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THESIS Entitled Studies on Fraction 1 Protein of Beta Vulgaris Submitted for the Degree of DOCTOR of PHILOSOPHY by Kenneth Edwar

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THESIS Entitled Studies on Fraction 1 Protein of Beta Vulgaris Submitted for the Degree of DOCTOR of PHILOSOPHY by Kenneth Edwar THESIS entitled Studies on Fraction 1 Protein of Beta vulgaris Submitted for the degree of DOCTOR OF PHILOSOPHY by Kenneth Edward Moon 1 970 TABLE OF CONTENTS Acknowledgements i Abbreviations i Summary ii Chapter 1 Introduction 1.1 Carbon Dioxide Fixation in the Calvin • 1 Cycle 1.2 Some Properties of RuDPcase and Similarity 2 to Fraction 1 Protein 1.3 Cellular Location of Fraction 1 Protein 3 1.4 The Mechanism of Action of RuDPcase 4 1.5 Structure of Fraction 1 Protein from 13 Higher Plants 1.6 Relationship of Structure to Enzymic 14 Activity and Role of Sulphydryl Groups 1.7 Studies on RuDPcase from Sources other 17 than the Higher Plants 1.8 Comparison of Some Kinetic and Physical 21 Constants of RuDPcase from Different Sources 1.9 Aim of This Research Project 23 Chapter 2 Methods and Materials 2.1 Plant Material 24 2.2 Preparation of Chloroplasts and Isolation 24 of Fraction 1 Protein 2.3 Concentrating Protein Solutions 26 2.4 Preparation of Reduced and Carboxy- 27 methylated Fraction 1 Protein 2.5 Preparation of Maleyl-carboxymethyl 28 Fraction 1 Protein 2.6 Removal of Maleyl Groups 29 2.7 Gel Electrophoresis 30 2.7 (i) Polyacrylamide Disk Electrophoresis 30 (ii) Polyacrylamide Electrophoresis in 31 the presence of SDS (iii) Slab Acrylamide Gel Electrophoresis 32 (iv) Isoelectric Focusing 33 2.8 Ribulose-1,5-diphosphate Carboxylase 3^ Assay 2.9 Preparation of the S-Sulphenylsulphanate 34 Derivative of Fraction 1 Protein 2.10 Edman Degradations 35 2.11 Hydrazinolysis 35 2.12 Isolation of Blocked N-Terminal Peptides 36 2.13 Tryptic Digestion and Peptide Mapping 3® 2.14 Amino Acid Analysis 2.15 (i) Carboxypeptidase B Digestion 39 (ii) Carboxypeptidase A Digestion 39 2.16 Titration of Sulphydryl Groups 40 2.17 Protein Determinations 41 2.18 Chemicals 41 Chapter 3 Results 3.1 Preparation and Purity of Fraction 43 1 Protein 3.1 (i) Isoelectric Focuaing -of--Fraction 46 1 Protein 3.2 Amino Acid Analyses 50 3.3 Titration of Native Fraction 1 Protein 55 with 5 t5!-Dithiobis(2-nitrobenzoic .acid) 3.4 Studies on the Relationship of Enzymic 57 Activity to Sulphydryl Groups and Structure 3.5 Subunit Structure of Fraction 1 Protein 65 (i) Electrophoretic Studies on SCM- 65 Fraction 1 Protein (ii) Molecular Weight Estimation of the 68 Subunits Resolved on Columns of Sephadex G-200 (iii) Amino Acid Composition of the 70 Subunit Components (iv) Studies on Maleylated Fraction 1 72 Protein (v) Further Characterisation of the 75 Subunits 3.6 Peptide Mapping of the Isolated Subunits 80 3.7 NH^-Terminal Amino Acid Analysis of 87 Subunit A 3.7 (i) Isolation of a Peptide with a Blocked 88 Amino Group 3.8 C-Terminal Residue 9k (i) Hydrazinolysis 9^ (ii) Carboxypeptidase B Digestion 95 (iii) Carboxypeptidase A Digestion 96 3.9 End Group Analysis of Subunit B 96 Chapter k Discussion 97 References 107 Publication 116 ACKNOWLEDGEMENTS I wish to thank my supervisors, Professor E.O.P. Thompson and Professor H. N. Barber, for their help and encouragement during the course of this study, I am also grateful to my wife for typing this thesis and to Mr. P. M. Long for preparing the photo­ graphs appearing in the thesis. ABBREVIATIONS RuDPcase ribulose-1,5-diphosphate carboxylase PCMB p-chloromercuribenzoic acid SDS sodium dodecyl sulphate DTNB 5,5!-dithiobis(2-nitrobenzoic acid) SCM-cys teine S-carboxymethyl cysteine ii SUMMARY Fraction 1 protein isolated from spinach beet chloroplasts was purified by ammonium sulphate fraction ation and Sephadex G-200 gel filtration. The protein was essentially homogeneous as judged by acrylamide gel electrophoresis. The RuDPcase activity was abolished by low or concentrations of urea and by exposure to a sulphydryl blocking reagent, sodium tetrathionate. In this latter case full RuDPcase activity was restored by incubating the inactive protein with cysteine. Structural studies have shown that if the isolated protein was reduced and S-carboxymethylated in the presence of 8M urea then two distinct subunits could be resolved by gel filtration on Sephadex G-200 in an 8M urea buffer at pH 10.0. A comparison of the amino acid composition of the two subunits showed distinct differences between them. The molecular weights of the two protein subunits were estimated tobe 5^,000 and 16,000 by comparison of their elution volume on gel filtration with elution volumes of reduced carboxymethylated proteins of known molecular weight. iii Further studies showed that when the protein was treated with maleic anhydride, after S-carboxymethlation, then the subunits of the protein could be separated on a Sephadex G-100 column without the use of 8M urea in the column. Electrophoretic examination of these two components in a Gradipore gel confirmed molecular weight dissimilarities. Precise values for their molecular weights were determined by electrophoresis in acrylamide gels containing SDS. Corrected values, i.e. allowances made for the introduction of the maleyl group, are 55»^00 and 12,000 for the large and small subunit respectively. On an average dry weight basis the large subunit accounts for 68$ and the smaller subunit 32$. Using these values a tentative model of Fraction 1 protein consisting of six large subunits and twelve small subunits is proposed. It is suggested that the six larger subunits could lie on the six faces of a cube with the twelve smaller subunits occupying the twelve edges. Peptide mapping of the tryptic digests of the isolated subunits gave different fingerprints, however in the case of the larger subunits the number of ninhydrin positive spots was only one half of the theoretical number expected. End group analysis, both amino and carboxyl, did not yield conclusive information. Evidence is presented to suggest that the amino end of the larger subunit has its amino function masked. The techniques used in the analysis of the carboxyl end of the protein would not have detected a C-terminal basic residue which had as its pentultimate residue proline. 1 CHAPTER 1. INTRODUCTION 1.1 Carbon Dioxide Fixation in the Calvin Cycle Ribulose 1,5-diphosphate carboxylase EC 4.1.1.39 occupies an important niche in the photosynthetic carbon reduction cycle of higher plants and other photosynthetic organisms. It is responsible for catalysing the carboxylation of ribulose 1,5-diphosphate to produce phosphoglyceric acid as shown in Reaction 1. (Calvin, 1962; Bassham, 1963; 1965). H_COPO.H~ HoC0P0-H“ I 3 2. 3 c = 0 + CO ^2^ + 2H+ Reaction 1. I U2 2 HCOH H-C-OH I Mg2+ C00~ H-C-OH h2copo3h The isolation from Chlorella of a cell-free extract that was capable of catalysing the above reaction was reported by Quayle et al. in 1954. Shortly after this, Weissbach et al. (1956) isolated the enzyme from spinach leaves and since its detection it has been the object of several investigations (Trown, 1965; Paulsen and Lane, 1966; Akoyunoglou and Calvin, 1963)* 2 1.2 Some Properties of RuDPcase and Similarity to Fraction 1 Protein. Wildman and Bonner (1947) investigating the soluble cytoplasmic proteins of spinach leaves found that after electrophoresis, two major groups of protein could be distinguished. One of these behaved homogeneously during electrophoretic and ultracentrifugation studies and was called Fraction 1 protein. Subsequent studies (Eggman et al. 1953) showed that Fraction 1 protein exhibited a sedimentation constant of 18 to 19 Svedberg units. The enzyme purified by Weissbach et al» (1956) was reported by them to behave as an almost homogeneous protein on ultracentrifugation and electrophoresis and its molecular weight was estimated to be about 300,000 and having a sedimentation coefficient of 17S. The similarity of the physical properties of these two proteins was noted by Dorner et al. (1957) and later workers confirmed this observation and demonstrated that RuDPcase activity was associated with the Fraction 1 protein (Lyttleton and T!so, 1958; Park and Pon, 1961; Mendiola and Akazawa, 1964; Van Noort and Wildman, 1964; Trown, 1965; Thornber et al. 1967)* 3 1.3 Cellular Location of Fraction 1 Protein It has been known for many years that chloroplasts isolated in an aqueous medium are leaky, i.e. a suspension of chloroplasts on standing will lose their outer membrane and virtually all the electron dense material (mainly protein) of the stroma (Kahn and von Wettstein, *196*1; and Jacobi and Perner, 1961). If however the chloroplasts are protected from swelling during isolation by a gentle method, a large proportion of Fraction 1 protein is found to be still within the chloroplasts (Lyttleton and Tfso, 1958; Park and Pon, 1961). Using non-aqueous medium it is possible to isolate chloroplasts which, when viewed under the electron microscope, are seen to have lost their outer membrane but retained their stroma contents. By means of this technique it has been shown that higher plant chloro­ plasts and chloroplasts isolated from the alga Euglena gracilis contain most, and possibly all, of the RuDPcase (Smillie and Fuller, 1959; Heber et al. 1963; Smillie, 1963)* Furthermore, Ridley et al» (19^7) have produced results that indicate that the bulk of the Fraction 1 protein occurs in the stroma, and is not 4. associated with the lamellar structure. 1.4 The Mechanism of Action of RuDPcase The stoichiometry of the reaction catalysed (Reaction 1 ) as well a.s many of the properties of the carboxylase have been known since the original study by Weissbach et al. (1956). Many of these findings have been verified by subsequent workers, most recent studies being those of Trown (1965) and Paulsen and Lane (1966). Despite these studies, information on the carboxylation mechanism or structural characteristics of the enzyme is meagre.
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