Biomedical subjects
Publications and source records attributed to H Rüdiger.
The alpha-mannosidase from Canavalia ensiformis is devoid of cysteine and cystine. Nevertheless, its cleavage into subunits is facilitated by the presence of mercaptoethanol in the denaturing buffer.
The immobilized lectin from the lentil (Lens culinaris) specifically binds two fractions out of the L. culinaris seed globulins. Both fractions are displaced from the lectin at low pH values. In addition, fraction I fails to interact at high ionic strengths, and fraction II in the presence of glucose or other lectin-specific sugars. The behaviour in zonal isoelectric precipitation and electrophoretical patterns indicate that both fractions represent subpopulations of the storage proteins. The interaction as demonstrated by affinity chromatography is corroborated by nephelometry: If the dissolved proteins (lectin plus fraction I or fraction II) are mixed under proper conditions the solutions become turbid. An even more pronounced interaction is observed if the lectin is reacted with both fractions at the same time. Seed albumins able to interact with the immobilized lectin include the dissolved lectin and two glycosidases (alpha-mannosidase, alpha-galactosidase) all of which are located in the protein bodies. A third glycosidase (beta-galactosidase) from outside of the protein bodies does not bind to the lectin. The results are discussed in view of the possibility that lectins may serve as packaging aids for other proteins in the protein bodies.
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Previous work from this laboratory had shown that Leguminosa seed extracts contain lectin-bound proteins. In the present paper, the isolation of protein bodies from the seeds of 7 Leguminosa species (Canavalia ensiformis, Lens culinaris, Pisum sativum, Glycine max, Sophora japonica, Wisteria floribunda and Phaseolus vulgaris) is described. Protein bodies were characterized microscopically and by their constituents, storage proteins, lectins and some glycosidases. From the protein bodies, lectin-bound proteins were isolated and were shown to be identical with those from whole seed extracts. This indicates a common localization of lectin-bound proteins and of lectins. Lectin-bound proteins belong to the storage proteins and to the proteins with glycosidase activity. The common localization of these proteins and interactions between them suggest a biological role of seed lectins: during maturation they may act as a packaging aid for storage proteins and enzymes into developing protein bodies. Lectins thus may contribute to an ordered construction and degradation of protein bodies.
Two lectins from the seeds of Robinia pseudoacacia were isolated and resolved in a single affinity chromatography run using immobilized ovomucoid by successive elution with different buffers. The lectins were characterized by their subunit sizes, total molecular masses and isoelectric points as well as by their ability to bind to ConA-Sepharose and to stimulate mitosis in murine lymphocytes.
A method is described for the purification of the alpha-mannosidase from Canavalia ensiformis. By three consecutive steps, a more than 500-fold purification is achieved and the pure enzyme obtained in 75% yield. One of these steps utilizes the specific interaction of the alpha-mannosidase with concanavalin A, the lectin from the same plant. This interaction is dependent on pH and ionic strength but does not involve the sugar binding site of the lectin. The interaction between both proteins may be important also in vivo.
The specific interaction between the lectin concanavalin A and the alpha-mannosidase from the Leguminosa Canavalia ensiformis was studied by means of laser nephelometry and affinity chromatography. Both proteins react optimally within a certain stoichiometrical range. Interaction is restricted to a narrow pH interval (around pH 5) and to low ionic strengths (less than 10mM NaCl). Neither the sugar-binding site of the lectin nor the catalytic and the hydrophobic sites of the enzyme participate in the interaction. The conformation of the enzyme at pH 5 which favours the interaction can be arrested by immobilization. After this, the enzyme is able to bind the lectin even at pH 8 where no interaction takes place between the dissolved proteins.
The lectin-binding protein (lectin binder) from the garden pea (Pisum sativum) was studied. It is a glycoprotein composed of four subunits of about 50 000 Da. Its amino-acid composition and molecular mass differ from those of lectin and of storage proteins. The interaction between lectin and lectin binder is demonstrated and quantified by several different methods and is shown to be specifically sugar-dependent. A biological function of lectin binders and lectins is discussed.
Two affinity adsorbents of general applicability for isolating of plant lectins are presented. The isolation of 30 lectins from 27 plants is described. The method works quickly and is inexpensive. In particular, mixtures of different lectins occurring in some plants may be resolved in one run.
A new fast-working one-step method for the resolution of the Phaseolus vulgaris isolectins is described which requires inexpensive materials.
Circulating insulin binding capacity was studied for 10 to 24 months in forty diabetic patients who were being treated with insulin for the first time. They received either neutral highly purified pork insulin, bovine or porcine desphe insulin containing formulations, semisynthetic human insulin, or "conventional" acid chromatographed bovine surfen insulin over the entire duration of the study. In only 6 of the 23 patients on pork insulin, porcine desphe insulin, or human insulin were minimal antibody concentrations (less than or equal to 2 IU/l) observed. The bovine desphe insulin containing beef insulin, was markedly more immunogenic: only 3 out of 12 patients did not produce any antibodies. All five patients treated with bovine surfen insulin reacted with a strong antibody formation, which was, on average, six times as high as that in the patients on bovine desphe insulin (approximately 90 vs 14 IU/l). According to these results, bovine desphe insulins should be considered more valuable in insulin therapy than formulations of unmodified beef or of mixed beef/pork insulins.
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It was found that lectin-binding protein (LB) from leguminosae seeds can serve as mitogens B lymphocytes from athymic nu/nu mice. Most of the experiments were performed using a LB from Vicia faba as this was the most potent mitogen among all LB tested. When mixed B and T lymphocyte populations were stimulated by LB a bell-shaped dose-response curve resulted which is also characteristic of the corresponding lectins; LB, however, act at much higher concentrations than the lectins. In control experiments, using lymphocytes from C3H/HeJ mice which have a genetic defect with respect to lipopolysaccharide responsiveness, the enhancement of DNA synthesis by LB was comparable to that observed with other strains. This indicates that stimulation by LB does not result from contamination by lipopolysaccharides. B lymphocytes from nu/nu mice were stimulated by LB as efficiently as mixed T and B lymphocyte populations whereas lectins per se had no effect on nu/nu lymphocytes. Cyclosporin A, a fungal metabolite, is known to specifically suppress T cell responses. Cyclosporin A did not influence the mitogenic activity of the LB from Vicia faba on unseparated spleen cells. Cell populations enriched for T cells (lymph node cells: nylon wool-passed or nylon wool-passed and anti-Ia plus complement-treated) responded poorly to LB, if at all, even in the presence of interleukin 2-containing supernatants.
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The seeds of Vicia cracca are known to contain two lectins: an N-acetylgalactosamine-binding lectin, which reacts specifically with human erythrocytes of blood group A, and a mannose/glucose-binding lectin unspecific towards human blood groups. The second lectin is the object of the present work. Purification was achieved by affinity chromatography on Sephadex G-100. The native lectin has a molecular weight of 44,000. It is composed of two small (Mr 5,700) and two large subunits (Mr 17,500). Binding of sugars to the lectin was assayed by equilibrium dialysis and spectrophotometrically; the association constants for glucose are 1.38 X 10(3) l/mol and 2.5 X 10(2) l/mol respectively. The primary structure of the small subunit was determined by analyses of the whole chain, of tryptic and CNBr peptides. It was shown to consist of 53 amino acid residues. The mannose/glucose-binding lectin from Vicia cracca is highly homologous to the lectins from Vicia sativa, Vicia faba, Pisum sativum and Lens culinaris. There is, however, only limited homology of the N-acetylgalactosamine-specific lectin from Vicia cracca.
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The complete amino acid sequence of the alpha chain of the mitogenic lectin from Vicia sativa has been determined. The polypeptide was digested by trypsin and chymotrypsin. The fragments were isolated and most of them were sequenced by automatic solid-phase Edman degradation. A total sequence of 52 amino acid residues was obtained which is homologous to the alpha subunits of the lectins from Pisum sativum, Lens culinaris and Vicia faba. The central part (residues 13-32) in particular is completely conserved in all four proteins. In contrast to the three other known sequences, the V. sativa alpha subunit has an additional serine at the N terminus. The differences of the related amino acid sequences of these lectins and concanavalin A, the lectin from Canavalia ensiformis, have been compared using the relative substitution frequency found in homologous proteins by McLachlan. The degree of homology decreases in the order: V. sativa greater than P. sativum greater than V. faba greater than L. culinaris greater than C, ensiformis. Prediction of the secondary structure according to Chou and Fasman reveals that the lectin alpha chain is similar to concanavalin A in the first half of the molecule whereas the C-terminal part apparently tends to form an alpha helix.