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69-10599 GOMES, Benedict, 1933 - - ~- ... - - - .- - _. - - .. ~. - - - I' This dissertation has been 69-10,599 microfilmed exactly as received GOMES, Benedict, 1933- BEEF LIVER MITOCHONDRIAL AMINE OXIDASE; PURIFICATION AND STUDIES ON SOME PHYSICAL AND CHEMICAL PROPERTIES. University of Hawaii, Ph.D., 1968 Biochemistry University Microfilms, Inc., Ann Arbor, Michigan BEEF LIVER MITOCHONDRIAL AMINE OXIDASE; PURIFICATION AND STUDIES ON SOME PHYSICAL AND CHEMICAL PROPERTIES A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY SEPTEMBER 1968 by BENEDICT GOMES Dissertation Committee: Kerry T. Yasunobu, Chairman Morton Mandel Lawrence H. Piette Robert H. McKay John B. Hall DEDICATION TO MY MOTHER Acknowledgements To the East-West Center of the University of Hawaii; the National Institute of Health; and the Hawaii Heart Association for fellowships. To Drs. I. Igaue and H. J. Kloepfer for their assistance in the enzyme purification. To Mrs. Tomi Haehnlen and Kazi Sirazul Islam for drawing figures. TABLE OF CONTENTS LIST OF TABLES ••••••••••••••••••••••.•••••••••• vi LIST OF FIGURES •••••.•.••••••••••.••••••••••••. viii ABBREVIATIONS .••••••.•.•.••••••••••.••••••••••• xi ABSTRACT •..•.•.•.••.•••••••••••••••.••••••••••. xii I. INTRODUCTION. •••.•.•••.••••••••..•••••••••. 1 A. Historical Background of Amine 2 Oxidase Studies •••..••••••••••.•••• B. Physiological Significance •.••••••• 5 C. Statement of the Problem........... 6 II. MATERIALS AND METHODS .•••••••.••••••••••.•• 8 A. Ma t e ria 1 s •••••••••••••••••.••••••.. 8 1. Materials and Reagents Obtained Comme r cia11y ••••••••••••••••••• 8 2. Materials Obtained as Gifts ••.• 10 B. Methods............................ 12 1. Preparation of Adsorbents and Ionexchange Materials •••••••••• 12 (a) Alumina C/'................ 12 (b) Calcium phosphate gel..... 12 (c) Diethy1aminoethy1 (DEAE)- cellulose ••••.•••.•.•••••• 12 (d) Hydroxy1ap a t i te ••••••••••• 12 (e) Starch (for Electrophoresis) 12 (f) Sephadex G-200 ••••••••••••• 13 (g) Agarose (Bio-Gel A-1.5) gel. 13 ii 2. Electrophoresis .••.•.••••.. 13 (a) Starch Block Electrophoresis 12 (b) Polyacrylamide Gel Electrophoresis .•.•••• 14 3. Ultracentrifuge Studies •••• lS (a) Sedimentation Velocity. lS (b) Sucrose Density Gradient •.......•.••.• 16 4. Preparation of Mitochondria •. 17 S. Measurement of Enzymatic Activity ..•.•...•....••••.•. 17 6. Determination of Hydrogen Peroxide •••••••.•.•.•••••••• 18 7. Determination of the Partial Specific Volume, V •..•••••••• 20 8. Determination of Molecular We igh t •....•....•...••••..•. 20 (a) Mol. Wt. by gel filtration method 20 (b) Mol. Wt. from sedimen­ tation coefficient, Stoke'~ radius, and the partial specific vo 1 ume ...••.....••••••. 21 9. Metal Analyses ..•..•••••••.•• 23 (a) Copper ................. 23 (b) Cobalt ................. 23 (c) Iron ................... 23 (d) Manganese .............. 23 ( e) Molybdenum •••• 0 •••••••• 23 iii 10. Determination of Riboflavin ••••• 23 11. Determination of Purine •••••••• 24 12. Determination of Adenine ••••••• 25 13. Determination of Ribose ••••.••• 26 14. Determination of Phosphorus •••• 26 15. Analysis of Phospholipid .•••••• 27 16. Determination of the Sulfhydryl Groups......................... 27 III. RESULTS 29 A. Purification and Purity Studies ••.•• 29 1. Purification of the Mitochon­ drial Amine Oxidase •.••••••••••• 29 Calcium phosphate gel t rea tmen t. ••.•.••••••••.....••.•• 29 DEAE-cellulose column chromatography 31 Hydroxylapatite column chromatography.................. 31 2. Studies on the Purity of the En z ym e .•••••.••••••••••.•••••••• 40 (a) Rechromatography on DEAE- cellulose ••.••••••••.•••••• 40 (b) Rechromatography on hydroxylapatite column 43 (c) Sephadex gel filtration 43 (d) Analytical starch block electrophoresis •••••••••••• 43 (e) Free boundary electrophoresis 52 (f) Polyacrylamide gel electrophoresis •.•.•••••••. 52 (g) Ultracentrifuge studies •••• 52 iv B. Kinetic Properties ••••••••.•••••.••. 52 1. Activity of the Enzyme •.•••••••• 52 2. Effect of Temperature on the Enzyme Activity................. 59 3. Effect of pH on the Enzyme Activity........................ 64 4. Substrate Specificity 64 5 • Inhibitor Specificity 64 (a) Product inhibition ..••.•.•• 64 (b) Inhibition by sulfhydryl reagents ••••••••••••.•.•••• 68 (c) Inhibition by metal chelating agents .••.•.•.••.•••••.••.. 76 (d) Inhibition by aldehyde reagents •..•••••••••••••••. 76 C. Physical Properties .••..•.•••.•••••. 85 1. Spectral Properties ••••••.••.••. 85 2. Sedimentation Coefficients •••••• 85 3. Partial Specific Volumes •.•••••• 92 4. Molecular Weights .•••••••••••••. 92 (a) Molecular weights determined by Agarose gel filtration.. 92 (b) Molecular weights determined from Stoke's radii, sedimen­ tation coefficients, and par t i a 1 s p e c ifi c volum e s ••. 9 6 (c) Molecular weights determined from sedimentation-diffusion coefficients and Stoke~s radii •••..••••.•••..•.•••.. 102 v 5. Frictional Ratios .••.••••••.•••• 102 D. Chemical Properties ••.••••••••.•••.. 106 1. Metal Content ••••••••••••.•••.•. 106 2. Phosphorus Content ••••.••••••••. 106 (a) To ta 1 pho sphorus ••••.•••••• 106 (b) Phospholipid Phosphorus 112 ( c) Flavin dinucleotide phosphorus •••••••••.•.••••• 112 3 • Organic Prosthetic Group •••••••• 114 4. Sulfhydryl Groups .••.••••••••••• 118 IV. DISCUSSIONS.AND.CONCLUSION •.••••.••••.••••• 131 V. SUMMARy ••••••••••.•.•••••..••••••••••••• o. 150 VI. BIBLIOGRAPHy.............................. 153 LIST OF TABLES I. Purification of Beef Liver Mitochondrial Amine Oxidase .•.•••••••.••••••••••.••.••• 37 Modified Procedure for the Preparation of Amine Oxidase (FLOW SHEET) ••••••••••.• 38- 39 II. Substrate Specificities of the two Amine Oxidase Components ••••••.•••••••••.•••••• 67 III. A. Inhibition of Amine Oxidase by Sul- fhydryl Reagents ••••••••••••••••.•••• 73 III. B. Inhibition of Amine Oxidase by Sul- fhydryl Reagents ...•.•.••.••••...•••• 74 III. C. Inhibition of Amine Oxidase by Sul- fhydryl Reagents •••••••••••••..•.•••• 75 IV. Inhibition of Amine Oxidase by Metal Che1ating Agents •••••.•.•••••••••..•••••• 81 V. The Effect of Aldehyde Reagents on the Enzyme Activity ••••••..••••.•••••.••••••• 84 VI. A. Sedimentation Coefficients at Different Protein Concentrations of the Mito- c h 0 n dria1 Am ine 0 x ida s e ••••....•••••• 93 VI. B. Sedimentation Coefficients by Sucrose Density Gradient ••••••••••••••.•.•••• 94 VII. Agarose Gel Filtration Data of Standard Proteins, Blue Dextran 2000, and Amine Oxidase Components ••.•..••••••••...•.•••• 95 VIII. Molecular Parameters Obtained from Gel Filtration Data •.•••••••••.••••••..•.•••• 101 IX. Physical Parameters of the Mitochondrial Amine Oxidase •••.••••••••••.•.••••.••.••• 103 X. Molecular Weights of the Amine Oxidase Components by three Methods •••••••.•••••• 104 XI. Frictional Ratios of the Amine Oxidase Comp 0 n e n t s •..••••••••.•••..•••••...•••••• 105 vii XII. Metal Content of Amine Oxidase ••••••••••• 111 XIII. Phosphorus content of Mitochondrial Amine Oxidase 113 XIV. A. Riboflavin, Adenine, Ribose, and Nucleotide Phosphorus Content of Mitochondrial Amine Oxidase......................... 119 XIV. B. Riboflavin, Adenine, Ribose, and Nucleotide Phosphorus Content of Mitochondrial Amine Oxidase •••.•••.•••.•.••••.••.•• 120 XIV. C. Pyridoxal Content of Phosphorylase a and of the Mitochondrial Amine Oxidase Components •••••.•••..•••••.••••.•.••• 121 XV. Number of Titratab1e Sulfhydryl Groups in the Mitochondrial Amine Oxidase Components 128 XVI. A. Properties: 1a. Kinetic Parameters of Mitochondrial Amine Oxidase •••.••• 145 XVI. B. Properties: lb. Kinetic Parameters of Mitochondrial Amine Oxidase •.••••• 146 XVI. C. Properties: 2. Molecular Parameters of Mitocnondria1 Amine Oxidase ••••••• 147 XVI. D. Properties: 3. Chemical Parameters of Mitochondrial Amine Oxidase ••••••• 148 LIST OF FIGURES 1. Chromatography of the partially purified amine oxidase on the DEAE-cellulose column. 33 2. Hydroxylapatite column chromatography of the partially purified mitochondrial amine oxidase 0....... 36 3. Rechromatography of the purified enzyme component. 2 on the DEAE-cellulose col urn n ......•. 0•••••••••••••••••••••••••••• 42 4. Rechromatography of purified component 2 on hydroxylapatite •.•••.•.••••••••••••.•••• 45 5a. Chromatography of amine oxidase component 1 on Sephadex G-200 ••••••.••••••••••••••••••• 47 5b. Chromatography of amine oxidase component 2 on Sephadex G-200 •.•••••••.•••••..••.•••..• 49 6. Migration of the amine oxidase .components on starch block electrophoresis •••.••••••.•••• 51 7. Electrophoretic pattern of component 2 54 8. Polyacrylamide gel electrophoresis of . amine oxidase components 1 and 2 •••.••••••• 56 9. Sedimentation pattern of the amine oxidase component 1 •••.•••••••••••.•••••••••••••••• 58 lOa. Effect of temperature on the enzymatic activity 61 lOb. Effect of temperature on the activity of the amine oxidase •.••••.•••.••••••••••••••• 63 11. Effect of pH variation on the activity of the enzyme components 1 and 2 ••••••.••.•••••••. 66 12. Product inhibition studies •••••••••.••••••• 70 l3a. Inhibition of amine oxidase by sulfhydryl reagents ....•.•.................•.......... 72 l3b. Lineweaver-Burk plot of benzylamine oxidation in the absence and presence of p-CMB •••••.• 78 ix 13c. Lineweaver-Burk plot of the benzy1amine oxidation in the presence and absence of p - CMB ••••••••.•.••••••.••••••••••••••••••• 80 14. Lineweaver-Burk plot of benzy1amine oxidation
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