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

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.

Bibliography: leaves 161-213. / xviii, 219, [48] leaves of plates : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Analyses of six rare alpha 1-antitrypsin alleles were undertaken to determine their molecular basis, assess the biosynthesis directed by these alleles and, examine their clinical correlation. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 1992

Soy flour was inoculated with Lactobacillus delbrueckii ssp. lattis R0187, and incubated for 8 h, to evaluate the protein hydrolysis and identify peptides generated by this fermentation, and the impact on ACE and trypsin inhibitory activities. Aqueous protein extracts prepared from different fermentation time periods showed a decrease in soluble protein content (from 2.83 to 0.02 mg/mL), while soluble inorganic nitrogen and free amino acid contents increased (from 0.029 to 0.062% w/w, and from 0.75 to 0.90% w/w, respectively). The protein extracts were analyzed by SDS-PAGE; proteolysis was observed after 5 h incubation of inoculated soy flour, suggesting that glycinin, beta-conglycinin, and trypsin inhibitors, were hydrolyzed. Peptides were isolated by tricine-SDS-PAGE, and analyzed by MS/MS; fragments of soy anti-nutritional factors (Kunitz and Bowman-Birk trypsir, inhibitors), as well as of other soybean proteins, were identified, confirming that these proteins were hydrolyzed. The protein extracts at time 0 h and 8 h were analyzed by RP-HPLC; one fraction was analyzed by MS/MS, which identified peptides from Lactobacillus species. Determination of trypsin inhibitory activity showed less inhibition of the enzyme with inoculated soy flour compared to the control (un-inoculated soy flour), confirming the deactivation of trypsin inhibitors by fermentation. Determination of ACE inhibitory activity showed a higher inhibition with the control (86% +/- 3.0) compared to inoculated soy flour (66% +/- 7.6).

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 135-140). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves 135-140). Also available on the Internet.

Thesis (Ph.D) - University of Queensland, 2006. / Includes bibliography.

Orientador: Maria Ligia Rodrigues Macedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T15:21:13Z (GMT). No. of bitstreams: 1 Guimaraes_LaysCordeiro_M.pdf: 5932501 bytes, checksum: 2972555d649882421e88bba9ccd5dfea (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Mestrado / Bioquimica / Mestra em Biologia Funcional e Molecular