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