Studies of lectins XXXVI. Properties of some lectins prepared by affinity chromatography on O-glycosyl polyacrylamide gels

299

Biochimica et Biophysica Acta, 5 3 8 ( 1 9 7 8 ) 2 9 9 - - 3 1 5 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 28395

STUDIES ON LECTINS XXXVI. PROPERTIES OF SOME LECTINS PREPARED BY AFFINITY CHROMATOGRAPHY ON O-GLYCOSYL POLYACRYLAMIDE GELS * V

•

V. H O R E J ~ I a n d J. K O C O U R E K

Department of Biochemistry, Charles University, Albertov 2030, 128 40 Praha 2 (Czechoslovakia) (Received

May 23rd, 1977)

Summary A number of lectins has been purified by affinity chromatography on O-glycosyl polyacrylamide gels. The lectins isolated (and the particular sugar ligands used in the affinity carriers) are as follows: Anguilla anguilla, serum (~-L-fucosyl-), Vicia cracca, seeds; Phaseolus lunatus, seeds; Glycine soja, seeds; Dolichos biflorus, seeds; Maclura pomifera, seeds; Sarothamnus scoparius, seeds; Helix pomatia, ablumin glands; Clitocybe nebularis, fruiting bodies (all N-acetyl-~-Dgalactosaminyl-); Ricinus communis, seeds (~-lactosyl-); Ononis spinosa, root; Fomes fomentarius, fruiting bodies; Marasmius oreades, fruiting bodies (all s-Dgalactosyl-), Canavalia ensiformis, seeds, (i.e., concanavalin A) (~-D-glucosyl-). Physicochemical properties of Glycine soja, Dolichos biflorus, Phaseolus lunatus, Helix pomatia and Ricinus communis lectins corresponded well to properties of the preparations studied earlier by other workers. For the other purified lectins the essential physicochemical data (sedimentation coefficient, molecular weight, subunit composition, electrophoretic patterns, amino acid composition, carbohydrate content, isoelectric point) were established and their precipitating, hemagglutinating and mitogenic activities determined.

Introduction

Affinity chromatography on various synthetic or semisynthetic affinity adsorbents became an invaluable tool for purification of lectins [1--3]. The present communication shows a wide applicability of O-glycosyl polyacrylamide gels [4,5] for isolation of lectins from plants, mushrooms, snails and fish sera. * A preliminary note of part of this c o m m u n i c a t i o n w a s published earlier [6].

300 Materials and Methods

O-Glycosyl polyacrylamide gels The O-glycosyl polyacrylamide gels used as affinity adsorbents were prepared by radical copolymerization of a water solution of acrylamide, N,N'methylene bisacrylamide and the appropriate allyl glycoside [4,5]. For large scale experiments it was not always necessary to prepare pure crystalline allyl glycosides; instead, the syrupy residue obtained after evaporation of allyl alcohol from the reaction mixture could be successfully used. (As determined by thin layer chromatography on silica gel or by paper chromatography, the syrup contained usually about 40--70% of the unreacted original sugar.) The properties of the gels have been described earlier [4,5]. Sources o f lectins Eel serum (Anguilla anguilla L., pooled from 70 animals) was obtained from eels caught during the summer months of 1975 in Lu~nice river, Southern Bohemia, Czechoslovakia. Jack bean meal (from Canavalia ensiformis D.C. seeds) was obtained from Serva, Heidelberg, West Germany. Clitocybe nebularis (Batsch, ex Fr.) Kumm. and Marasmius oreades (Bolt.)Fr. fruiting bodies were collected in Central Bohemia. Dolichos biflorus L. seeds were purchased from United Chemical and Allied Products, Calcutta, India. Fomes fomentarius (L. ex Fr.) Kickx (syn. Polyporus fomentarius L. ex Fr.) fruiting bodies were collected on fallen beech trees in a single locality in Northern Bohemia. Glycine soja (L.) Sieb. et Zucc. seeds were obtained from the agricultural cooperative farm J Z D Libochovice, Czechoslovakia. Helix pomatia L. vineyard (or Roman) snails were collected in Central Bohemia. Maclura pomifera (Raf.) Schneid. seeds were obtained from F.W. Schumacher Co., Sandwich, Mass., U.S.A. Ononis spinosa L. dried roots were obtained from L ~ i v ~ rostliny n.p., Zbraslav, Czechoslovakia. Phaseolus lunatus L. seeds were purchased from W. Atlee Burpee, Clinton, Iowa, U.S.A. Ricinus communis L. seeds were purchased from Sempra n.p., Praha, Czechoslovakia. Sarothamnus scoparius (L.) Wimm. and Vicia cracca L. seeds were collected from wildly-growing plants in Central Bohemia. Preparation o f crude extracts and protein fractions The crude protein fractions from Vicia cracca, Phaseolus lunatus, Maclura pomifera, Glycine so]a and Sarothamnus scoparius seeds were prepared by an overnight extraction of finely ground seeds with 10 vol. saline (0.9% NaC1); to the extract clarified by centrifugation and filtration through a diatomaceous earth layer, (NH4)2SO4 was added (700 g/1000 ml). The precipitate obtained by centrifugation was suspended in 4 vol. water and extensively dialysed against deionized water. The precipitate formed during dialysis was removed and discarded, the supernatant was lyophilized. Marasmius oreades and Clitocybe nebularis fruiting bodies were frozen at --20°C and then quickly thawed, and the expressed sap was treated as the above extracts. The w o o d y fruiting bodies of Fomes fomentarius were cut into small pieces (about 2 × 5 cm), then homogenized in a homogenizer with 4 vol. saline and stirred overnight. The mixture was filtered through a nylon cloth

301 and then through a thin layer of diatomaceous earth. The clarified extract was then treated as the above extracts. The lyophilized crude preparations were dissolved in saline and applied to the affinity columns as described in the next paragraph; the saline extract from jack bean meal (1 : 5) was applied directly. The albumin glands of vineyard snails were homogenized with 5 vol. saline. The homogenate was centrifuged, clarified by filtration through diatomaceous earth layer and applied to the affinity column. Eel serum was preserved for 2 days frozen at --20°C and after thawing it was applied directly to the affinity column.

Affinity chromatography Crude lyophilized preparations were dissolved in 50 vol. saline (if the dissolution were incomplete, the insoluble material was removed by centrifugation) and the solution was applied to the affinity gel columns. After the sample solution entered the gel, the column was washed with saline. Small columns (20 X 1.5 cm) were used in preliminary experiments, for large scale purifications larger ones (20 X 4 cm) were applied. In both cases the approximate flow rate was 4 ml/h per cm 2 of the column cross section; 15 min or 30 min fractions were collected. After the complete elution of inactive proteins (as monitored by following absorbance of the fractions at 280 nm on a MOM 202 spectrophotometer), the eluant (either a solution of a carbohydrate or 0.05 M glycine/HCl buffer, pH 3.0, in saline) was applied. Fractions of the effluent containing the desorbed lectin (detected by hemagglutination activity and optical absorbance at 280 nm) were combined, dialysed and lyophilized. When lectins had been eluted with a carbohydrate solution, the columns were washed with an acidic buffer to remove last traces of proteins. The gels were regenerated by stirring with 8 M urea solution overnight and then washing with saline. The regenerated gels could be reused at least five times without significant loss of capacity.

Gel Chromatography on Sephadex G-I O0 The mixture of Ricinus communis lectins (3.5 g) obtained by affinity chromatography on O-~-lactosyl polyacrylamide gel was dissolved in 100 ml 0.05 M phosphate buffer (pH 7.5) and applied onto a column of Sephadex G-100 (10 X 100 cm) equilibrated with the same buffer. After elution (100 ml/ h) the combined fractions corresponding to the separated lectins were dialysed against deionized water and lyophilized.

Polyacry lamide gel electrophoresis Disc polyacrylamide gel electrophoresis was performed in an apparatus designed by Davis [7] in alkaline [8] or acidic buffers [9]. Alkaline disc electrophoresis (pH 8.9) was run for 1.5 h at a current density 5 mA per tube (75 X 5 mm), acidic electrophoresis (pH 4.5) for 2 h at 7 mA per tube. The gels were stained with Amido Black 10B. If not otherwise stated, 75 pg of protein per tube were applied. Polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate was performed according to Weber and Osborn [10] in a 7.5% gel. Gel rods

302

75 X 5 mm were used at a current density of 7 mA per tube for 2.5 h; 40 t~g protein samples (non-reduced or treated with 2% mercaptoethanol or dithiothreitol) were analyzed, or 80 pg samples when staining was performed with Schiff reagent. Molecular weights of subunits were calculated from the comparison of their relative electrophoretic mobilities with those of lysozyme and its covalent oligomers [11]. The glycoprotein staining of periodate oxidized gels with Schiff reagent was performed according to Zacharius et al. [12]. The discontinual electrophoresis in the presence of sodium dodecyl sulfate at pH 9.5 was carried out according to Neville [13].

Isoelectric focusing and isoelectric point estimation Analytical isoelectric focusing in polyacrylamide gel was performed according to Righetti and Drysdale [ 14], using 5% polyacrylamide gel, 2% Ampholine (LKB, pH range 3--10) and glass tubes 110 X 5 mm. The protein sample (0.5 mg) was dissolved directly in the total volume of the polymerization mixture used for preparation of one gel rod (2 ml). The current density of 1 mA per tube was maintained until the voltage reached 350 V (about 4 h); the voltage was maintained for 12 h. Then some gels were cut into 5 mm long segments, the segments homogenized in small test tubes with 0.5 ml of water and after 1 h standing at room temperature with occasional stirring, the supernatants were tested for hemagglutination. Water was then added to a total volume of 2 ml in each test tube and the pH was estimated by a pH meter (M 120, Mikrotechna, Praha). Other gels were fixed in 5% trichloroacetic acid, then thoroughly washed with 5% acetic acid and stained with Amido Black 10B.

Estimation of molecular weight by gel filtration Thin layer gel filtration on Sephadex G-200 Superfine in saline (if not stated otherwise) was performed on the Pharmacia TLG apparatus [15]. Bovine serum albumine (dimer and monomer), ovalbumine, hen egg lysozyme and, in some cases, also aldolase and trypsin were used as reference proteins.

Ultracentrifugal analysis Sedimentation coefficients were measured on a MOM 3170 ultracentrifuge (MOM, Budapest, Hungary) using 0.1--0.6% protein concentration in saline.

Amino acid analysis Protein samples were hydrolyzed in sealed tubes under N2 at 110°C with 6 N HC1 for 20 and 70 h, respectively, and analyzed on an amino acid analyzer (Model AAA 881, Mikrotechna, Praha, Czechoslovakia). Values for serine and threonine were obtained by extrapolation to zero time hydrolysis and the maximum values were taken for the remaining amino acids. Cysteine and methionine were estimated as cysteic acid or methionine sulfone, respectively, after oxidation of a sample with performic acid [16] and hydrolysis (6 N HC1, l l 0 ° C , 20 h). The tryptophan content was estimated spectrophotometrically [171. N-terminal amino acids were determined by the dansylation technique [ 18].

Sugar analysis Total neutral sugar content was estimated by the phenol/sulfuric acid meth-

303 od [19], using D-glucose as reference sugar. Amino sugars were estimated on amino acid analyzer in a sample hydrolyzed with 4 N HC1 for 16 h. Qualitative neutral sugar composition was studied by paper chromatography in the solvent system ethyl acetate/acetic acid/formic acid/water (9 : 1.5 : 0.5 : 2) [21]. The sample was hydrolyzed with 2 M trifluoroacetic acid for 4 h at 105°C, evaporated in vacuo, dissolved in a drop of water and applied to the Whatman No. 4 paper sheet.

Metal content Metal content was estimated by atomic absorption spectroscopy on a Varian Techtron (Model AA-4) atomic absorption spectrophotometer. Estimation of hemagglutinating activity and its inhibition Hemagglutinating activity was assayed by a test tube serial dilution method referred to by Tobi~ka [22]. To 0.1-ml aliquots obtained by two-fold serial dilution of a 1% lectin solution, an equal volume of a 2% suspension of thricewashed red blood cells in saline was added. After 15 min the tubes were centrifuged and examined for agglutination. Trypsinized erythrocytes were prepared by incubation of 2 ml of 20% suspension of washed erythrocytes with 2 mg of trypsin ( L ~ i v a , Praha) at 37°C for 30 min. The suspension was then washed three times with 20 ml saline and diluted to 20 ml. Inhibitory activity of sugars was estimated using a lectin solution eight times more concentrated than the solution allowing for the first perceptible hemagglutination. To 0.1 ml aliquots obtained by 2-fold serial dilution of a 0.1 M sugar solution, 0.1 ml of the lectin solution was added. After 30 min, 0.2 ml of the 2% erythrocyte suspension was added and after another 15 min hemagglutination was observed as described above. If not otherwise stated, following sugars were tested for inhibitory activity: D-arabinose, L-arabinose, L-fucose, L-rhamnose, D-galactose, D-glucose, N-acetyl-D-galactosamine, N-acetyl-Dglucosamine and lactose. Mitogenic activity Mitogenic activity was estimated by measuring the incorporation of 3H-labelled uridine into the rabbit lymph node l y m p h o c y t e s [23]. The assay was carried out at three different concentrations of the lectin (0.5, 5 and 50 pg/ml). Concanavalin A was used as standard mitogen. Two-dimensional diffusion in cellulose acetate Immunodiffusion was carried o u t on cellulose acetate strips (Cellogel) equilibrated with saline. The lectins and precipitinogens (natural or synthetic carbohydrate-containing macrimolecules), respectively, were applied on the points a b o u t 7 mm apart and were allowed to diffuse in a paraffin oil bath for 24 h at room temperature. The precipitation bands were stained with Amido Black 10B. Synthetic precipitinogens, water soluble O-glycosyl polyacrylamide copolymers, were prepared by copolymerization of acrylamide with allyl glycosides, followed by dialysis and lyophilization [20].

304 Results

Isolation o f lectins Data concerning the isolation of lectins are summarized in Table I. The mixture of R. communis lectins obtained by affinity chromatography was further separated by gel filtration on Sephadex G-100, yielding two major peaks corresponding to the 'agglutinin' and the 'toxin' [24,25]. These two substances could be separated also by affinity chromatography on O-(N-acetyl-~D-galactosaminyl) polyacrylamide gel. The toxin which is specifically adsorbed on this gel may be subsequently eluted by 0.1 M D-galactose solution, whereas the agglutinin passes through the column unretarded. Properties o f the isolated lectins Electrophoretic patterns obtained under different conditions of some of the isolated lectins are shown in Fig. 1, ultracentrifuge patterns in Fig. 2. Results of amino acid analyses are summarized in Table II and other physicochemical properties are given in Table III. Data concerning biological activities and sugar specificities are in Table IV. In the following paragraphs are presented only those details not given in the figures and tables. The properties of concanavalin A and the lectins of Glycine soja [32,33], Helix pomatia [35,36] and Phaseolus lunatus [37,38] corresponded to those described earlier. Properties of Dolichos biflorus lectin are discussed elsewhere in detail [34]. Also the properties of both "agglutinin" and " t o x i n " of the R. communis seeds were essentially in a good agreement with literature data published earlier [24--27,39--41]. Atomic absorption spectroscopy of these substances indicated a content of Ca, Co, Mg, Mn, Ni and Zn lower than 0.1 atom/ mol. In both the agglutinin and the toxin alanine, proline and isoleucine were found as N-terminal amino acids. TABLE I I S O L A T I O N O F L E C T I N S BY A F F I N I T Y C H R O M A T O G R A P H Y Source

Affinity ligand

A n g u i l l a anguilla Canavalia e n s i f o r m i s C l i t o c y b e nebularis Dolichos biflorus Fomes fomentarius G l y e i n e soja H e l i x pornatia Maclura p o m i f e r a M a r a s m i u s oreades O n o n i s spinosa Phaseolus lunatus Riclnus communis Sarothamnus scoparius Vicia cracca

c~-L-Fuc c~-D-Glc ~-D-GalNAc a-D-GalNAc c~-D-Gal ~-D-GalNAc a-D-GalNAc ~-D-GalNAc a-D-Gal c~-D-Gal a-D-GalNAc ~-Lac ~-D-GalNAc c~-D-GalNAc

Capacity a <10 4 n.d. 2.7 15 7 4.5 <20 15 22 9 1 <20 3.5

Eluant

Yield

L - F u c (0.2 M) D-Glc (0.1 M) D-Gal b (0.2 M) Acid buffer c D-Gal ( 0 . 2 M) D - G a l (0.1 M) D - G l c N A c (0.1 M) D - G a l (0.2 M) D-Gal (0.2 M) D - G a l (0.2 M) Acid buffer c L a c (0.1 M) D-Gal (0.1 M) Acid buffer c

25 r a g / 1 0 0 m l s e r u m 1.1 g / 1 0 0 g seeds 7 m g / k g fruiting b o d y 1 2 5 r a g / 1 0 0 g seeds 14 rag/1 kg f r u i t i n g b o d y 1 1 0 r a g / 1 0 0 g seeds 35 m g / 1 0 g a l b u m i n g l a n d s 9 m g / 1 0 0 g seeds 28 m g / k g f r u i t i n g b o d y 45 m g / k g r o o t 45 r a g / 1 0 0 g seeds 0 . 5 g / 1 0 0 g seeds 4 0 r a g / 1 0 0 g seeds 1 4 0 r a g / 1 0 0 g seeds

a m l o f gel s u f f i c i e n t f o r specific a d s o r p t i o n of 10 m g of t h e lectin. b E l u t i o n w i t h acidic b u f f e r y i e l d e d a d d i t i o n a l 5 m g o f l e c t i n p e r kg of t h e f r u i t i n g bodies. c 0 . 0 5 M glycine/HC1 b u f f e r ( p H 3 . 0 ) in saline. n.d., n o t d e t e r m i n e d .

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1, disc e l e c t r o p h o r e s i s (pH 8.9); 2, disc e l e c t r o p h o r e s i s ( p H 4.5); 3, d o d e c y l sulfate e l e c t r o p h o r e s i s ( n o n - r e d u c e d sample); 4, d o d e c y l sulfate e l e c t r o p h o r e s i s ( r e d u c e d sample); 5, isoeleetric focusing.

b . A n g u i l l a anguilla.

c. Vicia cracca. 1, disc e l e c t r o p h o r e s i s ( p H 4.5); 2, d o d e c y l sulfate e l e c t r o p h o r e s i s ; 3, disc d o d e c y l sulfate

e l e c t r o p h o r e s i s ( p H 9.5); 4, isoelectric focusing.

307

Anguilla anguilla An anomalously low value of molecular weight, with regard to the calculated s20,w value was obtained by thin layer gel filtration on Sephadex G-200 (Table III), possibly caused by an interaction of the lectin with Sephadex gel. All the zones (isolectins) observed on disc electrophoresis in alkaline or acidic buffer systems (Fig. l b ) interacted with immobilized L-fucosyl residues under the conditions of affinity electrophoresis [30,31].

Maclura pomifera During electrophoresis in dodecyl sulfate medium the non-reduced protein migrated as a large molecule yielding a very diffuse band at the t o p of the gel rod; the corresponding Mr could n o t be evaluated. After reduction a single band was observed (Fig. l d , Table III), which was not stained by Schiff reagent after periodate oxidation.

Fomes fomentar~us Investigation of this lectin was in some instances difficult due to an extremely high viscosity of its more concentrated solutions (>0.1%). As revealed by paper chromatographic analysis of the hydrolysate, main components of the carbohydrate moiety of the lectin are galactose, glucose and fucose.

Marasmius oreades The lectin interacted weakly with Sephadex gel; in the absence of D-galactose it migrated with a mobility corresponding to a Mr value lower than 14 000. Addition of 0.2 M D-galactose into the gel and elution solution resulted in an increase of the migration rate (apparent Mr = 50 000). Both zones observed after electrQphoresis in the presence of dodecyl sulfate (Fig. li) were stained by Schiff reagent after periodate oxidation.

d. Maclura p o m i f e r a . 1, disc electrophoresis (pH 8.9); 2, dodecyl sulfate electrophoresis (reduced sample);

3, isoelectric focusing. e. ~ a r o t h a r n n u s scoparius. 1, disc electrophoresis (pH 8.9); 2, dodecyl sulfate electrophoresis; 3, dodecyl sulfate electrophoresis, stained with Schiff reagent; 4, disc dodecyl sulfate electrophoresis (pH 9.5); 5, isoelectric focusing. f. O n o n i s spinosa. 1, disc electrophoresis (pH 8.9); 2~ disc electrophoresis (pH 4.5); 3, dodecyl sulfate electrophoresis; 4, dodecyl sulfate electrophoresis, staining with Schiff reagent; 5, disc dodecyl sulfate electrophoresis (pH 9.5); 6, isoelectric focusing. g. C l i t o c y b e nebularis. 1~ dodecyl sulfate electrophoresis; 2, dodecyl sulfate electrophoresis, staining with Schiff reagent; 3, dodecyl sulfate electrophoresis (lectin eluted with acidic buffer); 4, isoelectric focusing (1, 2, 4, lectin eluted with D-gaiactose). h. Fornes fomentari~is. 1, disc electrophoresis (pH 8.9); 2, disc electrophoresis (pH 4.5); 3, dodecyl sul-

fate electrophoresis; 4, dodecyl sulfate electrophoresis, staining with Schiff reagent; 5, isoelectric focusing. i. Marasrnius oreadis. 1, disc electrophoresis (pH 8.9); 2, disc electrophoresis (pH 4.5); 3, dodecyl sulfate

electrophoresis; 4, isoeleetric focusing.

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312 Discussion Isolation o f lectins The great differences in capacity of the O-glycosyl polyacrylamide gels for different lectins are remarkable. Especially concanavalin A, Ricinus communis, Helix pomatia, Dolichos biflorus and Vicia cracca lectins were adsorbed very effectively. These differences may be probably due to a combination of effects of the molecular weight, affinity toward the immobilized sugar, number of sugar binding sites in the lectin molecule and lectin content in the crude preparation. In addition to the lectins given in Table I, a number of other lectins could be purified successfully on O-glycosyl polyacrylamide gels. Pisum sativum [4], Vicia faba, Lathyrus sativus and Lens esculenta lectins (Ho~ej§f, V., unpublished results) could be purified on O-a-D-mannosyl- or O-a-D-glucosyl polyacrylamide gels; Bandeiraea simplicifolia (syn. Griffonia simplicifolia) [4], Streptomyces sp. (Fujita, Y., personal communication) and Erythrina edulis lectins (Perez, G., personal communication) can be purified by affinity chromatography on the O-a-D-galactosyl polyacrylamide gel. O-a-L-Fucosyl polyacrylamide gel was used for purification of Ulex europaeus lectins [4,28] and of some fish roe lectins (Krajhanzl, A., Ho~ej§f, V. and Kocourek, J., [65]), the O-fl-lactosyl polyacrylamide gel for preparation of Bauhinia purpurea var. alba lectin (Ho~ej~f, V., unpublished observation) and O-a-D-galactosyl- and O-a-L-rhamnosyl polyacrylamide gels were successfully applied for the purification of a number of fish roe lectins [65]. On the other hand, O-glycosyl polyacrylamide gels do not absorb some bacterial sugar binding proteins (Hogg, R.W. and Boos, W., personal communications). The sugar residues in our gels were attached to the basic polyacrylamide backbone by a very short spacer (one methylene group only); the general applicability of these gels as effective affinity sorbents for lectins indicates a good sterical accessibility of the sugar binding sites in lectin molecules. In fact, recent results support this assumption, at least in the case of concanavalin A [29]. Properties o f the lectins Anguilla anguilla. This lectin is in many respects similar to the lectin from A. rostrata [42,43] and from an unspecified eel species [44]. The main differences are in subunit molecular weight and in the presence of several isolectins in our preparation. Similar electrophoretic heterogeneity was observed also in the case of a fructosan specific protein (lectin) from nurse shark serum [45]. Vicia cracca. Although the V. cracca lectin has been isolated several times by affinity chromatography [46,47], its physicochemical properties were not reported. During preparation of the manuscript of this paper Riidiger [48] published a paper dealing with some properties of this lectin. His data are consistent with ours. Maclura pomifera. Some properties of this lectin were reported by Ulevitch et al. [49]. These authors did not report the subunit structure; most of their data are similar to ours.

313

Sarothamnus scoparius. Brossmer et al. [51] have purified two similar lectins from the seeds of Cytisus scoparius, (a s y n o n y m o u s designation for S. scoparius). Our preparation seems to be identical with their CS I lectin; molecular weight, subunit composition and inhibition with simple sugars are similar for these t w o preparations. Ononis spinosa. The only a t t e m p t to purify the lectin from O. spinosa root was made by Herzog and Sou5ek [52], who also first recognized the inhibition of this lectin by D-galactose and lactose [50]. Their preparation, obtained by preparative electrophoresis, was evidently only partially purified. The lectins from plant roots have been studied only exceptionally so far. Only the mitogenic lectins from the root of Phytolacca americana [53] and Phytolacca esculenta [54] were obtained in purified form. It should be noted that the physicochemical properties of the O. spinosa root lectin are similar to most leguminosae seed lectins. Although it is relatively anti-O hemagglutination specific, it is best inhibited by N-acetyl-D-galactosamine and D-galactose; other anti-O specific lectins are usually inhibited either by L-fucose or chitin derived oligosaccharides. A lectin practically identical with the present one is contained also in the root of Ononis hircina [55]. Clitocybe nebularis. Our work was based on the observation by Coulet et al. [56], who reported inhibition of C. nebularis lectin by N-acetyl-D-galactosamine. No physicochemical properties of this mushroom lectin were reported as yet. Fomes fomentarius. So far, F. fomentarius B-specific lectin was studied mainly serologically in crude extracts [58,59]. Anstee [60] estimated the molecular weight of this lectin as 61 000--67 000, its hemagglutinating activity being resistant to mercaptoethanol and more sensitive to inhibition by N-acetyl-D-galactosamine than by D-galactose; these results are consistent with ours. The F. fomentarius lectin is interesting due to its high specificity for B-group erythrocytes; in fact none of the lectins purified so far is comparably anti-B specific. Unusual is also its high sugar content and extreme viscosity of its solutions. Marasmius oreades. To our knowledge, only serological studies on M. oreades crude lectin preparations were performed up to the present [61,62]. It should be noted that none of the three mushroom lectins described in this paper resembles the lectin of Agaricus campestris, the only mushroom lectin studied in purified state so far [63]. Acknowledgements The authors are indebted to Mr. J. Melena, Plan~ nad Lu~nicf, for providing eel serum; to Ing. P. Smolek, Department of Biochemistry, Charles University, Praha, for sedimentation coefficients and amino acid analyses; to Dr. M. Tich:~, Institute of Hygiene and Epidemiology, Praha, for metal content estimation by atomic absorption spectroscopy; and to Dr. C. Ha§kovec, Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Praha, for mitogenic activity determination.

314

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