, pp. 653-659, Academic Press, San Diego. 13. Shao, M. C., Chin, C. C., Caprioli, Biol. Chem. 262, 2973-2979.

R. M., and Wold, F. (1987) J.

14. Shao, M. C., and Wold, F. (1989) J. Biol. Chem. 264,6245-6251.

15. Green, N. M. (1965) Biochem. J. 94, 23C. 16. Wilson, M. B., and Nakane, P. K. (1978) in Immunofluorescence and Related Staining Techniques, (Knapp, W., Holubar, K., and Wick, G., Eds.), pp. 215-224, Elsevier/North-Holland Biomedical Press, Amsterdam. 17. Guo, C. Q., and Guo, X. Q. (1983) Shanghai 100.

18. Bhavanandan, 4000-4008.

Immunol.

J. 3, 97-

V. P., and Katlic, A. W. (1979) J. Biol. Chem. 254,

19. Yamashita, K., Totani, K., Ohkura, T., Takasaki, S., Goldstein, I. J., and Kobata, A. (1987) J. Biol. Chem. 262, 1602-1607.