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Microfilms International 300 N INFORMATION TO USERS This reproduction was made from a copy of a document sent to us for microfilming. While the most advanced technology has been used to photograph and reproduce this document, the quality of the reproduction is heavily dependent upon the quality of the material submitted. The following explanation of techniques is provided to help clarify markings or notations 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 through an image and duplicating adjacent pages to assure complete continuity. 2. When an image on the film is obliterated with a round black mark, it is an indication of either blurred copy because of movement during exposure, duplicate copy, or copyrighted materials that should not have been filmed. For blurred pages, a good image of the page can be found in the adjacent frame. If copyrighted materials were deleted, a target note will appear listing the pages in the adjacent frame. 3. When a map, drawing or chart, etc., is part of the material being photographed, a definite method of “sectioning” the material has been followed. It is customary to begin filming at the upper left hand comer of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning is continued again—beginning below the first row and continuing on until complete. 4. For illustrations that cannot be satisfactorily reproduced by xerographic means, photographic prints can be purchased at additional cost and inserted into your xerographic copy. These prints are available upon request from the Dissertations Customer Services Department. 5. Some pages in any document may have indistinct print. In all cases the best available copy has been filmed. University Microfilms International 300 N. Zeeb Road Ann Arbor, Ml 48106 8503994 Chang, Sulie Lin STUDIES OF A DE NOVO PATHWAY FOR GDP-L-FUCOSE SYNTHESIS IN PORCINE THYROID GLANDS The Ohio State University Ph.D. 1984 University Microfilms International300 N. Zeeb Road, Ann Arbor, Ml 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V . 1. Glossy photographs or pages 2. Colored illustrations, paper or print ______ 3. Photographs with dark background ______ 4. Illustrations are poor co p y _______ 5. Pages with black marks, not original copy_______ 6. Print shows through as there is text on both sides of page_____ 7. Indistinct, broken or small print on several pages ______ 8. Print exceeds margin requirements______ 9. Tightly bound copy with print lost in spine_______ 10. Computer printout pages with indistinct print ______ 11. Page(s)____________ lacking when material received, and not available from school or author. 12. P ag e(s)____________ seem to be missing in numbering only as text follows. 13. Two pages numbered ____________ . Text follows. 14. Curling and wrinkled pages _______ 15. O therDissertat ion contains pages with print at a slant, filmed as received. University Microfilms International STUDIES OF A DE NOVO PATHWAY FOR GDP-L-FUCOSE SYNTHESIS IN PORCINE THYROID GLANDS DISSERTATION Presented in Partial fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sulie Lin Chang, B.A., M.A. The Ohio State University 1984 Reading Committee: Approved By Dr. George A . Barber Dr. Elizabeth L. Gross Dr. George S. Serif Department of Biochemistry To the Memory of My Mother, Lien, to Whom I Owe All My Achievement. To My Father, Sam, Who Is Always Proud of Me. ii ACKNOWLEDGMENTS I wish to express my deep gratitude to my respected advisor and his wife. Dr. and Mrs. George S. Serif, for all the things they have done to support me for this study during years. The benefits I get from them in word and deed are immeasurable. I pray for them to be healthy and happy forever. There are several people who played a major role in this study. First is my husband, without his constinued support and encouragement, life would have been much more difficult. I also want to thank my sister in-law and her husband, Linda and Chang-An, who encouraged and supported me to switch field from Sociology to Biochemistry. iii VITA December 2, 1953 Born - Chalyl,Taiwan 1975 B.A., National Chengchi University, Taipei, Taiwan 1980 M.A., State University of New York at Albany, Albany New York 1980 - 1984 Teaching and Research Associate, Department of Biochemistry, The Ohio State University, Columbus, Ohio PUBLICATION Sulie Chang, Kay 0. Broschat and George S. Serif "An Assay for GDP-D-Mannose-4,6-Dehydratase" (1984) Analyti­ cal Biochemistry, in press. Sulie Lin “Leisure Activities of Workers in the Developing Industry: a field study report of a textile factory in Huasang, Taiwan." (1977) Tribune of Opinion in China, Vol. 3, No. 5, 37-41. Sulie Lin “Dilemma of the Working Girls in Industry in Taiwan." (1976) Tribune of Opinions in China, Vol.- 2, No. 6, 47-48. Sulie Lin “Field Methods in the Study of Organization" (1976) The Words and Thought, Vol. 13, No. 5, 327-337. FIELD OF STUDY Major Field: Biochemistry iv TABLE OF CONTENTS DEDICATION ............................................ ii ACKNOWLEDGMENTS ....................................... ill VITA ................................................... iv LIST OF TABLES ....................................... viii LIST OF FIGURES ...................................... ix LIST OF PLATES .......................... xi LIST OF ABBREVIATIONS ................................ xii INTRODUCTION ............................ 1 MATERIALS ............................................. 18 METHODS Enzymatic Synthesis of Radiolabelled GDP-D-mannose ................................. 20 1. Hexokinaae Incubation ..................... 20 2. Yeaat GDP-D-Mannose Synthaae Reaction .... 21 3. Isolation of Radiolabelled GDP-D-Mannoae . 22 4. Identification of Synthetic GDP-D-Mannoae 22 Protein Aaaaya ................................... 23 Preparation of Buffers ........................... 23 Paper Chromatography Solvents ................... 24 Scintillation "Cocktail" ........................ 24 Aaaaya for GDP-D-Mannose-4,6-Dehydratase Activity ....................................... 25 1. Assay D .1: GDP-D-(U-l^C)Mannose as Substrate .... 25 2. Assay D.2: GDP-D-<5-3h )Mannose as Substrate .... 27 3. Comparative atudy of Assay D.l and D.2 ... 29 Purification of GDP-D-Mannose-4,6-Dehydratase .. 30 Homogenization and Extraction ............... 30 Centrifugation ................................ 30 Blue Sepharose CL-6B Chromatography ......... 31 v DEAE-Sephadex A-50 Column Chromatography .... 32 Hydroxyapatite I Column Chromatography ...... 33 Combined Blue Dextran Agarose- Hydroxyapatite II Cgromatography ......... 34 Aaaaya for Eplmeraae Activity and Reductase Activity ........ 35 1. Isolation of Dehydratase Activity from Reductase on a FPLC TSK-G3000 SW Column .. 35 2. Assay E: Assay for Eplmeraae Activity .... 37 3. Assay R: Assay for Reductase Activity .... 38 Activation of Eplmeraae ......................... 40 1. Activation by NADH and NAD4- ...... 40 2. Activation by NADH and ADP-riboae ... 41 a. Experiment I ...................... 42 b. Experiment II .......................... 43 pH Optimum Determination for Eplmeraae and Reductase ............................... 43 Michaelia Constanta Determinations ........... 44 1. Michaelia Constant for GDP-4-Keto-6-Deoxy-D-Mannose ............. 44 2. Michael is Constant for NADH .............. 45 Sucrose Velocity Sedimentation .................. 45 1. Sedimentation Procedures .................. 45 2. Detection of Marker Enzymes ..... 46 a. Yeast Alcohol Dehydrogenase ........... 46 b. Yeast Hexokinaae ....................... 46 c. Horseradish Peroxidase ................ 47 3. Calculation of Relative Mobility.......... 47 Preparation of Polyacrylamide Gels ............. 48 1. Discontinuous Denatured Gels ............. 48 2. Continuous Non-denatured Gels ............ 49 3. Electrophoretic Conditions ......... 49 4. Protein Visualization Procedures.......... 50 5. Detection of Reductase Activity on Non-denatured Gels ........................ 50 RESULTS Identification of Synthesized Radiolabelled GDP-D-Mannose ................. 52 Comparative Study of Assay D.l and D.2.......... 55 Purification of GDP-D-Mannoae-4,6-Dehydratase .. 55 Development of Assay for Eplmeraae and Reductase 68 1. Isolation of Dehydratase Activity from Reductase on a FPLC TSK-G3000 SW columm .. 69 2. Location of Eplmeraae Activity in TSK-R .. 72 Activation of Eplmeraae ......................... 73 1. Activation by NADH ........................ 73 2. Activation by ADP-riboae .................. 76 vi Purification of Epimeraae and Reductaae ........... 80 1. Homogenization and Extraction ............ 80 2. Centifugation ............................. 80 3. Blue Sepharoae Chromatography ............ 80 4. DEAE-Sephadex A-50 Chromatography ....... 81 5. FPLC on Mono Q HR S/S Column ......... 84 6. FPLC on TSK-G3000 SW Column .............. 93 Gel Electrophoreaia of Epimeraae-Reductaae Preparationa .................................. 94 pH Optimum ........................................ 99 Michaelia Conatanta Determinationa ............. 99 Monomer and Oligomer Molecular Weighta of Epimeraa
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