ASPECTS RELATING TO THE OCCURRENCE OF AN INHIBITOR OF TISSUE PLASMINOGEN ACTIVATOR IN ERYTHRINA CAFFRA THUNB. PLANTS AND IN VITRO CULTURES by HENDRIK JOHANNES HEYER Submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Department of Botany, Faculty of Science, University of Natal, Pietermaritzburg. May 1990 i - PREFACE The experimental work described in this thesis was carried out in the Department of Botany, University of Natal, Pietermaritzburg, from January 1986 to December 1989 under the supervision of professor J. van Staden. These studies, except the acknowledged work of others are the - result of my own investigations. H. J. MEYER May 1990 - i PREFACE The experimental work described in this thesis was carried out in the Department of Botany, University of Natal, Pietermaritzburg, from January 1986 to December 1989 under the supervision of professor J. van Staden. These studies, except the acknowledged work of others are the result of my own investigations . H. J. MEYER May 1990 -ii ACKNOYLEDGEMENTS I would like to express my sincere appreciation to the following people and organisations for their help to make this study possible: Professor J. van Staden for his guidance and advice during the course of this study . Professor E. F. Hennessy of the Department of Botany, University of Durban-Westville for the provision of taxonomic information on the genus Erythrina and specimens ot Erythrina caffra for this study. Dr. J. D. Conradie of the Natal Blood Transfusion Services for his guidance and expert advise in the development of the enzyme-,linked immunosorbent assay. Dr. C. Heussen and Professor E. B. Dowdle of the Department of Clinical Science, University of Cape Town for the provision of monoclonal antibodies and information on the enzyme-linked immunosorbent assay. Dr. P. J. Hofman formerly of the Department of Horticulture, University of Natal, Pietermaritzburg for his ~upport in the development of the enzyme-linked immunosorbent assay. Dr. O. Safriel for financial assistance and the provision of t-PA inhibitor and seeds of Erythrina caffra. Dr. I. D. Wh itt 0 n for the provision of seeds and plant - iii - material of various members of the genus Erythrina. Mrs. A. Batten for the provision of a print of her fine aquarelle of Erythrina caffra. Mr. I. Crouch for his help wi th the use of various computer programmes. The consultants of the Computer Services, University of Natal, Pietermaritzburg for their assistance with the use of comptiter programmes. Mr. H. M. Dicks of the Department of Statisics and Biometry for his advice and help with the use of the Genstat computer programme for the statistical analysis of the data . The F.R.D for financial assistance. A special word of appreciation to my wife for her love and support during the course of the study. - iv - ABSTRACT A double sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify the proteinaceous inhibitor of tissue plasminogen activator (t-PA) which occur in the tissue of Erythrina caffra Thunb. Using the ELISA the t-PA inhibitor could be detected in nanogramme quantities on the micro titer plate. The concentration of the t-PA inhibitor was determined in different tissues of Erythrina caffra. t-PA inhibitor concentrations in the order of 1 000 microgrammes per gramme protein were found in the seeds. Relatively small quantities of t - PA inhibitor, in the order of 10 to 50 microgrammes per gramme protein , occurred in root, shoot, leaf and living bark material. The t-PA inhibitor was found to accumulate in a similar way to the storage proteins in developing seeds. The accumulation of the inhibitor is at a relatively low level during the e~rly period of seed development but increases exponentially just before the seeds reach their maximum size. The t-PA inhibitor content of the cotyledons decreased drastically during the process of germination and subsequent seedling development. The disappearance of the inhibitor can be the result of total degradation of the molecule or - v - partial proteolysis with the modified molecule still being present in the tissue. An attempt was made to increase the t-PA inhibitor content of excised leaves of Erythrina caffra with protein inducing substances such as polyamines, precursors of ethylene and phytic acid. The protein inducing compounds included cell wall hydrolysates of Erythrina caffra, the marine alga Ecklonia maxima Osbeck (Papenfuss) as well as Lye 0 per sic 0 n esc u 1 en tum MilL wh i chi n d u c edt he, s y nth e sis of proteinase inhibitors suggested to be involved in the defense mechanism of plants. None of the substances used, increased the t-PA inhibitor content of excised leaves or in vitro cultures of Erythrina caffra. It is suggested that the t-PA inhibitor is probably not involved in a defense mechanism of Erythrina caffra. A callus and suspension culture derived from shoot tissue was developed to determine the occurrence of the t-PA inhibitor in vitro. The optimal nutrient medium for the growth of callus was the salts and vitamins of MURASHIGE and SKOOG (1962). The medium was supplemented with 3 % sucrose, 0 . 1 gramme per litre meso - inositol, 10 micromoles per litre benzyl adenine and 5 micromoles per litre 2,4- dichlorophenoxyacetic acid . Different auxins and cytokinins had a similar growth stimulatory effect on the growth of callus derived from a number of organs of Erythrina caffra. The callus from different organs did however, grow at different rates on the same nutrient medium. Callus derived - vi - from leaf, shoot, and cotyledonary tissue grew at similar rates on the nutrient media of MURASHIGE and SKOOG' (1962), SCHENK and HILDEBRANDT (1972) and B5 (GAMBORG, MILLER and OJIMA, 1968) despite large differences in the concentration of the nutrients in the three nutri.ent media. The source of nitrogen and ratio of nitrate to ammonium was critical to the growth of callus cultures . The optimal concentration of nitrate and ammonium was 30 millimoles per litre . The growth of callus from different organs was significantly affected by the concentration of sucrose in the nutrient medium. A concentration of 3% was optimal for callus growth. Temperature had a significant effect on the growth of callus. The optimal temperature for callus growth was 25 °C. A shoot cell suspension culture was established and maintained at the same temperature and on the same medium as the callus cultures but with a ten times lower concentration of growth regulators. A low shake speed was essential for the growth of the suspension culture. Maximum growth was obtained at a shake speed of 60 rpm. Relatively low quantities of t-PA inhibitor, in the order of 1 to 5 microgrammes per gramme protein, was detected in the suspension cultures. An attempt was made to increase the t-PA inhibitor content of the suspension cultures with the pro te in i nduc i ng compounds used on excised leaves, but without success. However, the t-PA inhibitor content of the suspension culture was significantly increased with a ten times increase in the sulphate content of the nutrient medium. - vii - CONTENTS Page PREFACE i ACKNOWLEDGEMENTS ii ABSTRACT iv CONTENTS vii LIST OF TABLES xiv LIST OF FIGURES xxx LIST PLATES xl CHAPTER 1 INTRODUCTION 1 1.1 Description of Erythrina caffra 1 1.1.2 Distribution 2 1 . 1 . 3 Economic and medicinal importance 2 1.2 Trypsin and other proteinase inhibitors .3 1.2 . 1 Description 3 1.2.2 The occurrence of proteinase inhibitors in living organisms 15 1.2.3 The distribution of proteinase inhibitors in plants 17 1.2.4 The physiological significance of proteinase inhibitors 21 - viii - Page 1 . 2 . 5 The nutritional significance of proteinase inhibitors 30 1.2.6 The significance of proteinase inhibitors in human nutrition 32 1.3 Tis~ue plasminogen activator 33 l. 3 . 1 Description 33 l. 3 . 2 The site of synthesis of tissue plasminogen activator 34 1.3 . 3 The physiological significance of tissue plasminogen activator 36 1.3.4 Assay for tissue plasminogen activator 38 1.4 Purpose of the cur rent research 39 CHAPTER 2 MATERIALS AND METH ODS 41 2 . 1 Collection of plant material 41 2.2 Purifcation of cell walls for elicitor recovery 43 2.3 Hydrolysation of cell walls 45 2.4 Defatting of seed material 45 2.5 Extraction of protein 47 2.6 Determination of protein 50 2.7 Germination of seeds and growth of seedlings 50 - ix - Page CHAPTER 3 THE ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION AND QUANTIFICATION OF t-PA INHIBITOR 53 3.1 Introduction 53 3.2 Acquisition of the t-PA inhibitor 54 3 . 3 Quantification of the t-PA inhibitor 54 3.4 Raising of antibodies against t-PA inhibitor 54 3.4.1 Preparation of the antigen emulsion 54 3.4.2 Immunization of the rabbits 55 3 . 4.3 Bleeding of the rabbits 55 3 . 4 . 4 Preparation of the serum 56 3.4 . 5 Partial purification of the immuno- globulins 56 3 . 5 The conjugation of peroxidase with the immunoglobulins 57 3 . 6 Preparation of standards and samples 61 3.7 The enzyme-linked immunosorbent assay for t-PA inhibitor 62 3.8 Determination of the standard working range of the ELISA for the t-PA inhibitor 68 3.9 Non-specific reaction in the ELISA 69 3 . 10 Blocking of the solid phase 71 3.11 Coating of the solid phase . 78 3 . 12 The effect of the rinsing intensity on the ELISA 81 Page - x - 3.13 The effect of the incubation period on the 84 ELISA 3 . 14 The effect of the microtiter plate on the ELISA 87 3.15 Reproducibility of the ELISA 89 3.16 Cross-reaction in the ELISA 91 3.17 Conclusions 95 CHAPTER 4 THE IN VITRO CULTURE OF ERYTHRINA CAFFRA 97 4.1 Introduction 97 4.2 Nutrient medium and growth conditions 102 4.3 Source of explants and callus 105 4 .