V. Hořejší

Studies on lectins. XXXV. Water-soluble O-glycosyl polyacrylamide derivatives for specific precipitation of lectins.

By copolymerization of acrylamide and allyl glycosides of various sugars, O-glycosyl derivatives of polyacrylamide copolymers were prepared. The sugar content of the copolymers can be varied in the range 0--40%, their sedimentation coefficient shows the vales of 2.5-5.7 S; the molecular weight of an O-alpha-D-mannopyranosyl polyacrylamide copolymer (29% mannose, so20,w = 2.9 S) was estimated as 44 500. Copolymers with incorporated glycosyl residues interacting specifically with lectins yield precipitates with them upon immunodiffusion in cellulose acetate. The quantitative precipitin curves obtained with these copolymers are similar to those produced by quantitative precipitation of lectins with natural polysaccharides. The copolymers may serve as model substances of natural polysaccharides.

Studies on lectins. XXXI. Determination of dissociation constants of lectin. Sugar complexes by means of affinity electrophoresis.

A modification of affinity electrophoresis in polyacrylamide gels containing immobilized sugar residues is described. The immobilization of sugar residues is achieved by addition of a water soluble O-glycosyl polyacrylamide copolymer to the polymerization mixture, which serves for the preparation of gels for commonly used discontinuous polyacrylamide gel electrophoresis. The electrophoretic mobility of lectins in affinity gels containing specific immobilized sugar decreases with increasing sugar concentration. The addition of free sugar into the affinity gel abolishes retardation of the electrophoretic mobility caused by immobilized sugar. The relationship between electrophoretic mobility of lectins and concentration of immobilized and free sugar was used for the calculation of dissociation constants of the various complexes lectin-immobilized sugar and lectin-free sugar.

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

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 (alpha-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-alpha-D-galactosaminyl-); Ricinus communis, seeds (beta-lactosyl-); Ononis spinosa, root; Fomes fomentarius, fruiting bodies; Marasmius oreades, fruiting bodies (all alpha-D-galactosyl-), Canavalia ensiformis, seeds, (i.e., concanavalin A) (alpha-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 physiochemical 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.

Studies on phytohemagglutinins XII. O-glycosyl polyacrylamide gels for affinity chromatography of phytohemagglutinins

Abstract Copolymerization of alkenyl O -glycosides with acrylamide and N , N ′-methylene bisacrylamide produces neutral hydrophilic gels containing sugars having O -glycosidic links with the alkyl side chains of the polymer matrix. The preparation of these copolymers and their application as affinity adsorbents for the specific separation of phytohemagglutinins from other proteins is described.

Studies on lectins

SummaryThe occurrence of endogenous lectins in the ovaries of four fish species has been studied by indirect immunofluorescence staining with antibodies against individual lectins. Paraffin sections of the ovary of perch (Perca fluviatilis L.) were treated with an antibody against perch lectin. In cryostat sections of the tench (Tinca tinca L.) ovary, the l-rhamnose-specific lectin “I” was detected with a specific antibody. In cryostat sections of both roach (Rutilus rutilus L.) and rudd (Scardinius erythrophthalmus L.) ovaries, lectins were localized using a single antibody against roach lectin. The isolation of tench lectins is briefly described. In the fish species employed for this study, lectins are associated exclusively with the content and surrounding membrane of cortical vesicles situated within the cytoplasm of maturing oocytes. The positive reaction with lectin antibody was observed almost immediately after the formation of the first cortical vesicles in the peripheral cytoplasm of early previtellogenic oocytes. Their lectin content increases during the later stages when cortical granules fill the whole cytoplasm before moving towards the cell periphery, as the oocyte starts to accumulate yolk. The presence of lectins within cortical vesicles is significant also in view of the polysaccharide content of these structures. In the vitellogenic oocytes lectins seem to move towards the cell periphery and accumulate beneath the plasma membrane. Our observations are discussed in view of the present ideas on the intracellular function of lectins, and with respect to the role of cortical vesicles in fertilization and in postfertilization modifications of the egg envelopes.

Studies on phytohemagglutinins XVIII. Affinity electrophoresis of phytohemagglutinins

Abstract A new separation technique has been developed combining the principles of a affinity chromatography and electrophoresis. On a polyacrylamide gel column composed of layers of large-pore gel, gel with covalently linked affinity ligands (affinity gel) and small-pore gel, it is possible to “filter off” on the affinity gel proteins with combining sites for the corresponding ligand and to separate the remaining components on the small-pore gel. Several examples are given for the separation of phytohemagglutinins from the plant protein mixtures using O-glycosyl polyacrylamide derivatives as affinity gels.

Affinity electrophoresis of proteins interacting with blue dextran

Interaction of several enzymes (pyruvate kinase, myokinase, creatine kinase, aldolase, malate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase) and other proteins (bovine serum albumin and ovalbumin) with Blue Dextran was studied by means of affinity electrophoresis in polyacrylamide gels. A decrease of electrophoretic mobility of enzymes in affinity gels was dependent on Blue Dextran concentration and in some cases, dissociation constants of the protein-immobilized dye complexes could be calculated. Affinity electrophoresis in the presence of Blue Dextran reveals in some cases additional bands of isoenzymes, as compared with the control gels (without Blue Dextran).

Studies on phytohemagglutinins XVII. Some properties of the anti-H specific phytohemagglutinin of the furze seeds (Ulex europaeus L.)

The anti-H specific hemagglutinin has been isolated from the seeds of Ulex europaeus L. by saline extraction, (NH4)2SO4 precipitation and affinity chromatography of the active protein fraction on an O-α-l-fucosyl polyacrylamide adsorbent. The phytohemagglutinin reveals a single symmetrical peak on ultracentrifugation and a s20,w of 4.1 S. Sedimentation equilibrium data and the results of the polyacrylamide electrophoresis in sodium dodecylsulfate medium indicate a molecular weight of 46 000 and 40 000, respectively. There is no observable dissociation in the sodium dodecylsulfate medium of the phytohemagglutinin treated with 4 M urea and/or mercaptoethanol. The substance appears homogeneous also by electrophoresis on cellulose acetate strips and its mobility indicates an approximate pl of 6.0–6.1 in the phosphate buffer. On disc polyacrylamide gel electrophoresis at pH 8.9 a minor component separates from the bulk of the substance, presumably an isophytohemagglutinin. The amino acid analysis demonstrates the presence of high amounts of aspartic acid, serine and glycine and of only small amounts of typptophan, cysteine and methionine. Serine was shown to be the only N-terminal amino acid detectable by the dansylation technique. The phytohemagglutinin contains 2 atoms of Ca and 1 atom of Zn or Mn per molecule. The minimum hemagglutinating dose of the phytohemagglutinin is 1.2 μg/ml against O-type erythrocytes, 10 μg/ml against A2 type, 40 μg/ml against B-type and 50 μg/ml against A1-type. It is only slightly activated by Zn2+ and Mn2+ and there is the inhibition of its activity by EDTA. Prolonged photooxidation in the presence of ethylene blue likewise shows no effect on the activity of the phytohemagglutinin.

Studies on lectins: XLII. Isolation, partial characterization and comparison of lectins from the roe of five fish species

Abstract Lectins from the roe of four species of the Cyprinidae family, the roach (Rutilus rutilus), the vimba or zahrte (Vimba vimba), the rudd (Scardinus erythrophtalmus) and the bream (Abramis brama) were isolated by affinity chromatography on O-α- d - galactosyl polyacrylamide gel. The lectin from the roe of the perch (Perca fluviatilis, family Percidae) was isolated by affinity chromatography on O-α- l - fucosyl polyacrylamide gel. The lectin isolated from R. rutilus consists of subunits (mol. wt. approx. 25 000) and their various associates. Sedimentation coefficients of the main components are s20,w = 2.1 S and 7.7 S. The degree of association depends on pH, ionic strength and urea concentration. The lectin contains much cysteine and little aromatic amino acids, 2.6% of neutral sugar and 0.145% of Zn. It precipitates water soluble O-glycosyl polyacrylamide copolymers and agglutinates almost equally erythrocytes of all groups; the agglutination is inhibited best by l -rhamnose. The association constant of the complex lectin · l - rhamnose (KA) was determined from dependence of fluorescence quenching (fluorimetric titration). Two KA were found (KA = 2.1 · 103 M−1 and 1.4 · 102 M−1). The association constants of the complex with d -galactose are lower (KA = 1.1 · 102 M−1 and 1.7 · 101 M−1). The properties of lectins from V. vimba and S. erythrophtalmus are very similar to those of R. rutilus. The lectin from A. brama consists probably of subunits of molecular weight 15 000, it is relatively B-specific and is inhibited by l -rahmnose and d -galactose. The lectin isolated from P. fluviatilis roe consists of different associates of a basic subunit (Mr = 13 000). Sedimentation coefficients of the main components are s20,w = 3.3 S and 7.6 S. The lectin contains much cysteine and phenylalanine as well as tryptophan, 2% neutral sugar and 0.57% Zn. The lectin is relatively O-specific, its agglutination is inhibited almost equally by l -fucose, d -glucose and d -mannose. EDTA is strongly inhibitory, also Zn2+ and Mn2+ (0.1 M) partially inhibit the agglutination. The association constant of the complex lectin · l - fucose is KA = 3.4 · 102 M−1, as determined from concentration dependence of fluorescence enhancement.

Studies of lectins XXXII. Application of affinity electrophoresis to the study of the interaction of lectins and their derivatives with sugars

Affinity electrophoresis was used to study the sugar binding heterogeneity of lectins or their derivatives. Commercial and demetallized preparations of concanavalin A could be resolved by affinity electrophoresis into three components with different affinity to immobilized sugar. Similarly the Vicia cracca lectin obtained by affinity chromatography behaved on affinity gels as a mixture of active and inactive molecular species. Affinity electrophoresis has shown that the nonhemagglutinating acetylated lentil lectin and photo-oxidized or sulfenylated pea lectin retain their sugar binding properties; dissociation constants of saccharide complexes of these derivatives are similar to those of native lectins. The presence of specific immobilized sugar in the affinity gel improved the resolution of isolectins from Dolichos biflorus and Ricinus communis seeds.