Universiẗat, Diss., 2003--Tübingen.

Universiẗat, Veterinärmedizinische Fakultät, Diss., 2005--Leipzig.

Submitted in partial fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban Institute of Technology, 2003. / South Africa produces 190 000 tonnes of soybean per annum. Seed producing companies require knowledge of the diversity of the germplasm to produce hybrids that will be competitive in local and overseas markets. Furthermore, they need to ascertain the presence/absence of the anti-nutritional factor, Kunitz trypsin inhibitor protein. Currently, seed producing companies plant the seed and wait for the grow-out in order to select desirable traits. This process is time-consuming, tedious and does not necessarily ensure the selection of the best genetic stability as it is based on phenotypic expression alone. This study was undertaken to evaluate a molecular method to determine the genetic diversity among soybean parent lines and optimize a method which can be used to evaluate seeds for the Kunitz trypsin inhibitor protein / M

Bacteroides fragilis strain Bf-1 produces an extracellular bacteriocin at the beginning of the stationary growth phase. Production is not inducible by either ultraviolet light or mitomycin C. The low molecular weight bacteriocin (MW estimates of 13 500 and 18 800 obtained from Sephadex G-100 chromatography and SDS-PAGE electrophoresis respecively) is stable between pH 7 - 9 and is inactivated on incubation with trypsin and pronase. An unusual feature of the Bf-1 bacteriocin is its apparent biphasic temperature stability: while the majority of the activity (97%) is destroyed by heating at 60ºC (t [subscript] 1/2 = 2.5 min at 60ºC), a small proportion (3%) is stable even after autoclaving at 121ºC for 15 min. The killing of sensitive cells occurs in 2 stages and the killing action is reversed by incubation with trypsin. The transition from stage I to stage II is dependent on the temperature of incubation and the growth state of sensitive cells. 2,4-Dinitrophenol prevents this transition. The Bf-1 bacteriocin has an unusual mode of action. It specifically inhibits RNA synthesis whilst having no effect on protein or DNA synthesis. No effect on intracellular ATP levels were observed. The heat-stable (3%) fraction had a similar biochemical effect. In vitro studies involving RNA polymerase indicated that the bacteriocin and the antibiotic rifampicin have similar effects on RNA synthesis. The bacteriocinogenic strain (Bf-1) is insensitive to its own bacteriocin both in vivo and in vitro, although this immunity is overcome in vitro by the addition of higher concentrations of the Bf-1 bacteriocin. The bacteriocinogenic strain (Bf-1) harbors a cryptic plasmid (or plasmids) which on a neutral sucrose gradient, sediments faster than the Col E1 marker plasmid DNA. Attempts to cure this strain of its bacteriocinogenic phenotype were unsuccessful.

Tryptases are trypsin‐like serine proteinases found in the granules of mast cells. Although they show 40% sequence identity with trypsin and contain only 20 or 21 additional residues, tryptases display several unusual features. Unlike trypsin, the tryptases only make limited cleavages in a few proteins and are not inhibited by natural trypsin inhibitors, they form tetramers, bind heparin, and their activity on synthetic substrates is progressively inhibited as the concentration of salt increases above 0.2 M. Unique sequence features of seven tryptases were identified by comparison to other serine proteinases. The three‐dimensional structures of the tryptases were then predicted by molecular modeling based on the crystal structure of bovine trypsin. The models show two large insertions to lie on either side of the active‐site cleft, suggesting an explanation for the limited activity of tryptases on protein substrates and the lack of inhibition by natural inhibitors. A group of conserved Trp residues and a unique proline‐rich region make two surface hydrophobic patches that may account for the formation of tetramers and/or inhibition with increasing salt. Although they contain no consensus heparin‐binding sequence, the tryptases have 10–13 more His residues than trypsin, and these are positioned on the surface of the model. In addition, clustering of Arg and Lys residues may also contribute to heparin binding. Putative Asn‐linked glycosylation sites are found on the opposite side of the model from the active site. The model provides structural explanations for some to the unusual characteristics of the tryptases and a rational basis for future experiments, such as site‐directed mutagenesis.

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Cheung, Hang Kei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 138-149). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.vi / List of Abbreviations --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / Chapter Chapter 1: --- Introduction of Lectins --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.1.1 --- Definition and History of Lectins --- p.1 / Chapter 1.1.2 --- More than Just Carbohydrate Binding --- p.2 / Chapter 1.1.3 --- Classification of Lectins --- p.3 / Chapter 1.2 --- Plant Lectins --- p.4 / Chapter 1.2.1 --- History of Plant Lectins --- p.4 / Chapter 1.2.2 --- Occurrence of Plant Lectins --- p.5 / Chapter 1.3 --- Physiological Roles of Plant Lectins --- p.6 / Chapter 1.3.1 --- Lectins as Storage Proteins --- p.6 / Chapter 1.3.2 --- Lectins as Defense Proteins --- p.7 / Chapter 1.3.3 --- Lectins as mediator in symbiosis with bacteria --- p.8 / Chapter 1.4 --- Biological Activities of Plant Lectins --- p.9 / Chapter 1.4.1 --- Immunomodulatory Activity --- p.9 / Chapter 1.4.2 --- Lectins and Cancer --- p.10 / Chapter 1.4.3 --- A ntiviral A ctivity --- p.12 / Chapter 1.5 --- Lectins in Glycomic Study --- p.14 / Chapter 1.5.1 --- Background --- p.14 / Chapter 1.5.2 --- Glyco-catch method --- p.15 / Chapter 1.5.3 --- Lectin Blot Analysis --- p.16 / Chapter 1.6 --- Aim of current study --- p.17 / Chapter Chapter 2: --- Purification and Characterization of a Lectin from Musa acuminata --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Materials and Methods --- p.20 / Chapter 2.2.1 --- Purification Scheme --- p.20 / Chapter 2.2.2 --- Assay of Hemagglutinating A ctivity --- p.21 / Chapter 2.2.3 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.22 / Chapter 2.2.4 --- Molecular Mass Determination by FPLC Gel Filtration --- p.22 / Chapter 2.2.5 --- Protein Concentration Determination --- p.22 / Chapter 2.2.6 --- N-terminal amino acid sequence analysis --- p.22 / Chapter 2.2.7 --- Inhibition of Lectin-induced Hemagglutination by Carbohydrates --- p.23 / Chapter 2.2.8 --- Effect of Temperature and pH on Lectin-induced Hemagglutination --- p.23 / Chapter 2.2.9 --- Assay of Mitogenic Activity on Murine Splenocytes --- p.24 / Chapter 2.2.10 --- Assay of Nitric Oxide Production by Murine Peritoneal Macrophages --- p.25 / Chapter 2.2.11 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.25 / Chapter 2.2.12 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.26 / Chapter 2.2.13 --- RNA Extraction --- p.27 / Chapter 2.2.14 --- Reverse Transcription: First Strand cDNA Synthesis --- p.28 / Chapter 2.2.15 --- Polymerasae Chain Reaction (PCR) --- p.28 / Chapter 2.3 --- Results --- p.32 / Chapter 2.4 --- Discussion --- p.46 / Chapter Chapter 3: --- Purification and Characterization of a Lectin from Gymnocladus chinensis Baill. --- p.49 / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Material and Methods --- p.50 / Chapter 3.2.1 --- Purification Scheme --- p.50 / Chapter 3.2.2 --- Assay of Hemaggl utinating Activity --- p.51 / Chapter 3.2.3 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.51 / Chapter 3.2.4 --- Molecular Mass Determination by FPLC Gel Filtration --- p.51 / Chapter 3.2.5 --- Protein Concentration Determination --- p.51 / Chapter 3.2.6 --- N-terminal amino acid sequence analysis --- p.52 / Chapter 3.2.7 --- Inhibition of Lectin-induced Hemagglutination by Carbohydrates --- p.52 / Chapter 3.2.8 --- Effect of Temperature and pH on Lectin-induced Hemagglutination --- p.52 / Chapter 3.2.9 --- Assay of Mitogenic Activity on Murine Splenocytes --- p.52 / Chapter 3.2.10 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.52 / Chapter 3.2.11 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.53 / Chapter 3.2.12 --- Assay of Anti-fungal Activity --- p.53 / Chapter 3.3 --- Results --- p.56 / Chapter 3.4 --- Discussion --- p.67 / Chapter Chapter 4: --- Introduction to Protease Inhibitors --- p.70 / Chapter 4.1 --- General Introduction --- p.70 / Chapter 4.2 --- Serine Protease Inhibitors --- p.71 / Chapter 4.2.1 --- Kunitz Type Serine Protease Inhibitors --- p.73 / Chapter 4.2.2 --- Bowman-Birk Type Serine Protease Inhibitors (BBI) --- p.74 / Chapter 4.2.3 --- Squash Type Serine Protease Inhibitors --- p.75 / Chapter 4.3 --- Roles of Pis in Plants --- p.76 / Chapter 4.3.1 --- Pis as a defense protein --- p.76 / Chapter 4.3.2 --- Pis in seed germination --- p.78 / Chapter 4.4 --- Applications of Protease Inhibitors --- p.79 / Chapter 4.4.1 --- Pis in Cancer Prevention --- p.79 / Chapter 4.4.2 --- Pis in Crop Protection --- p.81 / Chapter 4.5 --- Aim of Current Study --- p.83 / Chapter Chapter 5: --- Isolation and Characterization of a Trypsin Inhibitor from the seeds of Lens culinaris --- p.84 / Chapter 5.1 --- Introduction --- p.84 / Chapter 5.2 --- Materials and Methods --- p.86 / Chapter 5.2.1 --- Purification Scheme --- p.86 / Chapter 5.2.2 --- Assay of Trypsin-Inhibitory Activity --- p.87 / Chapter 5.2.3 --- Assay of Chymotrypsin-Inhibitory Activity --- p.88 / Chapter 5.2.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.88 / Chapter 5.2.5 --- Molecular Mass Determination by FPLC Gel Filtration --- p.88 / Chapter 5.2.6 --- Protein Concentration Determination --- p.89 / Chapter 5.2.7 --- N-terminal amino acid sequence analysis --- p.89 / Chapter 5.2.8 --- Effect of DTT on the inhibitory activity of trypsin inhibitor --- p.89 / Chapter 5.2.9 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.90 / Chapter 5.2.10 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.90 / Chapter 5.2.11 --- Assay of Anti-fungal Activity --- p.90 / Chapter 5.3 --- Results --- p.93 / Chapter 5.4 --- Discussion --- p.103 / Chapter Chapter 6: --- Isolation and Characterization of trypsin inhibitors trom the seeds of Vigna mungo (L.) Hepper --- p.106 / Chapter 6.1 --- Introduction --- p.106 / Chapter 6.2 --- Materials and Methods --- p.107 / Chapter 6.2.1 --- Purification Scheme --- p.107 / Chapter 6.2.2 --- Assay of Trypsin-Inhibitory Activity --- p.109 / Chapter 6.2.3 --- Assay of Chymotrypsin-Inhibitory Activity --- p.109 / Chapter 6.2.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.109 / Chapter 6.2.5 --- Molecular Mass Determination by FPLC Gel Filtration --- p.109 / Chapter 6.2.6 --- Protein Concentration Determination --- p.109 / Chapter 6.2.7 --- N-terminal amino acid sequence analysis --- p.110 / Chapter 6.2.8 --- Effect of DTT on the inhibitory activity of trypsin inhibitor --- p.110 / Chapter 6.2.9 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.110 / Chapter 6.2.10 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.110 / Chapter 6.2.11 --- Assay of Anti-fungal Activity --- p.110 / Chapter 6.3 --- Results --- p.113 / Chapter 6.4 --- Discussion --- p.132 / Chapter Chapter 7: --- General Discussion --- p.135 / References --- p.138

Trypsin inhibitory activity from the hemolymph of the tobacco hornworm, Manduca sexta, was purified by affinity chromatography on immobilized trypsin and resolved into two fractions with molecular weights of 13700 (inhibitor A) and 8000 (inhibitor B) by Sephadex G-75 gel filtration. SDS-polyacrylamide gel electrophoresis under non-reducing conditions gave a molecular weight estimate of 15000 for inhibitor A and 8500 for inhibitor B. Electrophoresis of these inhibitors under reducing conditions on polyacrylamide gels gave molecular weight estimates of 8300 and 9100 for inhibitor A and inhibitor B, respectively, suggesting that inhibitor A is a dimer. Isoelectro-focusing on polyacrylamide gels focused inhibitor A as a single band with pI of 5.7, whereas inhibitor B was resolved into two components with pIs of 5.3 and 7.1. Both inhibitors A and B are stable at 100° C and at pH 1.0 for at least 30 minutes, but both are inactivated by dithiothreitol even at room temperature and non-denaturing conditions. Inhibitors A and B inhibit trypsin, chymotrypsin, plasmin, and thrombin but they do not inhibit elastase, papain, pepsin, subtilisin BPN' and thermolysin. In fact, subtilisin BPN' completely inactivated both inhibitors A and B. Inhibitor A and inhibitor B form stable complexes with trypsin. Stoichiometric studies showed that inhibitor A combines with trypsin and chymotrypsin in a 1:1 molar ratio. The inhibition constants (Ki) for trypsin and chymotrypsin inhibition by inhibitor A were estimated to be 1.45 x 10-8 M and 1.7 x 10-8M, respectively. Inhibitor A in complex with chymotrypsin does not inhibit trypsin (and vice versa) suggesting that inhibitor A has a common binding site for trypsin and chymotrypsin. The amino terminal amino acid sequences of inhibitors A and B revealed that both these inhibitors are homologous to the bovine pancreatic trypsin inhibitor (Kunitz) . Quantitation of the trypsin inhibitory activity in the hemolymph of the larval and the pupal stages of Manduca sexta showed that the trypsin inhibitory activity decreased from larval to the pupal stage. Further, inhibitor A at the concentration tested caused approximately 50% reduction in the rate of proteolytic activation of prophenoloxidase in a hemocyte lysate preparation from Manduca sexta, suggesting that inhibitor A may be involved in the regulation of prophenoloxidase activation. However, inhibitor B was not effective even at three times the concentration of inhibitor A. Since activation of prophenoloxidase has been suggested to resemble the activation of alternative pathway of complement, the effect of inhibitors A and B and the hemolymph of Manduca sexta on human serum alternative pathway complement activity was evaluated. The results showed that, although inhibitors A and B do not affect human serum alternative complement pathway, other proteinaceous component(s) in Manduca sexta hemolymph interact(s) and cause(s) an inhibition of human serum alternative complement pathway when tested using rabbit erythrocyte hemolytic assay.