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Effects of Membrane Association and Membrane Phospholipid Composition on Atpase Activity in Bacillus Stearothermophilus

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Effects of Membrane Association and Membrane Phospholipid Composition on Atpase Activity in Bacillus Stearothermophilus University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 1975 Effects of membrane association and membrane phospholipid composition on ATPase activity in Bacillus stearothermophilus Paul Edwin Orndorff The University of Montana Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation Orndorff, Paul Edwin, "Effects of membrane association and membrane phospholipid composition on ATPase activity in Bacillus stearothermophilus" (1975). Graduate Student Theses, Dissertations, & Professional Papers. 6863. https://scholarworks.umt.edu/etd/6863 This Thesis is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. EFFECTS OF MEMBRANE ASSOCIATION AND MEMBRANE PHOSPHOLIPID COMPOSITION ON ATPASE ACTIVITY IN BACILLUS STEAROTHERMOPHILUS By Paul E. Orndorff B.A., Knox College, 1972 Presented in partial fulfillment of the requirements for the degree of Master of Science UNIVERSITY OF MONTANA 1975 Approved by: ^ ..LÂ ûit pnairm^i, Board of Examiners D e a p ^ Gradua-^ School , I97S Date ^ Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: EP37664 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI OlssartaEioft Publishing UMI EP37664 Published by ProQuest LLC (2013). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Orndorfft Paul £., M.S., December, 1975» Microbiology Effects of Membrane Association and Membrane Phospholipid Compo­ sition on ATPase Activity in Bacillus stearothermophilus ( 79 pp.) Director, Dr. G. L. Card Membrane phospholipid composition in whole cells of Bacillus stearothermophilus was altered by a 20-min period of anaerobiosis. During this period there was a decrease in phosphatidylglycerol and corresponding increase in cardiolipin (CL). Membrane- associated ATPase was solubilized by dialysis of the membrane fractions against 10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA. The optimum conditions for ATPase activity in all fractions was at pH 8.4 in the presence of 1.0 mM MgCl2 * The temperature optimum for the membrane-bound fractions (high CL and control) was at 64°C, whereas soluble ATPase exhibited maximum activity at 59°C. Enzyme activity remained stable in the control membrane fraction, whereas the soluble enzyme lost 100% activity after JO-min incuba­ tion at 80°C; and the activity of the high CL membrane fraction was reduced 50%. The high CL membrane fraction retained three times more ATPase than the control membrane fraction when the fractions were washed successively with (i) 10 mM Tris-HCl (pH 7*5)» 5.0 mM MgCl2 , (ii) 2 M LiCl, 0.25 M Tris-HCl (pH 7-5) and (iii) dialyeed against 10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA. Membrane CL concentration did not influence the reassociation of solubil­ ized ATPase to ATPase-depleted membranes. A of 1.8 mM was determined for both the soluble and membrane fractions at 60 C. The Km remained constant in the soluble fraction, whereas the decreased to 1.1 mM in the control membrane fraction and decreased to 0.32 mM in the high CL membrane fraction at 45 C. The determina­ tion of the activation energy of membrane and soluble fractions revealed that the soluble enzyme underwent a change in activation energy at 56 C . The temperature at which there occurred a change in activation energy was displaced slightly from 56 C in the membrane-bound enzyme preparations. High CL membrane prepara­ tions showed a lower activation energy over a temperature range of 45°-5&^C than did the control membrane preparations. These findings suggest that membrane association and membrane phospho­ lipid composition affect both the physical and kinetic properties of ATPase in Bacillus stearothermophilus * 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS I wish to express my sincere thanks and appreciation to Dr. G. L. Card for his encouragement and guidance in the preparation of this thesis and in all aspects of my graduate work. In addition, I would like to thank Kalene De Vore for her help and extraordinary patience in typing this thesis. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CONTENTS Page ABSTRACT................... ii ACKNOWLEDGMENTS............................................ iii LIST OF T A B L E S ....................... vi LIST OF FIGURES ................................vii LIST OF......... ABBREVIATIONS.......................... ix CHAPTER I. INTRODUCTION....... ......... ........................ 1 II. MATERIALS AND M E T H O D S ........................... 17 O r g a n i s m .......... 17 Growth of Bacillus stearothermophilus ..... 17 Treatment of Cells to Achieve the Phosphatidyl­ glycerol (PG) to Cardiolipin (CL) Conversion. 18 Fractionation of Cells and the Preparation of Crude E n z y m e ............................. l8 Solubilization of Membrane-Bound ATPase .... 20 Reassociation of Solubilized ATPase with the Depleted Membrane Fraction ................. 22 Conditions and Calculations for Determining the Activation Energy of Membrane-Associated and Solubilized ATPase ..................... 23 Assay of ATPase A c t i v i t y ..................... 26 Dry Weight Determ inatio n ..................... 27 Determination of P r o t e i n ................ 27 R e a g e n t s ..................................... 29 III. RE S U L T S .............................................. 30 Enzyme Characterization ....................... 30 Localization of the Enzyme; Effects of DNase vs. Sonication in Preparing Crude Fractions......................... 30 Determination of Optimum Conditions for Enzyme Activity ............. 30 pH O p t i m u m ........................... 30 Determination of Optimum Divalent Cation Concentration ............... 33 Determination of Optimum Temperature for Activity ....................... 33 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Page Thermal Stability of the Crude Membrane- Bound and Soluble Enzyme: the Effects of CL Concentration and Mg lone ..... 53 Solubilization and Heaseociation of ATPase from High CL and Control Membrane Preparations ......................... .. 41 Solubilization of Membrane-Associated A T P a s e ............................. 4i Peassociation of Solubilized ATPase with ATPase-Depleted Membrane Fractions . 45 Kinetic Properties of Membrane-Bound and Soluble ATPase at 60®C and 45°C .... 43 The Effects of Temperature on the Kinetics of Membrane-Bound and Soluble ATPase . 49 IV. DISCUSSION 56 V. SÜMMAEY . 71 LITERATURE CITED 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table Page 1. Composition of the Fisk-Subbarow Reagent .......... 28 2. The Effects of Sonication vs. DNase on the Distribution of ATPase Activity in Crude Soluble and Crude Membrane Fractions from High CL and Control C e l l s ......................... 31 3. ATPase Activities of Fractions S^-8^ and . 44 4. Reassociation of Soluble ATPase with Depleted Membranes from High CL and Control Cells ..... 46 5. Values of K and V from Figures l6 and IV» and the ComparisoÊ*of the Differences in K and V Between the High CL and Control Preparations at 60*C and 4$^C 50 6 . Activation Energies for the Fractions Shown in Figures l8 and 1 9 ..................................55 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure Page 1. Flow Sheet for the Preparation of "Soluble ATPaee" and "Depleted Membranes" ..................... 21 2. Energy Diagram for an Exothermic Reaction ....... 25 3* Preparation and Centrifugation of High CL and Control Cell Lysates; The Effects of Sonication on the Distribution of ATPase Activity ............ 32 4. Determination of Optimum pH for ATPaee Activity in High CL end Control Crude Membrane Fractions . 3^ 3* Optimum Divalent Cation Concentration for ATPase Activity in High CL and Control Crude Membrane F r a c t i o n s .......................................... 33 6. Optimum Temperature for ATPase Activity in Crude Soluble and Crude Membrane Fractions ............ 36 7 . The Effects of Mg^* on ATPase Activity During Prior Incubation of Control and High CL Crude Membrane Fractions at 8 0 ® C ..................................38 8. The Effects of Mg^* on ATPase Activity During Prior Incubation of Control and High CL Crude Soluble Fractions at 8 0 ° C ..................................38 9 . The Effects of Mg^* on
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