Eur. J. Biochem. 98, 39-45 (1979)

Properties of Succinylated Wheat-Germ Agglutinin

Michel MONSIGNY, Claude SENE, Angele OBRENOVITCH, Annie-Claude ROCHE, Francis DELMOTTE, and Egisto BOSCHETTI

Centre de Biophysique Moleculaire, Centre National de la Recherche Scientifique, Orleans, and Pharmindustrie, IBF-Reactifs, Clichy

(Received January 11 /March 12, 1979)

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a p l of 4.0 0.2 while the native lectin is basic, p l of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 pg/ml with both lectins and the concentrations of AT-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins.

The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins.

The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.

Wheat germ agglutinin is a plant lectin which is capable of agglutinating erythrocytes and some other types of cells [ l ] (for recent reviews see [2,3]).

Agglutination is inhibited by N-acetylglucosamine and its (gl-4)oligomers [4- 61. Wheat germ agglutinin is a basic protein with an isoelectric point of 8.7 & 0.3 [7]. At neutral pH, wheat germ agglutinin is a dimer; in acidic medium, it dissociates into two subunits of M , 18000 [8,9].

Subunit dissociation also occurs upon acetylation [7] or tryptophan oxidation with N-bromosuccinimide [lo]; this dissociation is concommitent with a total loss of agglutination properties. However upon succinyla- tion, neither subunit dissociation nor loss of A+ human red-blood-cell agglutinating activity occur [7]. In the present paper, the physicochemical properties and the sugar binding properties of succinylated wheat germ agglutinin are compared with those of the unmodified lectin.

MATERIALS AND METHODS

Materials

Wheat germ agglutinin prepared as in [ l l ] was purchased from Pharmindustrie-IBF-Reactifs, Clichy

(France). Tri-N-acetylchitotriose and tetra-N-acetyl- chitotetraose were prepared by acetolysis of chitin, isolation of the per-0,N-acetylated oligosaccharides by silica gel column chromatography [12], de-0-ace- tylation by catalytic methanolysis with 0.01 M sodium methoxide in methanol at 4°C for 24 h. The tri-N- acetylchitotriose and the tetra-N-acetylchitotetraose were obtained by crystallization from their methanolic solution. Biogel P-60 was purchased from Biorad. Ultrogel ACA 54, Ultrogel A4 and HA-Ultrogel (hy- droxyapatite) were purchased from Pharmindustrie- IBF-Reactifs. Fluorescein isothiocyanate was ob- tained from Research Organics Inc. (Cleveland, U.S. A.).

Succinylation

Wheat germ agglutinin or its fluoresceinylthio- carbamyl derivative (20 mg) were dissolved in 5 ml of saturated sodium acetate at 4°C. Solid succinic anhydride (6 mg) was added to the stirred protein solution. After 90 min, the protein solution was dialyzed against distilled water and then freeze-dried. A second succinylation step was carried as described above.

40 Succinylated Wheat-Germ Agglutinin

Affinity Chromatography

Briefly, Ultrogel A4 was substituted with I-(thio- p-aminobenzy1)-N-acetyl-p-D-glucosamide [13]. Wheat germ agglutinin or its succinylated derivative were adsorbed on the affinity gel at pH 8.6 (0.15 M NaC1, 0.05 M Tris-HCI buffer) and eluted with 0.05 M HCI. The pH of the eluate was adjusted to 7 with 0.5 M NaOH and the neutral solution was dialysed thor- oughly against distilled water and freeze-dried.

Gel Filtration

Wheat germ agglutinin or its succinylated derivative were dissolved in 0.15 M NaCl, 0.05 M Tris-HC1, pH 7.4 (concentration 1 mg/ml) and applied to a column (1.6x60cm) of Biogel P-60 or of Ultrogel ACA 54 equilibrated in the above buffer. The fol- lowing markers (bovine serum albumin 67000, p-lacto- globulin 35 000, myoglobin 17 800 and cytochrome c 12400) were used to calibrate the columns.

Hydroxyapatite Column Chromatography

Wheat germ agglutinin or/and its succinylated derivative were dissolved in 0.01 M sodium phosphate buffer, pH 6.8 (5 mg protein in 2 ml), and applied to a column (6 x 1.6 cm) of HA-Ultrogel equilibrated in the same buffer. Proteins were eluted with the same buffer and with a gradient of increasing phosphate (pH 6.8) concentration up to 0.4 M.

Isoelectric Focusing

Isoelectric focusing was performed in polyacryl- amide gel according to Wrigley [I41 with 2% ampho- line, pH range 3.5-9.5 for 1.5 h a t 1500 V using LKB- Ampholine polyacrylamide gel plates. The anode solution was 1 M H3P04 and the cathode solution was 1 M NaOH. Samples containing 100 pg of protein in distilled water were applied into the template. After focusing, the gel was photographed without any fixing or staining treatment. The pH profile was obtained by fractionation of the gel into 2-mm sticks, each being eluted with 1 ml of 0.1 M KCl for 1 h prior to pH measurement.

Agglutination A ssa ys

A twofold serial dilution of the protein was made in 50 p1 of 0.15 M NaCl aqueous solution and 50 pl of a 3 % suspension of rabbit red blood cells, using a microtitrator plate (system Cooke, M 220 4 A).

After 1 h at room temperature, the degree of agglu- tination was assessed under a light microscope. The inhibition assays were carried out as previously de- scribed [15].

Sedimentation Studies

Velocity measurements were performed at a speed of 60000 rev./min, Spinco-Beckman model E ultra- centrifuge equipped with an ultraviolet optical system, at 280 nm. The protein concentration was 0.5 mg/ml. Values were corrected for conditions of temperature and solvent to density and viscosity of water at 20 "C. The following buffers containing 0.15 M NaCl were used: 0.05 M Tris-HC1, pH 7.5; 0.025 M sodium phosphate, pH 6.5; 0.05 M sodium acetate, pH 5.5; 0.05 M sodium acetate, pH 4.8 ; 0.05 M sodium acetate, pH 4.0; 0.05 M sodium acetate, pH 3.6; 0.05 M gly- cine/HCl, pH 3.2; 0.05 M glycine/HCl, pH 2.3; 0.05 M HCl, pH 1.5.

Fluorescence Measurements

Excitation and emission spectra were recorded with a Fica MK I1 (Fica-France) spectrofluorimeter. The solutions were contained in 5 x 5-mm quartz cells. All the solutions were filtered on Millipore HAWP 0.45-pm pore-diameter filters. The protein concentra- tion was about 1 pM, so that the absorbance was very low, A&' < 0.05, at the excitation wavelength. Sugar binding experiments were conducted with solu- tion of protein at a constant concentration in which was dissolved various amounts of the tested sugar. The binding constant was calculated as:

1

where Ll j2 is the ligand concentration giving half the change of fluorescence intensity, and P the total pro- tein concentration expressed as the binding site con- centration (four sites per dimer of 36000 molecular weight). L1/2 was determined by plotting log ( F - Fo)/ ( F , - F) versus the log of ligand concentration L ; Fo is the Iectin fluorescence in the absence of ligand, F is the ligand fluorescence in the presence of a given ligand concentration and F , is the fluorescence of the lectin in the presence of saturating concentration of ligand; F, was obtained by plotting 1 / ( F - Fo) versus the reciprocal value of the ligand concentration and extrapolating to L-' = 0.

Phosphorescence Measurements

Phosphorescence measurements were conducted in the conditions previously described [17]. The 1-(thio-

M . Monsigny, C. Sene, A. Obrenovitch, A.-C. Roche, F. Delmotle, and E. Boschetti 41

p-mercuribenzoate) of di-N-acetylchitobiose was used to assess the interaction between the ligand and the tryptophan residue present in the binding site of the lectins.

Fluoresc.einylthiocarbamy1 Derivatives

Wheat germ agglutinin was substituted on its free amino groups by reaction with fluoresceinylisothio- cyanate in the presence of N-acetylglucosamine (10 mg/ ml) according to Loor [16]. The fluorescent derivative was purified by affinity chromatography on Ultrogel A4 substituted with l-(thio-p-aminobenzyI)-2-acet- amido-l,2-dideoxy-~-~-glucopyranoside [ 1 1 1. The extent of substitution was estimated using the absor- bance ratio at 495 nm and 280 nm. Usually this ratio was close to 1.

Succinylated fluoresceinylthiocarbamyl agglutinin was prepared by succinylation of the fluoresceinyl- thiocarbamyl agglutinin as described above.

Quantitative Measurements of the Binding of Fluoresceinyl Derivatives of the Lectins [18/ on Tlzymus Cells o f B A L B / c Mice

Thymus cells from BALB/c mice were teased in RPMI medium. The suspension was passed through a nylon mesh in order to remove fat and clumps. The cells were pelleted by centrifugation (1 50 x g , 10 min) at 4'C. The pellet was suspended in a lysing buffer (150 mM NH4C1, 10 mM KHC03, 0.13 mM EDTA) for 4 min at 4°C. The cell suspension was then washed twice with fresh RPMI medium. Finally, thymus cells were suspended in phosphate-buffered saline pH 7.4, at a final concentration of 2 x lo6 cells/ ml. The incubation with fluoresceinylthiocarbamyl derivative of lectins was carried out at different lectin concentrations varying from 0.5 to 100 pg/ml for 1 h at 4'C. Lectin specifically bound to the cells was released by incubating labelled cells for 1 h at 4°C with 1 ml of 0.3 M N-acetyl-D-glucosamine and its concentration was determined by the spectrofluori- metric method, previously described [18]. The ap- parent number of binding sites per cell were then evaluated from a Scatchard plot.

RESULTS AND DISCUSSION

The main physicochemical properties of succinyl- ated wheat agglutinin are summarized in Table 1. Wheat germ agglutinin and its succinylated derivative were able to bind on Ultrogel A4 substituted with 1 - (thio -p - aminobenzyl) - N - acetyl- P - D -glucosaminide and were eluted from the affinity column by the use of an acidic solution. So, succinylated wheat germ agglu-

Table 1. Plzysicochemical properties of ,client g w m ngglutinin nntl of succinylated wheat germ agglutinin

Property Conditions Value for agglutinin

unmodified succinylated

s20. w

Apparent molecular weight

Absorbance

Isoelectric PH (PI)

Solubility

Binding constant

pH 7.5. 0.5 mgiml pH 1.5, 0.5 mg!ml

Biogel P-60 Ultrogel ACA 54

pH 7.5 ).,,,in Amax

A274iA249 0.1 M NaOH L,,,

3.6 s 2.1 s

9500 f 1000 7200 1000

249 nm 275 nm 1.77 296 nm

8.5

2.5 mg/ml

18000 M - ' 18000 M-'

3.6 s 2.1 s

18 000 & 1500 24000 f 2000

249 nm 274 nni 1.77 297 nm

4.0

200 mgiml

14000 M - ' 14000 M-'

a (GlcNAc)3 = tri-N-acetylchitotriose. (GIcNAc)~ = tetra-N-acetylchitotetraose

- 4.0

- 0.5

Fig. 1. Isoelectrofocusing of ( A ) wheat gc'rm agglutinin und ( B ) .sue- cinylated wheat germ agglutinin

tinin is still able to bind this immobilized ligand, in contrast with the results of Rice and Etzler [7] who reported that it did not bind to ovomucoid-Sepharose. This apparent discrepancy could be related to the facts that wheat germ agglutinin and its succinylated derivative have a similar affinity towards N-acetyl- glucosamide (Table 1) and that the succinylated lectin does not bind an acidic glycoprotein such as ovo- mucoid.

The absorption spectra of the wheat germ agglu- tinin and its succinylated derivative were very similar

42 Succinylated Wheat-Germ Agglutinin

?

2 1 3 5 7

PH

Fig. 2. Sedimentation coeffi'cients of'(- -@) wheat germ agglutinin (0.S mglml) and of (A-----A) succinylated wheat germ agglutinin (0.S mglml) at various p H , and at 20 "C. Concentration of suc- cinylated wheat germ agglutinin at pH 4.0 and at pH 1.5 was about 60 pg/ml

. 0.4 E. - aJ L

0 L Q

c

. 0.3 %

E . a2 s I

- 0.1

-0 0 10 20 30 40 50 60

Elution volume (ml)

Fig. 3 . Chromatography of ( A ) vvheat germ agglutinin (5 mg/2 ml) and of ( B ) succinylatrd wheat germ agglutinin (S mg/2 mlj on a column (1.6 x 6 cm) of HA-Ulrrogel (hydroxyapatitej in 0.01 M sodium phosphate bujyer p H 6.5. Elution with the same buffer, and then with a continuous gradient (0.01 M to 0.4 M) of sodium phosphate buffer, pH 6.8

at neutral pH as well as in an alkaline medium, showing that the aromatic residues were not in- volved in the succinylation procedure.

Wheat germ agglutinin is focused in a sharp band at pH 8.5 in gel isoelectric focusing experiments in good agreement with other results [7]. The succinylated lectin is focused in the pH range 3.8-4.2, forming several discrete bands (Fig. 1). So, succinylated wheat germ agglutinin is a slightly acidic protein, in contrast with the native agglutinin which is a strongly basic protein. The dramatic change in the electrical proper- ties of wheat germ agglutinin after succinylation was also indicated by solubility measurements and chro- matographic analysis.

The maximal solubility of native and succinylated wheat germ agglutinin was measured at 20 "C in 0.1 M NaCl, 0.05 M Tris-HC1 buffer, pH 7.2. In both cases, the starting material was a freeze-dried protein ob- tained after affinity chromatography purification and dialysis against distilled water. In these conditions, the solubility of wheat germ agglutinin was found to be 2 mg/ml, and the solubility of succinylated agglu- tinin was as high as 200 mg/ml, i.e. 100 times higher than the unmodified protein. However, the solubility of succinylated agglutinin at pH lower than 5 was very low, about 50 - 100 pg/ml.

The sedimentation coefficients of native and suc- cinylated wheat germ agglutinin were 3.4 S from neu- tral pH down to pH 4 and 2.1 S at pH below 2 (Fig. 2). The rapid decrease of the sedimentation coefficient of wheat germ agglutinin between pH 4.2 and 3.2 is related to the dimeric-monomeric transition, in agree- ment with the molecular weight determinations at neu- tral and acitic pH [7]. Succinylated wheat germ agglu- tinin, which has a sedimentation coefficient of 3.4 S down to pH4.8 is in a stabledimeric stateat neutral pH. It was not possible to determine the sedimentation coefficient of succinylated wheat germ agglutinin at pH 4.0,3.6,3.2 and 2.3 because its solubility was below 100 pg/ml. However, in strongly acidic medium, as expected, the sedimentation coefficient was found to be 2.1 S.

Using gel filtration techniques, the apparent molec- ular weight of wheat germ agglutinin was very low (7200 or 9500), suggesting that it was retarded by some adsorption and/or ion-exchange phenomenon ; the apparent molecular weight of succinylated wheat germ agglutinin (1 8 000 or 24 000) was considerably greater than that observed for the native agglutinin in the same solvent and with the same gels but was lower than that the actual molecular weight which is 36000 [8,9].

On a column of hydroxyapatite equilibrated in a low-ionic-strength buffer, pH 6.8, it was found that succinylated wheat germ agglutinin was not retarded but that the native agglutinin was adsorbed; the latter was eluted with a higher-ionic-strength buffer (Fig. 3).

The minimal concentration required to agglutinate cells were 2 pg/ml with rabbit red blood cells, murine thymocytes and splenocytes, 10 pg/ml with A' human red blood cells using succinylated wheat germ agglu- tinin as well as unmodified agglutinin, suggesting that the succinylation does not affect the biological activity of the protein. This result is in good agreement with the findings of Rice and Etzler [7]. However, it was found that horse red bloods cells were agglutinated with 10 pg/ml of the native agglutinin but were not agglutinated with up to 500 pg/ml of the succinylated lectin.

The agglutination activities of native and succinyl- ated wheat germ agglutinin were inhibited to the same

M. Monsigny, C. Sene, A. Obrenovitch, A.-C. Roche, F. Delmotte, and E. Boschetti 43

extent by N-acetylglucosamine (Table 2). However, the agglutination of rabbit red blood cells induced by both lectins were not inhibited by N-acetylneuraminic acid up to 50 mM, while a concentration of 30 mM inhibited horse red blood agglutination in the presence of the native agglutinin.

The similar binding properties of N-acetylglucos- amine and di-N-acetylchitobiose on both native and succinylated wheat germ agglutinin was also shown by quantitative fluorescence studies of the sugar binding. The two lectins upon excitation at 295 nm, gave a maximum intensity of fluorescence at 348 nm (Fig. 4). Upon adding tri-N-acetylchitotriose or tetra- N-acetylchitotetraose, the maximum wavelength shifted downwards to 338 nm and the intensity increased markedly (Table 3). With the native agglutinin, the increase of fluorescence intensity upon sugar binding has been interpreted as a contact between the ligand and a tryptophan residue [17]. Therefore, in the suc- cinylated lectin, this specific interaction seems not to be disturbed. This conclusion is also supported by the phosphorescence experiments (Table 3). The phos- phorescence life time of succinylated wheat germ ag- glutinin is identical with that of the unmodified lectin.

The binding of the thiomercuribenzoate derivative of di-N-acetylchitobiose induced similar effects on the phosphorescence intensities and on the phospho- rescence life times of both unmodified and succinylated wheat germ agglutinins. The association constants calculated for the oligosaccharides of N-acetylglucos- amine with succinylated wheat germ agglutinin were slightly lower than the association constants calculated with the native lectin (Fig. 5, Table 2). Furthermore, the binding of N-acetylglucosamine derivatives onto native and succinylated wheat germ agglutinin were

Table 3. Fluorescence and phosphorescence characteristics of native and succinylated wheat germ agglutinin in the presence and in the absence of ligands (G1cNAc)d = tetra-N-acetylchitotetraose; If and I,' = fluorescence intensities in the absence and in the presence of 1.25 mM tetra- N-acetylchitotetraose, respectively; (G1cNAc)z = di-N-acetylchito- biose; (GlcNAc)zSHgBz = thiomercuribenzoate derivative of di- N-acetylchitobiose; I,' and I,' = phosphorescence intensities in the absence and in the presence of 0.5 mM thiomercuribenzoate derivative of di-N-acetylchitobiose. The lectin concentrations were 1 pM for the fluorescence experiments and 13 pM for the phos- phorescence experiments in 0.15 M NaCl, 0.05 M Tris-HC1, pH 7.2

Property Ligand Value for agglutinin

Table 2. Minimal concentration of free ligands required to inhibit the rabbit red blood cell agglutination induced by native and suc- cinylated wheat germ agglutinin NeuNAc = N-acetylneurarninic acid; n.i. = no inhibition at a concentration of 50 mM

Compound Concn to inhibit agglutinin

native succinylated

mM ~ ~ ~ _ _ _ _ _ _ ~ ~ ~ ~

GlcNAc 2.5 2.5 GlcNAc(/?l-4)GlcNAc 0.03 0.03 NeuNAc n.i. n.i.

native succinylated

Fluorescence emission at 25 ' C 348 nm 348 nm

(GIcNAc)~ 338 nm 338 nm - A n a x

I;lI; 1.67 1.62

Phosphorescence emission at 77 K

~ . m a x - 430 nm 430 nm t life time - 5.4 k 0.2 s 5.6 & 0.2 s

(GIcNAc)~ 5.9 i 0.1 s 5.9 & 0.1 s (G1cNAc)zSHgBz 0.014 0.014

k 0.002 s * 0.002 s I;lI,' 6 6

300 400 500 300 400 500 Wavelength (nm)

Fig.4. Fluorescence spectra of ( A ) wheat germ agglutinin and of ( B ) succinylated wheat germ agglutinin, in the absence (-) and in the presence (------) qf2.5 m M tetra-N-acetylchitotetraose. Excitation wavelength: 295 nm

44

2

- 1 \

9

L

. k0

rn - 0

-1 -5 -4 - 3 - 2

log L

Fig. 5. Fluorescencc intensity c.hanges of nheut germ agglutinin (o,@) and of succ,inylated wheat germ agglutinin in,.) in the presence of rri-N-acetylchitotrios~, (0,o) and of tetrcr-N-acetyl- chitotrtruose (@. W)

Succinylated Wheat-Germ Agglutinin

0.15

0.10 h Q

a05 h A 0.0 1 5

0.010 4

0.005

- 0 1 2

b (119)

Fig. 6. Scatchard plots of' the speciJic binding of ,/luoresceinqlthio- carhamj~l derivatives of ( A ) wheut germ agglutinin and ( B ) suc- sinyluted wheat germ agglutinin to thymus cells of BaIblc mouse. b = amount of the fluorescein lectin bound to 2 x 10' cells and specifically released in the presence of 0.3 M N-acetylglucosamine; f = concentration of the free fluoresceinylthiocarbamyl derivative of the lectin

very similar as shown by nuclear magnetic resonance studies (J. P. Grivet et al., unpublished results).

The fluoresceinylthiocarbamyl derivatives of na- tive and succinylated wheat germ agglutinin were found quite suitable to visualize the membrane glyco- conjugates of cells such as lymphocytes or baby hamster kidney (BHK) cells by fluorescence micro- scopy (Obrenovitch et al., unpublished results). The quantitative studies of the binding of native and succinylated wheat germ agglutinin to murine thymo- cytes as well as to BHK cells (wild type and various variants) and to murine splenocytes (unpublished

results) showed a similar heterogeneity of the binding sites. The Scatchard plots (Fig. 6) exhibited two slopes corresponding to high-affinity and low-affinity binding sites. However, the total number of succinylated ag- glutinin molecules bound to murine thymocytes (2.1 f 0.6 x 106/cell) or to BHK cells (8 f 2 x 106/cell) was much lower than that of native agglutinin mole- cules (17 * 2 x lo6 and 62 f 5 x 106/cell, respectively). Because the actual binding constants of both native and succinylated wheat germ agglutinin with oligo- saccharides containing N-acetylglucosamine (Table 1) are very similar, it is suggested that succinylated agglu- tinin binds the membrane glycoconjugates containing N-acetylglucosamine as the native agglutinin does and that, in addition, the native agglutinin binds glyco- conjugates containing N-acetylneuraminic acid. In- deed, wheat germ agglutinin does bind free N-acetyl- neuraminic acid [19 - 21 ] and N-acetylneuraminic acids present in cell-surface glycoconjugates [4,22], but suc- cinylated wheat germ agglutinin does not. This view is further supported by the fact that horse red blood cells are agglutinated by the unmodified lectin but are not agglutinated by the succinylated lectin and that the horse red blood cell agglutinination by the un- modified lectin can be inhibited by N-acetylneuraminic acid as well as by N-acetylglucosamine (Monsigny et al., unpublished results); rabbit red blood cells are ag- glutinated by both lectins but this agglutination is not inhibited by N-acetylneuraminic acid (Table2). Further studies are needed to decide whether succinylated wheat germ agglutinin cannot bind cell-surface glyco- conjugates containing N-acetylneuraminic acid or if these acidic glycoconjugates prevent succinylated wheat germ agglutinin from reaching glycoconjugates containing N-acetylglucosamine.

We thank Mr Philippe Bouchard and Ms Genevieve Serros for valuable technical assistance. This work was supported by grants C. L. 75.4.074.3. from Institut Nationulde In Suntl et de la Recherche mkdicale; A.C.C. 77.7.0252; from Dblegation Ginirale d la Reckerche Scicnt(fique et Technique, and A. T.P. 76 2302 from Cenrre National dr la Recherche Scientifique.

REFERENCES

1 . Aub, J. C., Tieslau, C. & Lankester, A. (1963) Proc. Nut1 Acad. Sci. U.S.A. 50, 613-619.

2. Lis, H. Sr Sharon, N. (1977) in The Antigens (Sela, M., ed.) vol. 4, pp. 429 - 529, Academic Press, New York and London.

3. Monsigny, M., Roche, A. C. & Kieda, C. (1978) in Structure and Function of Biological Membranes. Molecular Aspects (Centre de Biophysique Moleculaire, ed.), pp. 161 -234. Commission of the European Communities, Brussels.

4. Burger, M. M. & Goldberg, A. R. (1967) Proc. Nut1 Acud. Sci. U.S.A. 57, 359-366.

5. Burger, M. M. (1969) Proc. Nail Acad. Scr. U.S.A. 62, 994- 1001.

131, 155-162.

4099.

6. Allen, A. K. , Neuberger, A. & Sharon, N. (1973) Biochem. J .

7. Rice, R. H. & Etzler, M. E. (1975) Biochemistrj: 14, 4093-

M. Monsigny, C. Sene, A. Obrenovitch, A.-C. Roche, F. Delmotte, and E. Boschetti 45

X. Nagata. Y . 91 Burger, M. M. (1974)J. Bid . Cl7em. 249, 3116-

9. Rice, R. H. 91 Etzler, M. E. (1974) Biocknz. Biophys. Rcs.

10. Privat. J . P.. Lotan, R., Bouchard, P.. Sharon, N . Sr Mon-

11. Bouchard, P., Moroux, Y., Tixier, R., Privat, J. P. & Mon-

12. Delmotte, F. 91 Monsigny, M. (1974) Corholij,dr. Res. 36,

13. Rafestin. M. E . , Obrenovitch, A,, Oblin, A. 91 Monsigny, M.

14. Wrigley, C. W. (1968) J . Chromutogr. 36, 362-372. 15. Roche, A. C. 91 Monsigny, M. (1974) Bioc,/7im. Biop l l~~s . Actrr,

3122.

Coiwtlim. 5Y, 414-419.

signy, M. (1976) Eur. J . Biocliem. 68, 563-572.

signy, M. (1976) Biochimie (Paris) 58, 1247- 1253.

219 -226.

( 1974) FEBS L,ett. 40, 62 - 66.

371. 242 - 254.

16. Loor, F. (1973) E.\p. Cell Res. 82, 415-425. 17. Monsigny, M., Delmotte, F. 91 Helene, C. (1978) Proc.. Ntrrl

Acad. Sci. U.S.A. 75, 1324-1328. 18. Monsigny, M., Sene, C. 91 Obrenovitch, A. (1979) E w . J .

Biorhem. 96, 295 - 300. 19. Greenaway, P. J. & Levine, D. (1973) Not. A’CII. Biol. 241.

191 - 192. 20. Jordan, F., Basset, E. & Redwood, W. R. (1977) Biorhcw.

Biophys. Res. Commun. 75. 1015- 1021. 21. Roche, A. C. (1978) T/ir;.se (k. D o m r a t c;.s-Scicwc,.c., Orlt;ans,

France. 22. Bhavanandan, U. P., Unietoto, J.. Banks, B. R. 91 Davidson.

E. A. (1977) Biocllen7isrry, 16, 4426-4437.

M. Monsigny, C. Sene, A. Obrenovitch, A.-C. Roche, and F. Delmotte, Centre de Biophysiquc Moleculaire du Centre National de la Recherche Scientifique, 1A Avenue de la Recherche Scientifique, La Source, F-45045 Orleans-Cedex, France

E. Boschetti, Pharmindustrie, IBF-Reactifs, F-92110 Cllchy, France