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J Kocourek

Publications and source records attributed to J Kocourek.

At least 55 records · Page 3Linked to original sources
PubMed1978

Studies on 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.

Agglutinins↗
PubMed1978

Additional precipitation reactions of lectins with human serum glycoproteins.

Highly purified human serum glycoproteins were treated with neuraminidase and examined for their cross reaction with several lectins with anti-galactosyl specificity: beta-D-galactosyl structures are thought to be the main terminal sugar residues that become attached de novo after removal of neuraminic acid. The following lectins were tested: Tridacnin from the bivalve clams Tridacna maxima and Tridacna gigas, the agglutinin from the sponge Axinella polypoides, the lectin from the roach Rutilus rutilus and the plant lectins from Ricinus communis, Ononis spinosa, Glycine soja and Abrus precatorius. In agar gel diffusion, these purified and precipitating lectins gave more or less strong or negative results against the different neuraminidase-treated serum glycoproteins, thus indicating subtle differences with respect to their anti-galactosyl combining specificity. On the other hand, serum glycoproteins which reacted with the same lectin, did not always show complete identity lines. Finally, as revealed by these lectins, the carbohydrate moiety of serum glycoproteins may reflect a complex and broad spectrum of heterogeneity. This could lead to a more detailed understanding of the topographical and steric arrangement of the chemical structure and of the biological role of carbohydrate groups in these glycosubstances.

Animals↗
PubMed1978

Studies on lectins. XXXIV. The effect of thermal treatment on the lectin of lentil seeds (Lens esuclenta Moench).

The lectin of lentil seeds (Lens esculenta Moench) dissociates into subunits upon incubation of its solution at temperatures above 50 degrees C. The dissociation is accompanied by precipitation of the heavy subunit, release of Mn2+ from the protein molecule and an almost complete loss of erythroagglutinating activity. Addition of Mn2+ or D-glucose to the lectin solutions has a partially protective effect on the dissociation and inactivation.

Glucose↗
PubMed1977

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.

Electrophoresis, Polyacrylamide Gel↗
PubMed1977

Studies on 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.

Acetylation↗
PubMed1977

alpha-Mannosidase and mannanase of some wood-rotting fungi.

Cultivation media from 11 wood-rotting fungi contained alpha-mannosidase and mannanase activity, alpha-Mannosidase was studied in more detail in Phellinus abietis and mannanase was studied more intimately in basidiomycetes Phellinus abietis, Trametes sanguinea and Pholiota aurivella. Suitable cultivation conditions and optimum conditions for the production of alpha-mannosidase and mannanase were determined. Both enzymes are constitutive; mannanase is extracellular, alpha-mannosidase was found in both mycelium and cultivation medium.

Basidiomycota↗
PubMed1977

Separation and properties of alpha-mannosidase and mannanase from the basidiomycete Phellinus abietis.

Proteins of a crude enzyme preparation obtained from the cultivation medium of the basidiomycete Phellinus abietis were separated by gel filtration and ion-exchange chromatography. The preparation contained a minimum of three enzymes capable of splitting alpha-D-mannosidic bonds: alpha-mannosidase, exomannanase, and endomannanase, which were separated. Some properties of the mannanase complex of the crude enzyme preparation, and of a partially purified alpha-mannosidase were examined. The mannanase complex exhibited two pH optima, its temperature optimum being at 45 degrees C. The pH optimum of purified alpha-mannosidase was at pH 5.0, the temperature optimum being at 45 degrees C. The pH optimum of purifed alpha-mannosidase was at pH 5.0, the temperature optimum at at 60 degrees C; the enzyme had a relatively high heat stability. The Km of alpha-mannosidase for p-nitrophenyl alpha-D-mannopyranoside was 1.5 X 10(-5) M. Pure alpha-mannosidase did not split mannan.

Basidiomycota↗
PubMed1976

Studies on phytohemagglutinins. XXVIII. Chemical modifications of the lectin from seeds of the lentil (Lens esculenta Moench.)

The reaction of lentil lectin with maleic anhydride yields maleyl derivatives with a degree of modification depending on the amount of used maleic anhydride. Hemagglutinating activity of maleyl derivatives decreases with the increasing number of modified lysine residues, but even at a maleylation of 90% of free -NH2 groups, the maleyl derivative of the lectin still retains a hemagglutinating activity. Maleylation of the lentil lectin is not accompanied by dissociation; the chemically modified lectin also retains its bound Mn. Maleyl derivatives of the lectin retain the ability to interact specifically with polysaccharides (Sephadex, mannan). After removal of maleyl groups in acidic medium, the hemagglutinating activity does not differ from that of the native lectin. Acetylation of the lentil lectin with N-acetylimidazole results in formation of a modified protein containing 3-5 acetylated -OH groups of the tyrosyl residues out of the total of 15 groups and 7-17 acetylated -NH2 groups out of the total of 26 free -NH2 groups, depending upon the reaction conditions. The presence of D-mannose in the reaction mixture does not affect the extent of modification of tyrosyl groups, but it decreases the modification of -NH2 groups. Acetyl derivatives of the lentil lectin do not possess hemagglutinating activity, but they retain the ability to interact with polysaccharides and to bind to erythrocytes. Acetylation does not affect the content of protein bound Mn. After de-O-acetylation, the hemagglutinating activity of the modified protein increases to the value of the native protein.

Acetates↗
PubMed1976

Studies on phytohemagglutinins. XXVII. A study of the pea lectin binding site.

Under defined mild conditions the reaction of the pea lectin with 2-nitrophenylsulfenyl chloride results in sulfenylation of only 2 of the 10 tryptophan residues of the lectin molecule with simultaneous loss of biological activity. Both sulfenylated tryptophan residues belong to the two heavy subunits of the lectin. Enzymic hydrolysis and separation of the tryptic peptides yields only one homogeneous yellow peptide containing the modified tryptophan residue. The isolated peptide has the following sequence (NPS, nitrophenylsulfenyl): HAsp-Val-Val-Pro-Glu-(2-NPS-Trp)-Val-ArgOH. The octapeptide is either directly a part of the pea lectin binding site or it plays an important role in maintaining the tertiary structure of the binding site. According to the amino acid composition and amino acid sequence, the octapeptide isolated from the pea lectin is almost identical with that part of the peptide chain of concanavalin A near to which the location of the sugar binding site is supposed to be.

Amino Acid Sequence↗
PubMed1975

Studies on phytohemagglutinins. XXV. Isolation and characterization of hemagglutinins of the spindle tree seeds (Evonymus europaea L.).

A mixture of isophytohemagglutinins has been isolated from the fleshy arils of the spindle tree seeds (Evonymus europaea L.) by fractional precipitation of the saline extract of the arils by (NH4)2SO4 at a 0.40% saturation. Successive preparative disc electrophoresis on polyacrylamide gel affords separation of one slower moving component, phytohemagglutinin I, from the mixture of other isophytohemagglutinins that have a very similar electrophoretic mobility. Phytohemagglutinin I has a sedimentation coefficient Sw,20 of 7.1 S and an approximate mol. wt of 127 000. Amino acid analysis shows a high amount of aspartic acid, alanine and glycine but also significant amounts of serine, threonine, cysteine and methionine. Aspartic acid is the only N-terminal amino acid found by the dansylation technique. Phytohemagglutinin I contains glucosamine and 4.7% neutral sugar. Its approximate pI in citrate/phosphate buffer is 4.4-4.5. The metal content amounts to 0.250% Ca, 0.019% Mg, 0.034% Zn and 0.026% Cu. Mn is not present. Ultracentrifugation analysis reveals homogeneity in the sedimentation behavior of the mixture of isophytohemagglutinin, an Sw,20 of 7.1 S and an approximate mol. wt of 119 000. The mixture has an amino acid composition closely resembling that of phytohemagglutinin I and an identical pI but contains only 1.9% neutral sugar. Two N-terminal amino acids were shown to be present, aspartic acid and tyrosine. With the exception of Cu which is absent, the metal content is almost the same as that of phytohemagglutinin I. Both phytohemagglutinin I and the mixture are devoid of anti-A1 activity and show detectable anti-H, anti-B and anti-A2 erythroagglutinating activity in approximate limit concentrations of 2.5, 5 and 10 mug/ml, respectively. This activity is not influenced by the presence of EDTA, Ca2+ or Mg2+, but is stimulated by Zn2+. Mn2+ and Co2+ have an inhibitory effect. None of the simple sugars tested inhibited the hemagglutination reactions.

Amino Acids↗
PubMed1975

Studies on phytohemagglutinins. XXVI. A new type of a lentil hemagglutinin isolated from Lens esculenta Moench., subsp. microsperma (Baumg.) Barulina.

In seeds of the lentil Lens esculenta Moench., subsp. microsperma (Baumg.) Barulina, a nonspecific phytohemagglutinin was found, the properties of which differ from those of the previously described lentil hemagglutinins. The phytohemagglutinin is not adsorbed to the Sephadex matrix; its hemagglutinating activity is different towards erythrocytes of different human blood groups of the ABO system. The isolated phytohemagglutinin preparation is homogeneous by disc polyacrylamide and starch gel electrophoreses and by gel chromatography on Bio-Gel P-100. Ultracentrifugation of the phytohemagglutinin yields a single symmetrical peak with S20,W of 3.9 S. From the sedimentation data, a molecular weight of 53 300 was calculated. Phytohemagglutinins of similar properties were shown to be present also in other cultivars of Lens esculenta, subsp. microsperma.

ABO Blood-Group System↗
PubMed1975

Studies on phytohemagglutinins. XXIV. Isoelectric point and hybridization of the pea (Pisum sativum L.) isophytohemagglutinins.

The isoelectric point of the two pea isophytohemagglutinins varies from pH 5.7 to pH 8.4 depending on the composition of the buffer used. Isoelectric focusing reveals three main molecular species with pI at pH 5.90, 6.35 and 7.00. Molecular species with pI at pH 5.9 and 7.0 correspond to the two pea isophytohemagglutinins which can be obtained by DEAE-cellulose chromatography (Entlicher, G., Kostír, J.V. and Kocurek, J. (1970) Biochim. Biophys. Acta 221, 272-281). A molecular species with pI at pH 6.35 is formed from the two pea isophytohemagglutinins by hybridization. According to the hybridization pattern and subunit composition of the pea isophytohemagglutinins the subunit composition AABB, AACC and AABC can be proposed for the three molecular species with respect to ionic properties of the subunits.

Buffers↗