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Studies on purification and characterization of ribosome-inactivating protein from the garden pea (pisum sativum).

by Lam Suet Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 109-121). / Acknowledgements --- p.i / Table of contents --- p.ii / Abstract --- p.vii / List of Abbreviations --- p.ix / List of Tables --- p.x / List of Figures --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ribosome-inactivating proteins (RIPs) --- p.3 / Chapter 1.1.1 --- Types of RIPs --- p.4 / Chapter 1.1.1.1 --- Type I RIPs --- p.5 / Chapter 1.1.1.2 --- Type II RIPs --- p.7 / Chapter 1.1.2 --- Physicochemical properties --- p.7 / Chapter 1.1.3 --- N-glycosidase activity of RIPs --- p.8 / Chapter 1.1.3.1 --- Specificity of N-glycosidase activity --- p.10 / Chapter 1.1.3.2 --- Inhibition of protein synthesis --- p.11 / Chapter 1.1.4 --- Other enzymatic and biological activities of RIPs --- p.11 / Chapter 1.1.4.1 --- Enzymatic activities --- p.11 / Chapter 1.1.4.2 --- Multiple depurination --- p.13 / Chapter 1.1.4.3 --- RNase activity --- p.14 / Chapter 1.1.4.4 --- DNase activity --- p.15 / Chapter 1.1.4.5 --- Biological activities --- p.16 / Chapter 1.1.5 --- Storage of RIPs in plant cells --- p.17 / Chapter 1.1.5.1 --- RIPs targeted to subcellular compartments --- p.18 / Chapter 1.1.5.2 --- Cytoplasmic RIPs --- p.20 / Chapter 1.1.6 --- Physiological roles of RIPs --- p.22 / Chapter 1.1.6.1 --- Defensive role in plants --- p.22 / Chapter 1.1.6.2 --- Metabolic role of RIPs --- p.26 / Chapter 1.1.6.3 --- RIPs as storage proteins --- p.26 / Chapter 1.1.7 --- Application of RIPs --- p.27 / Chapter 1.1.7.1 --- Therapeutic applications --- p.27 / Chapter 1.1.7.2 --- Possible use of RIPs in agriculture --- p.30 / Chapter 1.2 --- Objectives of the present study --- p.31 / Chapter 1.2.1 --- Rationale of the study --- p.31 / Chapter 1.2.2 --- Outline of the thesis --- p.32 / Chapter Chapter 2 --- Screening of hitherto unexplored plant species for RIPs --- p.33 / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.2 --- Materials and methods / Chapter 2.2.1 --- Materials --- p.36 / Chapter 2.2.2 --- Preparation of crude powder --- p.36 / Chapter 2.2.3 --- Protein determination --- p.38 / Chapter 2.2.4 --- Preparation of rabbit reticulocyte lysate --- p.38 / Chapter 2.2.5 --- Protein synthesis inhibition assay --- p.39 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Preparation of crude powder --- p.41 / Chapter 2.3.2 --- Protein synthesis inhibition assay --- p.41 / Chapter 2.4 --- Discussion --- p.43 / Chapter Chapter 3 --- Purification of RIP from garden pea (Pisum sativum) --- p.45 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Materials --- p.50 / Chapter 3.2.2 --- Purification of RIP from garden pea --- p.52 / Chapter 3.2.3 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.54 / Chapter 3.2.4 --- Precautions for working with RNA --- p.56 / Chapter 3.2.5 --- N-glycosidase assay --- p.57 / Chapter 3.2.6 --- Quantitation of RNA --- p.60 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Quantitation of RNA --- p.61 / Chapter 3.3.2 --- Affinity chromatography on Affi-gel Blue gel --- p.61 / Chapter 3.3.3 --- Iminodiacetic acid-agarose chromatography --- p.64 / Chapter 3.3.4 --- Cation exchange chromatography on Resource-S --- p.66 / Chapter 3.3.5 --- Gel filtration on Superose 12 HR 10/30 --- p.69 / Chapter 3.3.6 --- "Assessment of purity, yield and activity" --- p.72 / Chapter 3.4 --- Discussion --- p.74 / Chapter Chapter 4 --- Physicochemical and biological properties of garden pea RIP --- p.77 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and methods / Chapter 4.2.1 --- Materials --- p.81 / Chapter 4.2.2 --- Molecular weight determination --- p.82 / Chapter 4.2.3 --- Subunit composition --- p.82 / Chapter 4.2.4 --- Isoelectric focusing (IEF) --- p.83 / Chapter 4.2.5 --- Detection of glycoproteins --- p.84 / Chapter 4.2.6 --- N-terminal amino acid sequence --- p.84 / Chapter 4.2.7 --- Inhibition of cell-free protein synthesis --- p.86 / Chapter 4.2.8 --- N-glycosidase activity --- p.86 / Chapter 4.2.9 --- Deoxyribonuclease activity --- p.87 / Chapter 4.2.10 --- Activity towards tRNA --- p.88 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Molecular weight determination --- p.89 / Chapter 4.3.2 --- Subunit composition --- p.91 / Chapter 4.3.3 --- Isoelectric focusing (IEF) --- p.92 / Chapter 4.3.4 --- Detection of glycoproteins --- p.94 / Chapter 4.3.5 --- N-terminal amino acid sequence --- p.96 / Chapter 4.3.6 --- Inhibition of cell-free protein synthesis --- p.97 / Chapter 4.3.7 --- N-glycosidase activity --- p.99 / Chapter 4.3.8 --- Deoxyribonuclease activity --- p.101 / Chapter 4.3.9 --- Activity towards tRNA --- p.102 / Chapter 4.4 --- Discussion --- p.103 / Chapter Chapter 5 --- General discussion and conclusion --- p.106 / References --- p.109

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_321872
Date January 1997
ContributorsLam, Suet Ling., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xii, 121 leaves : ill. ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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