Brain Research, 83 (1975) 337-348 © Elsevier ScientificPublishingCompany,Amsterdam- Printed in The Netherlands

337

SYNAPTOSOMAL PLASMA MEMBRANE GLYCOPROTEINS: FRACTIONATION BY AFFINITY CHROMATOGRAPHY ON CONCANAVALIN A

J. P. ZANETTA*, I. G. MORGAN**,*** AND G. GOMBOS*** Centre de Neurochimie du CNRS, 11 rue Humann, 67085 Strasbourg (France) (Accepted September 13th, 1974)

SUMMARY

Synaptosomal plasma membrane glycoproteins were solubilized in 0.08~o sodium dodecyl sulfate (SDS) and separated by affinity chromatography on concanavalin A-Sepharose. Three fractions were obtained. Fraction CO (unadsorbed) contained 63 ~ of the protein, but only 23 ~o of the sugar and was rich in fucose, galactose and N-acetyl-neuraminic acid (NANA) relative to the other sugars. Many proteins were detected in this fraction by polyacrylamide gel electrophoresis, but only one band stained well for carbohydrate. Fraction CR (retarded) contained glycoproteins which reacted weakly with concanavalin A and stained poorly with periodic acid-Schiff reagent (PAS). There was no enrichment in total sugar/mg protein relative to the original fraction, but there was a marked enrichment in N-acetyl-glucosamine and NANA relative to the other sugars. The protein profile of this fraction was complex, but only one major PASpositive band was detected. However, most, if not all, ~he proteins seemed to be weakly PAS-positive. Fraction C1 (adsorbed) was markedly enriched (5-fold) in sugar/mg protein, particularly in mannose and N-acetyl-glucosamine. It had a relatively simple protein profile, and most of the protein bands stained well with the PAS reaction. Glucose was detected in the initial fraction, and in all the subfractions, but it could not be shown definitely to be either a contaminant or an intrinsic constituent of synaptosomal plasma membrane (SPM) glycoproteins. Minor sugars, if present, could, at most, account for less than 0.25 ~ of the carbohydrate.

* Attach6 de Recherche au CNRS. Present address: Department of BehaviouralBiology,Research School of BiologicalSciences. Australian National University, Canberra, A.C.T. 2600, Australia. *** Charg6s de Recherche au CNRS. **

338

J.P. ZANETTAet al.

1N T R O D U C T I O N

Synaptosomal plasma membranes (SPM), like other plasma membranes, contain concanavalin A (Con A) 'receptors'. In fact preincubation of synaptosomes with Con A modifies their electrophoretic mobility5 and Con A-ferritin binds to mobile 'receptors' on the synaptosomal surfacO 9, as well as to 'receptors' in synaptic clefts 3. Affinity chromatography on different lectins has been used to separate membrane-bound glycoproteins from different sources a,2,1',13 including brain z7. However, in most cases, the solubilization procedure did not necessarily monomerize the membrane glycoproteins. We have used sodium dodecyl sulfate (SDS) which, in spite of the claims of Katzmann16, monomerizes proteins if the formation of S-S bridges is avoided. This paper reports on the fractionation of SPM glycoproteins into three classes based on their ability to interact with Con A, as a step preliminary to the isolation of individual glycoproteins. MATERIAL AND METHODS

Chemicals Iodoacetamide was obtained from Sigma (St. Louis, Mo., U.S.A.), sodium dodecyl sulfate (SDS) from Roth (Karlsruhe, G.F.R.), a-methyl-glucoside (aMG) from Schuchardt (Mfinchen, G.F.R.), sodium periodate from Merck (Darmstadt, G.F.R.), Con A-Sepharose from Pharmacia (Uppsala, Sweden) and Coomassie Brilliant Blue R from Gurr (London, G.B.). All other chemicals were analytical grade except those used for GLC analyses which were the purest available aa,34. Apparatus Gas-liquid chromatographic (GLC) analysis was performed using a Varian Aerograph 2100 apparatus equipped with 4 columns and 4 flame-ionization detectors. Chromatograms were recorded on two Varian Aerograph A-25 dual channel recorders. Peak areas were determined either by planimetry or using a Vidar 6300 digital integrator. Methods Synaptosomal plasma membranes (SPM) from adult rat brain were prepared using the method of Morgan et aL zl, except that the final centrifugation of the SPM at 11,000 × g for 20 rain was omitted in order to increase yields. Thus the SPM fractions were slightly contaminated with myelin, which could introduce a glycoprotein of a molecular weight of around 100,000 as a minor component 28. Delipidation and solubilization procedure SPM were suspended in a small volume of water, and lipids were removed by

CoN-A POSITIVESPM GLYCOPROTEINS

339

two cycles of extraction with methanol-chloroform (1:2 v/v) and (2:1 v/v). The lipidfree pellet was washed with methanol, then with water, and finally with 10~o (v/v) 2-mercaptoethanol saturated with ethylene diamine tetraacetic acid (EDTA). The pellet was dissolved at room temperature in a small volume (1 ml/10 mg proteins) of a 4 ~o SDS solution containing 1 ~o 2-mercaptoethanol and brought to pH 8.0 with 1 M sodium hydroxide. Solid iodoacetamide, in 100-fold excess over that necessary to block the 2-mercaptoethanol, was added and the reaction was allowed to proceed for 6 h in the dark, at room temperature, under nitrogen, with gentle stirring. The solution was dialyzed for 48 h against 49 vol. of distilled water, then for 24 h against a large volume of 0.08 ~o SDS solution. The solution was centrifuged for 2 h at 30,000 × g and the pellet, which did not contain glycoprotein, was discarded. The dialysis steps eliminated excess iodoacetamide, and were calculated to decrease the SDS concentration to 0.08 ~o while maintaining the protein in solution. A low SDS concentration was necessary since at concentrations above 0.1 ~o it inactivated the lectins11.

Affinity chromatography on coneanavalin A bound to Sepharose Con A-Sepharose gel was exhaustively washed with pH 3.5 and pH 9.0 buffers, then with 0.08 ~o SDS. Large amounts of non-covalently bound Con A were eliminated by these steps. Before chromatography the gel was washed with water, then with 20 mM Tris-HC1 (pH 7.2). A mixture of CaCI~, MgCI2 and MnCI2 (0.025 M each) was added and the gel was kept for at least 2 h at room temperature. After exhaustive washing with 20 mM Tris-HC1 (pH 7.2), the gel was equilibrated with 0.08 ~ SDS, 20 mM Tris-HC1 (pH 6.7). All the affinity chromatography procedure was performed in this solution. The divalent cations normally used to stabilize Con A 14 had to be omitted from the SDS solutions, since they precipitated both SDS and the membrane proteins u. NaC1, often added to reduce non-specific adsorption, could not be used for the same reason. Proteins bound to the Con A gel were eluted with 0.25 M a-methyl-glucoside in 0.08 ~ SDS, 20 mM Tris-HCl (pH 6.7). One centimeter diameter columns were used. The height of the columns depended on the amount of protein applied (approximately 0.1 cm gel/mg protein). The flow rate was regulated in such a way that the sample remained for at least 2 h in the column. The volume of samples applied to the column was always less than 1.5 column volume. Elution was continuously monitored at 280 nm, with a Gilson spectrochrom. Quantitative precipitation and redissolution of proteins Fractions isolated by affinity chromatography were precipitated by adding 4 voi. methanol and 1.5 vol. ethyl acetate. The mixture was allowed to stand for 16 h at --20 °C. The precipitate was centrifuged for 30 min at 30,000 × g, and the pellet was washed with methanol. After drying under a stream of nitrogen, the pellet was solubilized in a small volume of 4 % SDS, and dialyzed against 49 vol. of water for 2 days at room temperature, then against a large volume of 0.07 % SDS. The low

340

J.P. ZANETTAel al.

concentration of SDS is necessary to obtain sharp electrophoretic protein profiles after precipitation and redissolution in SDS. Electrophoreses A discontinuous polyacrylamide gel electrophoresis systema2 was used. The acrylamide concentration in the gel was 12 ~ and all electrophoreses were performed in the presence of 0.1 ~ SDS. Proteins were stained overnight with 0.015 ~ Coomassie Brilliant Blue (CBB) in mixture A (acetic acid-methanol-water, 1:4:5 v/v/v) and destained by diffusion in the same mixture. Protein bound carbohydrates were stained by a periodic acid-Schiff (PAS) reaction as follows. (1) Gels were left overnight in a large volume (approximately 800 ml/gel) of mixture A to fix proteins and to wash out the bulk of the SDS and the sucrose added to the sample (possible sources of non-specific staining). (2) All the following steps were carried out in the dark, at room temperature. The gels were transferred to freshly prepared 2 ~ sodium periodate solution in mixture A and left for 6 h. Preliminary experiments showed that: (a) controlled pH was not necessary, and (b) in agreement with Kapitany and Zebrowski 15, concentrations of periodate between 1 and 2 ~ gave optimum staining without excessive background. (3) The gels were washed in mixture A for 2 days (10 changes) then placed overnight in a fuschin solution prepared according to Segrest and Jackson 25. At this step faintly stained bands were seen. (4) The intensity of the bands was increased several fold by immersing the gels in 50 Y/ootechnical methanol in tap water. After a few changes, the gels became a deep uniform purple color. The background color was slowly removed by diffusion in solution A, and intensely stained bands were revealed. The CBB and PAS stained gels were allowed to swell overnight to the same dimensions in 3 ~ acetic acid in water. Analytical methods Proteins were determined by the method of Lowry et al. 17. Sugars were determined using a method which involved methanolysis and separation of the trifluoroacetate derivatives 33. However, since SDS was strongly bound to proteins and could interfere with methanolysis and GLC analyses, the following procedure was used.

TABLE I DISTRIBUTION OF

SPM

PROTEIN IN THE VARIOUS SOLUTIONS USED DURING SOLUBILIZATION

Total membrane protein Water-soluble EDTA + 2-mercaptoethanol soluble Soluble in 0.08 ~ SDS Insoluble in 0.08 ~ SDS

SPM protein (rag)

% of total protein

367.9 1.3 2.4 362.0 2.2

100 0.35 0.65 98.1 0.60

C o N - A POSITIVE S P M GLYCOPROTEINS

341 SPM C1

-2.0 SPM

SPM CR

CO

A

-1.0

0.25M =m-Gluc

Sample

~6o

200

360

,~

5~o ELUTION VOLUME(ml)

Fig. 1. Affinitychromatographyof proteins from rat brain SPM on concanavalinA bound to Sepharose-4B in the presence of 0.08% SDS-20 mM Tris-HCI (pH 6.7). Column:60 cm × 1 cm; sample: 360 mg protein in 90 ml; flowrate: 0.5 ml/min.

As previously described, 100--300 #g of protein was precipitated. The pellet was extracted with a small volume (2 ml) of a methanol-chloroform mixture, then washed with methanol. The internal standard (mesoinositol) was added and the dried sample was submitted to methanolysis. The remaining trace amounts of SDS were eliminated by three extractions with hexane85. The suspension of protein in methanol was centrifuged, and the pellet washed with anhydrous methanol. The sugars were determined on the methanol extract. The sugar-free protein pellet was hydrolyzed (24 h, 115 °C, 6 M HCI) and the amino acid composition was determined by GLC 34 in the presence of pipecolic acid as internal standard. In expressing the composition of the samples analyzed, the protein content was calculated from the sum of the amino acids determined by GLC, which agreed with the protein determined by the method of Lowry

et al. 17. RESULTS

Solubilization and analysis of proteins from synaptosomal plasma membranes Using the procedure described, most of the proteins could be solubilized in 0.08 ~o SDS (Table I). The alkylation procedure avoids aggregation, due to formation of S-S bonds, giving a stable solution, even at high protein concentration (5-10 mg/ml). The washing before SDS solubilization extracted negligible amounts of proteins (Table I), less than in brain microsomal fractions11. Similarly the 0.08 ~o SDS insoluble residue contained very little protein (Table I). The sugar composition of the 0.08 ~o SDS soluble fraction was similar to that previously reported 3° for SPM (Table III). Only peaks corresponding to N-acetyl-

342

.J. i,. ZANE'IIA CI re,/.

TABLE I1 DISTRIBUTION OF PROTEIN AND CARBOHYDRATE IN THE FRACTIONS OBTAINED BY AFFINI'FY C H R O M A I O ( i RAPHY OF S P M

PROTEINS ON C O N A - S E P H A R O S E

Weight ~ of total protein *Weight ~ of total sugar *pg carbohydrate mg protein

Total

CO

CR

100 100

63 23

24 26

28.0

79.3

65.9

CI

YieM

11 50

98 99 o/,,

332.0

* Calculated from analytical results. All carbohydrates were taken into account.

neuraminic acid (NANA), fucose, N-acetyl-glucosamine, N-acetyl-galactosamine, mannose, galactose and glucose were detected in the GLC tracings of the solubilized glycoproteins. Other monosaccharides, such as xylose, rhamnose, arabinose and N-acetylmannosamine, which have been described by others 4 in the central nervous system, could at most account for 0.25 ~ of the sugars, and thus we are not able to establish their presence in the synaptosomal plasma membrane. Affinity chromatography on Con A-Sepharose in the presence of SDS As previously described, affinity chromatography on insolubilized lectins11 was possible in low concentrations of SDS. When SPM proteins were chromatographed on Con A-Sepharose in the presence of SDS, three peaks were obtained (Fig. 1). One fraction was eluted in the void volume (CO), one fraction (CR) was retarded but still eluted with the buffer, and one fraction (C1) was only eluted with a-methyl-glucoside.

TABLE III MOLAR RATIOS OF MONOSACCHARIDES RELATIVE TO N-ACETYL-GLUCOSAMINE IN THE FRACTIONS OBTAINED BY AFFINITY CHROMATOGRAPHY OF

Fucose Galactose Mannose Glucose N-acetyl-glucosamine N-acetyl-galactosamine N-acetyl-neuraminicacid Total

SPM

PROTEINS

SPM*

0.08 % S D S soluble

CO

0.22 0.34 0.91 n.d. 1.00 0.19 0.48

0.29 (5.27) 0.43 (1.35) 0.59(11.89) 0.91 (3.15) 1.09 (21.90) 0.66 (2.26) 0.76(15.07) 1.84 (6.28) 1.00 (24.52) 1.00 (4.17) 0 . 1 2 ( 2 . 9 6 ) 0.13 (0.56) 0.49(16.96) 0.77 (4.25) (100) (23.12)

CR

0.22 0.30 0.29 0.29 1.00 0.04 0.48

CI

(1.74) (2.74) (2.55) (2.52) (10.80) (0.48) (7.20) (25.50)

* According to Vincendon et al. ao for pure synaptosomal plasma membranes. Figures in brackets are the weight percent of the total carbohydrate in each fraction. n.d.: not determined.

0.31 0.52 1.45 0.45 1.00 0.12 0.31

(3.33) (5.85) (16.41) (5.06) (13.85) (1.62) (6.04) (50.33)

CoN-A POSITIVESPM GLYCOPROTEINS

343

TABLE IV AMINO ACID COMPOSITION (MOLE/100 MOLES) OF THE FRACTIONS ISOLATED BY AFFINITY CHROMATOGRAPHY ON INSOLUBILIZED CON A

Ala Arg Asp Cys* Glu Gly His Hy-Lys ** Hy-Pro** Ile Leu Lys Met Phe Pro Ser Thr Tyr Val

Total

CO

CR

CI

8.87 3.96 10.05 0.90 11.59 7.71 0.81 1.08 0.27 3.68 10.59 4.67 0.90 5.20 4.31 8.45 7.00 5.29 4.66

8.78 4.80 10.24 traces 11.14 8.13 traces 1.06 0.49 3.41 9.43 5.28 0.81 5.20 4.31 7.72 6.99 7.40 4.55

8.10 3.31 8.47 1.23 11.66 5.89 4.66 4.54 traces 2.82 8.10 5.15 1.84 6.01 3.07 9.33 6.50 5.52 4.55

7.56 4.46 11.41 traces 12.40 7.31 traces 1.48 0.25 2.73 9.18 4.84 1.24 5.95 4.46 8.68 8.55 4.59 4.84

* Determined as S-carboxymethyl-cysteine. ** Tentative identification.

To ensure that the fractions were really distinct, and that adsorption to Con A was complete, each fraction was precipitated, redissolved and repassed separately on identical columns. Each fraction migrated as during the first chromatography. It is important to note that the C R fraction was always retarded. However, if the affinity chromatography was performed in unbuffered 0.08 ~o SDS, which has a p H of 5.3-5.4 or in 0.08 ~o SDS-20 m M T r i s - H C l (pH 6.7) containing 0.25 M a-methyl-glucoside, the C R fraction disappeared and was eluted in the void volume of the column. These results suggest that the C R fraction interacts weakly with Con A in the optimum p H range for binding to Con A. The analytical results (see below) also favor this interpretation. Quantitative results

Most of the protein was found in the CO fraction which contained only 23 o f the total carbohydrate (Tables II and III). On the other hand, the C1 fraction which contained only 11 ~/o of the protein contained about 50 ~o o f the sugar, which corresponded to a 5-fold enrichment compared to the original fraction. Sugars were not concentrated in the C R fraction. It should be noted that the yields (with regard both to protein and carbohydrate) were quantitative, indicating that elution from the affinity column was complete. The carbohydrate compositions of the fractions eluted from the Con A column

344

CH

J. P. ZANETTA c l o[.

I~AS

~_.._v___/

GliB "-

PAS ~__.__..I

C1

CDB "~

PAS v

CR

~

PAS

J

CO

Fig. 2. Polyacrylamide gel electrophoresis (l 2 % acrylamide) in the presence of 0.1% SDS of fractions isolated from rat brain SPM by affinity chromatography on insolubilized Con A. Proteins (CBB) and glycoproteins (PAS).

were significantly different (see Table III). The C1 fraction was very rich in mannose (75 ~ of the total mannose) and N-acetyl-glucosamine. In addition this fraction contained other monosaccharides (fucose, glucose, galactose, N-acetyl-galactosamine and N-acetyl-neuraminic acid) in appreciable quantities. The CR fraction was particularly rich in N-acetyl-glucosamine, which could explain its chromatographic behavior since Con A weakly binds N-acetyl-glucosamine 9. In view of the relatively high N-acetyl-glucosamine-mannose ratio of this fraction, the presence of mucopolysaccharides cannot be excluded, although under the standard conditions of methanolysis, very little N-acetyl-glucosamine was released from commercial standard mucopolysaccharides. The CO fraction was particulacly rich in galactose, NANA, and fucose, but also contained appreciable quantities of mannose (10~ of the total mannose) and N-acetyl-glucosamine. The amino acid compositions of these fractions (Table IV) were different, but did not have great significance since these fractions were extremely heterogeneous.

Electrophoretic profiles of the glycoprotein fractions isolated from SPM by affinity ehromatography on insolubilized Con A As previously described 22,al, the protein profile of SPM is very complex. The

C o N - A POSITIVE

SPM GLYCOPROTEINS

345

glycoprotein profiles were similar to those previously reported 7,22,al, although more minor bands were seen due to the improved staining method. The CO and CR fraction had one major glycoprotein band, but very faint PAS-positive bands were seen all along the gels. Each of the faintly PAS-positive bands corresponded to a band well stained by CBB. For the C1 fraction, the protein profile was much simpler than for the other fractions, but a large number of polypeptide chains were seen, most of which stained strongly for glycoproteins. These results were in good agreement with the marked enrichment in carbohydrates found analytically. In particular the most strongly stained glycoprotein bands present in the original SPM were markedly enriched in this fraction. In addition minor bands which could not be seen in the original fraction, were detected. Although the complexity of the electrophoretic profile prevents a definite conclusion, our general impression is that most, if not all, the CBB-stained bands were also stained by PAS. DISCUSSION

The presence of glucose in all the fractions raised the possibility of the presence of glucose-containing glycoproteins which have been reported in brain 2a,2s and specifically in SPM 20. However, the glucose we have detected could in part be derived, as a contaminant, from either the Ficoll or the sucrose used during the preparation of SPM. In similar experiments on microsomal fractions which were prepared without Ficoll, glucose was detected, although at lower concentrations (unpublished results). The fact that Con A binds primarily to non-reducing terminal mannose 6,z6 does not mean that C1 glycoproteins should have only simple glycans, containing only, or mainly, mannose and N-acetyl-glucosamine, as do ovalbumin18 and Con A positive-glycopeptides from brain microsomal fractions10. Terminal mannose on side chains of complex glycans, or the presence of two types of glycans on the one polypeptide chain (as in thyroglobulinz0 and in some immunoglobulins8) could explain the presence of fucose, galactose and NANA in this fraction, as was in fact observed. On the other hand, mannose present in the CO fraction can be attributed to nonterminal mannose. The C1 fraction from SPM contains a large number of PAS stained bands. Susz et aL 27 found that crude membrane preparations from rat brain, solubilized and chromatographed on Con A with a slightly different method, separated into 8 major PAS-positive fractions during polyacrylamide gel electrophoresis. The large number of bands observed by us could be due to specific enrichment of minor glycoproteins in purified SPM fractions, to the higher sensitivity of our PAS procedure and, finally, to the fact that in the concentration gradient gel used by Susz et al. 27, all bands were separated in the 7 ~o region which is less discriminating than our 12~o gels. In fact Con A-positive fractions prepared from rat brain microsomal fractions and separated as in this paper gave electrophoretic profiles more complex than those of SPM 11. The rechromatography experiments indicate that the multiple band pattern of fraction C1 could not be explained by non-specific adsorption on Con A-Sepharose.

346

. J . P . ZAN['.'TTA Ct (/[.

Similarly, since polyacrylamide gel electrophoresis in SDS separates proteins according to molecular weight, not to charge, and since the molecular weights of these glycoproteins are very different, the complex electrophoretic profile cannot be attributed to microheterogeneity. Thus the 'Con A receptors" of synaptosomal plasma membranes contain a large glycoprotein population, even if we assume that a certain number of Con A binding glycans are not accessible in situ. However, before we can conclude that each synaptosome contains many 'Con A receptor" glycoproteins, we must take into account the fact that SPM are derived from a heterogeneous population of neurons, and each 'family' of neurons could have a more limited glycoprotein composition. But preliminary studies on the regional differences in the glycoproteins of SPM suggest that there are no major differences in the glycoprotein profiles of different populations of SPM. Due to the complexity of the electrophoretic profile, it is difficult to correlate the CBB- and PAS-stained bands. The intensity of a given band can be very different with the two stains. This is particularly evident for fraction CR, where the PAS stain shows one major band and several very faint bands. But, in this fraction, it is hard to say if all the CBB-stained bands have a PAS-positive equivalent. While in the case of CI it is clear that all the proteins should be glycoproteins, this is probably the case for CR, since rechromatography with and without a - M G suggests that the CR fraction interacts weakly, but specifically, under optimum conditions with the carbohydrate binding site of concanavalin A. The faint PAS staining of most of the bands, all of which are probably glycoproteins, could be explained as due to poor PAS staining of certain glycans, or to the low amounts of carbohydrate on many of the glycoproteins present. Further fractionation is necessary before drawing definite conclusions. ACKNOWLEDGEMENTS

This work was in part supported by a N A T O Research G r a n t No. 706, and forms part of the Th~se de Doctorat ~s-Sciences of J. P. Zanetta to be submitted to the Universit6 Louis Pasteur de Strasbourg. We thank Odile Lallemand and R a y m o n d Langs for skilled technical assistance.

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DAVISON, P. MANDELAND I. G. MORGAN(Eds)., Functional and Structural Proteins of the Nervous System, Plenum Press, New York, N.Y., 1972, pp. 39-52. 5 BOSMAN,H. B., Sialic acid on the synaptosome surface and the effect of concanavalin A and trypsin on synaptosome electrophoretic mobility, FEBS Letters, 22 (1972) 97-100. 6 BOURILLON, R., Interactions des phytoh6magglutinines avec les sites r6cepteurs glucidiques des membranes de surface des cellules normales et transform6es. In P. BOULANGER,M. F. JAYLEET J. ROCHE (Eds.), Expos. ann. Biochim. mdd., Masson, Paris, 1973, pp. 59-91. 7 BRECKENRIDGE,W. C., AND MORGAN, I. G., Common glycoproteins of synaptic vesicles and the synaptosomal plasma membrane, FEBS Letters, 22 (1972) 253-256. 8 CLAMP, J. R., AND JOHNSON, I., Immunoglobulins. In A. GOTTSCHALK(Ed.), Glycoproteins, their Composition, Structure and Function, Part A, Elsevier, Amsterdam, 1972, pp. 612-652. GOLDSTEIN, I. J., HOLLERMAN, C. E., AND SMITH, E. E., Protein-carbohydrate interaction. 11. Inhibition studies in the interaction on concanavalin A with polysaccharides, Biochemistry, 4 (1965) 876-883. 10 GOMaOS,G., HERMETET,J. C., REEBER,A., ZANETTA,J. P., AND TRESKA-CIESIELSKI,J., The composition of glycopeptides, derived from neural membranes, which affect neurite growth in vitro, FEBS Letters, 24 (1972) 247-250. l l GOMnOS, G., REEBER, A., ZANETTA, J. P., AND VINCENDON, G., Fractionation of nervous tissue membrane glycoproteins, Colloque International du CNRS sur les Glycoconjuguds, Lille, June 1973, Editions du CNRS, Paris, 1974, in press. 12 HAYMAN,M. J., AND CRUMPTON, M. J., Isolation of glycoproteins from pig lymphocyte plasma membrane using Lens culinaris phytohemagglutinin, Biochem. biophys. Res. Commun., 47 (1972) 923-930. 13 HAYMAN,M. J., SKEHEL,J. J., AND CRUMPTON,M. J., Purification of virus glycoproteins by affinity chromatography using Lens culinaris phytohemagglutinin, FEBS Letters, 29 (1973) 185-188. 14 KALB, A. J., AND LEVITZKI, A., Metal-binding sites of concanavalin A and their role in the binding of a-methyl-o-glucopyranoside, Biochem. J., 109 (1968) 669-672. 15 KAPITANY,R. A., AND ZEBROWSKI, E. J., A high resolution PAS stain for polyacrylamide gel electrophoresis, Analyt. Biochem., 56 (1973) 361-369. 16 KATZMANN,R. L., The inadequacy of sodium dodecyl sulfate as dissociative agent from brain proteins and glycoproteins, Biochim. biophys. Acta (Amst.), 266 (1972) 269-272. 17 LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-272. 18 MARSHALL,R. D., AND NEUaERGER, A., Hen's egg albumin. In A. GOTTSCHALK(Ed.), Glycoproteins, their Composition, Structure and Function, Part B, Elsevier, Amsterdam, 1972, pp. 732-761. 19 MATUS, A., DE PETRIS, S., AND RAFF, M. C., Mobility of concanavalin A receptors in myelin and synaptic membranes, Nature New Biol., 244 (1973) 278-279. 20 McQUILLAN, M. T., AND TRIKOJUS, V. M., Thyroglobulin. In A. GOTTSCHALK(Ed.), Glycoproteins, their Composition, Structure and Function, Part B, Elsevier, Amsterdam, 1972, pp. 926-963. 21 MORGAN,I. G., WOLFE, L. S., MANDEL,P., AND GOMnOS,G., Isolation of plasma membranes from rat brain, Biochim. biophys. Acta (Amst.), 241 (1971) 737-751. 22 MORGAN, I. G., ZANETTA,J. P., BRECKENRIDGE,W. C., VINCENDON, G., AND GOMBOS, G., The chemical structure of synaptic membranes, Brain Research, 62 (1973) 405-411. 23 QUARLES,R. H., EVERLY,J. L., AND BRADY, R. O., Evidence for the close association of a glycoprotein with myelin in rat brain, J. Neurochem., 21 (1973) 1177-1191. 24 RIDDELL,O., AND LEONARD,B. E., Some properties of a coma-producing material obtained from mammalian brain, Neuropharmacology, 9 (1970) 283-299. 25 SEGREST,J. P., AND JACKSON, R. L., Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In V. GINSBURG (Ed.), Methods in Enzymology, Vol. XXVII1, Academic Press, New York, N.Y., 1972, pp. 54-63. 26 SHARON, N., AND LIS, H., Lectins: cell-agglutinating and sugar-specific proteins, Science, 177 (1972) 949-959. 27 Susz, J. P., HOF, H. I., AND BRUNNGRABER,D. G., Isolation of concanavalin A-binding glycoproteins from rat brain, FEBS Letters, 32 (1973) 289-292. 28 VAN NIEUWAMERONGEN,A., VAN DEN EIJNDEN, D. H., HEIJLMAN,J., AND ROUKEMA,P. A., Isolation and characterization of a soluble glucose-containing sialoglycoprotein from the cortical grey matter of calf brain, J. Neurochem., 19 (1972) 2195-2205. 29 VAN N~EUWAMERONGEN,A., AND ROUKEMA,P. A., GP-350, a sialoglycoprotein from calf brain. Its subcellular localization and occurrence in various brain areas, J. Neurochem., 23 (1974) 85-89.

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