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A Cell-Free System for Protein Synthesis from Azotobacter Vinelandii

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A Cell-Free System for Protein Synthesis from Azotobacter Vinelandii Loyola University Chicago Loyola eCommons Master's Theses Theses and Dissertations 1968 A Cell-Free System for Protein Synthesis from Azotobacter vinelandii Julian Scheinbuks Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_theses Part of the Medicine and Health Sciences Commons Recommended Citation Scheinbuks, Julian, "A Cell-Free System for Protein Synthesis from Azotobacter vinelandii" (1968). Master's Theses. 2248. https://ecommons.luc.edu/luc_theses/2248 This Thesis is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Master's Theses by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 1968 Julian Scheinbuks I. CELL-FREE SYSTEM FOR FflOTElN S'YNTHESIS PROM kZai'OBACTm VINELANDII /~SitH sc~-; 0 ,· .. ~ LOYOLA 0 \ UNIVERSITY ,-j Q LIBRARY /J e~o-MJ~Q!~S~~ /· by Julian Scheinbuks k Dissertation Submitted to the Faculty of the Graduate School of Loyola University in PartiS!l FUlfillment of the Requirements for the Degree of Master of Science January 1968 TABLE OF CONTENTS Page No. INTRODUCTION 1 MATERIALS AND METHODS 4 Organism and conditions of culture 4 Preparation of cell-free extracts 4 Procedure for preincubationof ribosomes .[; Amino acid incorporating system 6 Precipitation and washing procedure 6 Preparation of tRNA 7 Conditions of culture 1 Extraction and isolation of tRNA 8 Procedure for determination of protein 9 Reagents 10 RESULTS ll Characteristics of the incorporating system 11 Ribosomes and supernatant fraction requirements 11 Cofactor requirements 11 Antagonists to protein synthesis 15 Magnesium requirement 15 Potassium requirement 15 Polyamine requirement 18 ATP requirement 18 pH dependence 18 Page No. Temperature dependence 18 Kinetics of endogenous incorporation 18 Characteristics· of poly U-stimulated incorporation 2ti of phenylalanine Effect of poly U in the stimulation of · phenylalainine incorporation 24 Participation of tRNA 24 Variable• aaaociatad with riboaomal activity Jl Dependence upon phase of growth Jl Dependence upon rapidity of halting of cell metabolism 31 Effects of preincubating ribosomes 34 Dependence upon the mode of cell rupture 34 Stability of cell-free extracts 37 Kinetics of poly U-directed polyphenylalanine synthesi~ 38 Characteristics of poly A-stimulated polylyaine , 38 incorporation Membrane bound ribosomes llll DISCUSSION 46 Antagonists to prote~n synthesis 46 Requirements for protein synthesis 46 Magnesium 46 Potassium 48 Polyamine& 49 ATP 49 Comparison of poly U and poly A-stimulation 5o Page No. Transfer RNA requirement: substitution of !• coli tRNA for Azotobacter tRNA. 50 Variables affecting ribosomal activity 51 Dependence upon phase of growth 51 Dependence upon rapidity of cessation of cell metabolism 52 Dependence upon stability of ribosomes 52 Kinetics of endogenous and poly U-directed phenylalanine incorporation 54 Membrane bound ribosomes 55 SUMMARY 56 ACKNOWLEDGEMENTS 57 LITERATURE CITED 58 LIST OF FIGURES Page No. Figure 1. The effect of the ribosomal concentration upon 12 phenylalnine incorporation. Figure 2. The effect of the S-140 protein fraction upon 13 phenylalanine incorporation. 2+ Figure 3. The effect of Mg concentration upon 16 phenylalanine incorporation. Figure 4. The effect of K+ upon phenylalanine 17 incorpora~ion. Figure 5. The effect of spermidine upon endogenous 19 phenylalanine incorporation. Figure 6. The effect of ATP concentration upon 20 phenylalanine incorporation. Figure 7. The effect of temperature upon phenylalanine incorporation. 14 Figure 8. Kinetics of endogenous C phenylalanine 23 incorporation. Figure 9. The effect of poly U upon the stimulation of 25 phenylalanine incorporation. Figure 10. The effect of tRNA upon the stimulation of 27 polyphenylalanine synthesis. Figure 11. The kinetics of incorporation of 14c 28 phenylalanine into hot and cold TCA precipitable material. Page No. Figure 12. Kinetics of poly U-stimulated phenylalanine 39 incorporation. Figure 13. Kinetics of phenylalanine incorporation using 40 preincubated ribosomes prepared according to the procedure described in Materials and Methods. Figure 14. A time course of phenylalanine incorporation in lll S-30 fractions stimulated by poly U and tRNA. Figure 15. A time course of phenylalanine incorporation following preincubation of ribosomes. Figure 16. A time course of phenylalanine incorporation 43 using preincubated ribosomes. ' ' ,. LIST OF TABLES Page No. Table 1. Requirements for }henylalanine incorporation. 14 Table 2. Requirements for the stimulation of polyPhenyla­ lanine synthesis. 26 Table 3. Substitutions of Azotobacter tRNA with E. coli 30 tRNA and the requirements for poly U and poly A­ stimulation. Table 4. Ribosomal activity of extracts from Azotobacter 3Z obtained by harvesting at several phases of growth. Table 5. The effect of the rapidity of halting cellular 33 metabolism upon ribosomal activity. Table 6. The effect of storage and preincubation of 35 ribosomes. Table 7. The effect of the mode of cell lysis upon the 36 incorporation of phenylalanine. Table 8. The comparison of ribosomal activity of free 45 ribosomes to activity of ribosomes extracted from hulls. 1 INTRODUCTION Nirenberg and Matthaeii (56) were among the first to demonstrate the requirements for in vitro protein synthesis in Escherichia Coli; the requirements include ribosomes, which are the structural site of amino acid incorporation and a dialyzed 100,000 x & supernatant fraction which contains the soluble components. Their important contribution provided the molecular basis for the study of the coding problem with their discovery that poly U (an oligonucleotide containing repeating units of 5' uridylic acid) acted as a messenger and that it coded for the synthesis of polyphenylalanine. These relationships were the basis for subsequent studies of the coding and translational problems. A number of bacterial, mammalian and plant cell-free systems (3-5, 14, 27-29, 37, 40, 44, 53, 60, 71, 78, 86, 87, 88) have since been developed with requirements similar to the !• coli system (55, .56, 58, 73, 83, 85). Amino acid incorporation is dependent upon ribosomes, 100,000 X A supernatant or pH 5 fraction, an ATP generating system, GTP, a critical concentration of magnesium, an optimal pH, and optimal concentrations of tRNA, polyamine, and KCl. Systems from E. coli and yeast have been of great use to biology due to the large body of information regarding biosynthetic pathways for cellular constituents as well as the large number of mutants readily available. These resources led to explosive advances in our knowledge of the mechanisms of protein synthesis, the action of antibiotics, the genetic 2 code, and the structure of protein synthesizing components. The system from Bacillus cereus may be useful for the information it may yield in conjunction with sporulation and germination, two processes which may be simple analogues of differentiation. Systems from plants and animals may yield information peculiar to processes observed in these advanced forms. A cell-free amino acid incorporating system was developed from Azotobacter for several reasons. First, in order to study protein synthesis directed by natural endogenous message, polyribosome& must be readily available and in reasonable quantities. Azotobacter may be disrupted by osmotic shock (64), an extremely gentle method which permits isolation of large quantities of polyribosome& (62). At least 80 to 90% of the cellular ribosomes are polyribosome&; four-fifths of which are larger than tetramers (62). Furthermore, various size classes of polyribosome& up to the octomer class can readily be separated. Individual size classes of larger polyribosome& are less well resolved and isolated but are readily separated from the smaller size classes (62). Thus we can examine the activities of individual size classes of polyribosome& as well as the kinds of nascent protein (enzymes) synthesized on a given size class of poly­ ribosome. Second, Azotobacter is an organism in which the study of membrane bound ribosomes may prove significant since Azotobacter has an internal membranous network (62) similar tothat found in animal cells to which ribosomes are attached (63) and whose function in protein synthesis is established (34). Thus it is possible to compare the incorporating 3 activity of "free cytoplasmic" ribosomes and polyribosomes with those associated with membranes. In bacteria, ribosomes attached to cellular membranes have been reported (6, 73) bu~ have not been demonstrated con- elusively in ~· An early report of a cell-free protein synthesizing system in Azotobacter vinelandii (1959, prior to that reported by Nirenberg) implicated a poorly defined fraction of cell wall and membrane 14 which activly incorporated Camino acids (20). However, this system did not exhibit the requirements presently associated .with cell-free systems. Therefore, the development of a cell-free system for protein synthesis with clearly defined requirements in Azotobacter is then a prerequisite to experiments involving the isolation of membrane bound ribosomes and the characterization of amino acid incorporation associated with these particles. Finally, Azotobacter forms cysts, 4 dormant, resistant stage, which is similar in some respects to spores. It would be of interest to determine
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