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The Effect of Carboxin on Complex-Ii from Beef Heart Mitochondria." (1973)

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The Effect of Carboxin on Complex-Ii from Beef Heart Mitochondria. Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1973 The ffecE t of Carboxin on Complex-Ii From Beef Heart Mitochondria. Mary Bradley Coleman Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Coleman, Mary Bradley, "The Effect of Carboxin on Complex-Ii From Beef Heart Mitochondria." (1973). LSU Historical Dissertations and Theses. 2528. https://digitalcommons.lsu.edu/gradschool_disstheses/2528 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS This material was produced from a microfilm copy of the original document. While the most advanced technologicalmeans to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted. The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction. 1. The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity. 2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. 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Xerox University Microfilms 300 NorthZ n b Road Ann Arbor, Michigan 48106 74-18,324 COLEMAN, Mary Bradley, 1943- THE EFFECT OF CARBOXIN ON COMPLEX II FROM BEEF HEART MITOCHONDRIA. The Louisiana State University and Agricultural and Mechanical College, Ph.D., 1973 Microbiology \ University Microfilms, A XEROXC om pany Ann, Arbor, Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. THE EFFECT OF CARBOXIN ON COMPLEX IT FROM BEEF HEART MITOCHONDRIA A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Microbiology by Mary Bradley Coleman B.S., Delta State College, 1963 December, 1973 ACKNOWLEDGMENT The author wishes to express sincere appreciation to Dr. A. D. r,arson for guidance and assistance through­ out the investigation and in preparation of this dissertation. Gratitude is expressed to Dr. H. D. Braymer and to Dr. George R. Newkome for encouragement and assistance during the course of this investigation. Appreciation is expressed to her sister, Marthalynn Bradley, for co-operation in the typing of this dissertation. The support and understanding of her family is also acknowledged. Acknowledgment is made to the Agricultural Experiment Station of Louisiana State University for the funds which supported this investigation. TABLE OF CONTENTS Page ACKNOWLEDGMENT i i TABLE OF CONTENTS.................................... iii LIST OF TABLES....................................... iv LIST OF FIGURES...................................... vi ABSTRACT.............................................. vii REVIEW OF LITERATURE................................ 1 MATERIALS AND METHODS............................... 25 Preparation of cell fractions................ 25 Assays........................................... 27 Manometric methods............................. 29 Preparation of derivatives.................... 29 Nuclear magnetic resonance spectra.......... 32 Electron paramagnetic resonance spectra 32 Chemicals....................................... 32 RESULTS............................................... 34 DISCUSSION............................................ 80 LITERATURE CITED..................................... 93 VITA................................................... 100 i i i LIST OF TABLES TABLE Page 1. Effect of carboxin on oxygen uptake by beef heart and rat liver mito­ chondria employing various TCA cycle intermediates as substrates.................... 3 5 2. Effect of carboxin on succinate oxidation by beef heart mitochondria (4 mg protein)....................... 4 3 3. Effect of 10”^ M carboxin on succinate dehydrogenase activity of beef heart mitochondria as measured by the phenazine methosulfate assay................ 44 4. Effect of carboxin on the reduction of coenzyme Q of beef heart mito­ chondria (8 mg protein) as measured by the neotetrazolium assay................. 4 5 5. Effect of carboxin on the activity of succinate-coenzyme Q reductase as measured by the DCIP assay.................. 47 6. DCIP, neotetrazolium, and ferricyanide assays of succinate-coenzyme Q reductase activity performed in the presence of added coenzyme Q (1.3 x 10"^ M ) ............. 48 7. Effects of carboxin on succinate and DPNH oxidation by acetone-extracted beef heart mitochondria as measured by the neotetrazolium assay..................... 50 8. Effect of increasing concentrations of coenzyme Q 2 on inhibition of succinate- coenzyme Q reductase by carboxin as measured by the DCIP assay system.......... 52 9. Inhibition of succinate oxidation by disrupted beef heart mitochondria (4 mg protein) by carboxin and carboxin analogs........................................ 65 10. Inhibition of succinate-coenzyme Q reductase activity by carboxin and carboxin analogs as measured by the DCIP assay system............................. 68 i v TABLE Page 11. Inhibition by carboxin and carboxin analogs of DPNH oxidation by disrupted beef heart mitochondria (4 mg protein) 70 v LIST OF FIGURES FIGURE Page 1. Structural formula of carboxin............... 3 2. Effect of carboxin on oxygen uptake of beef heart mitochondria.................... 37 3. Effect of carboxin on o<-ketoglutarate oxidation by disrupted beef heart mito­ chondria........................................ 39 4. Comparison of the effects of carboxin and malonate on o<-ketoglutarate oxidation by beef heart mitochondria........ 42 5. NMR spectrum of 2,3-dihydro-5-carboxy- 6-methyl-l,4 oxathiin......................... 54 6. NMR spectrum of ortho-phenyltoluate.......... 56 7. NMR spectrum of ortho-toluanilide............ 58 8. NMR spectrum of N-phenyl-3,3-dimethyl- acrylamide...................................... 60 9. NMR spectrum of 2,3-dihvdro-5-carboxylic acid methyl ester-6-methyl-l,4 oxathiin 62 10. NMR spectrum of 2,3-dihydro-6-methyl-5- carboxyisopropylamide-1,4 oxathiin.......... 64 11. Effect of 2,3-dihydro-5-carboxylic acid methyl ester-6-methyl-l,4 oxathiin on succinate oxidation by disrupted beef heart mitochondria............................. 67 12. Effect of ortho-phenyltoluate on DPNH oxidation by disrupted beef heart mito­ chondria........................................ 72 13. Effect of N-phenyl-3,3-dimethyl acrylamide on DPNH oxidation by disrupted beef heart mitochondria............................. 74 14. EPR spectrum of sub-mitochondrial particles reduced by succinate............... 77 15. Effect of carboxin on the EPR spectrum of sub-mitcv-hondrial particles reduced by succinate.................................... 79 vi ABSTRACT The site of action of carboxin (2,3-dihydro-6- methyl-'3-carboxanilido-l, 4 oxathiin) has previously been shown to be at the level of the succinoxidase system. The results reported in this dissertation confirm that inhibition lies within the succinoxidase system and furthermore show the site of inhibition within this system. The effect of carbcxii on intact and disrupted beef heart and rat liver mitochondria was determined by con­ ducting respirometry studies employing tricarboxylic acid (TCA) cycle intermediates as substrates. Results of these studies confirmed that carboxin specifically inhibits succinate oxidation. The absence of inhibition by carboxin of activity using phenazine methosulfate (PMS) assays and intact beef heart mitochondria ruled out inhibition at the level of the succinate dehydrogenase flavin moiety. Inhibition by carboxin of formazan formation by intact mitochondria and by succinate-coenzyme Q reductase and inhibition of dichlorophenolindolphenol (DCIP) and ferricyanide reduction by succinate-coenzyme Q reductase indicates that the site of action of carboxin corresponds to Complex II of the electron
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