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Information to Users INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9227S91 Separation of cytokinin nucleosidase from adenosine nucleosidase by affinity chromatography and the study of postharvest calcium treatments on sliced tomato Tamg, Jessica Jiashi, Ph.D. The Ohio State University, 1992 UMI 300 N. ZeebRd. Ann Arbor, MI 48106 SEPARATION OF CYTOKININ NUCLEOSIDASE FROM ADENOSINE NUCLEOSIDASE BY AFFINITY CHROMATOGRAPHY AND THE STUDY OF POSTHARVEST CALCIUM TREATMENTS ON SLICED TOMATO DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jessica J. Tarng, B.S., M.S. ***** The Ohio State University 1992 Dissertation Committee Approved by J.B. Allred M.E. Mangino Adviser A. Proctor Department of Food Science and Technology Copyright by Jessica Jiashi Tarng 1992 To My Parents and Dear Husband Jimmy ii ACKNOWLEDGMENTS I wish to express my sincere gratitude and appreciation to Dr. Grady W. Chism III, as my academic adviser, for inspiring and caring for me throughout my research and life in The Ohio State University. Thanks to the members of my reading committee, Drs. J.B. Allred, M.E. Mangino, and A. Proctor, and to the Department of Food Science and Technology for the financial assistance. To my colleague Madge Player, Daniele Korleskind, Isabelle Laye, and Serena Laroia, thank you for sharing, caring, and encouraging me. Special acknowledgment goes to all the faculty, staff, and fellow students who all have helped in their own way. VITA August 20,1962 Born - Taipei, Taiwan, Rep. of China 1980-1984 ......... B.S., The National Chung-Hsing University, Taichung, Taiwan, Rep. of China. 1984-1986 ......... M.S., The National Taiwan University, Taipei, Taiwan, Rep. of China. 1986-198 7........ Research Assistant, The National Taiwan University, Taipei, Taiwan, Rep. of China. 1987-Presen t.... Graduate Research Associate, The Ohio State University, Columbus, Ohio. Major Field: Food Science and Technology TABLE OF CONTENTS DEDICATION.............................................................................................................. i i ACKNOWLEDGEMENTS........................................................................................ iii VITA......................................................................................................................... iv LISTOFTABLES.................................................................................................... vi LIST OF FIGURES.................................................................................................... vii ABSTRACT................................................................................................................ x i CHAPTER I. ISOLATION OF CYTOKININ NUCLEOSIDASE FROM ADENOSINE NUCLEOSIDASE IN RIPE TOMATO FRUIT BY AFFINITY CHROMATOGRAPHY AND THEIR KINETIC CHARACTERISTICS................................................................................. 1 Introduction................................................................................. 1 Literature Review...................................................................... 3 Materials and Methods .............................................................. 20 Results and Discussion............................................................. 30 II. EFFECTS OF VACUUM INFILTRATION AND DIPPING OF CALCIUM SOLUTIONS ON THE QUALITY OF MINIMALLY PROCESSED TOMATO SLICES.............................................................. 67 Introduction................................................................................. 67 Literature Review...................................................................... 69 Materials and Methods .............................................................. 78 Results and Discussion............................................................. 81 LIST OF REFERENCES..............................................................................................103 v LIST OF TABLES TABLE PAGE 1. The protocol for silver staining.................................................. 29 2. Purification of adenosine nucleosidase in each peak fraction of affinity chromatography from ripe tomato fruit...................................................................................... 43 3. Kinetic constant for cytokinin nucleosidase and adenosine nucleosidase activity in the four peak fractions in Fig. 10 and 11........................................................... 49 4. Acceptability scores for calcium vacuum-infiltrated tomato slices.................................................................................... 102 LIST OF FIGURES FIGURE PAGE 1. Chemical structures, trivial names and abbreviations for some naturally occurring and synthetic cytokinins...................................................................... 4 2. Cytokinin biosynthesis interconversion and degradation in plant tissues: key role of N6-[isopent-2-enyl]adenosine.................................................... 6 3. Enzymes of N6-[isopent-2-enyl]adenosine metabolism........................................................................................ 8 4. The steps of affinity chromatography..................................... 16 5. Chemical structure of bicinchoninic acid .............................. 20 6. The reaction scheme shows how the BCA combines the biuret reaction to form the purple color complex.............................................................................................. 22 7. The comparison of protein recovery percentages attained by the widely used Bradford method and the recently developed BCA protein assay method 32 8. Affinity chromatography of cytokinin nucleosidase on an i6Ado-Agarose column (Elution gradient 1)......... 35 9. Affinity chromatography of adenosine nucleosidase on an i6Ado-Agarose column (Elution gradient 1)......... 37 10. Affinity chromatography of cytokinin nucleosidase on an i6Ado-Agarose column (Elution gradient 2)......... 39 vii 11. Affinity chromatography of adenosine nucleosidase on an i6Ado-Agarose column (Elution gradient 2).......... 41 12. Affinity chromatography of cytokinin nucleosidase on an Ado-Agarose column (Elution gradient 2............. ) 46 13. Affinity chromatography of adenosine nucleosidase on an Ado-Agarose column (Elution gradient 2)............. 48 14. Lineweaver-Burk plot of adenosine nucleosidase activity in peak fraction of NS I against adenosine concentration..................................................................................... 51 15. Lineweaver-Burk plot of cytokinin nucleosidase activity in peak fraction of NS I against cytokinin concentration..................................................................................... 53 16. Lineweaver-Burk plot of adenosine nucleosidase activity in peak fraction of NS II against adenosine concentration..................................................................................... 55 17. Lineweaver-Burk plot of cytokinin nucleosidase activity in peak fraction of NS II against cytokinin concentration..................................................................................... 57 18. Lineweaver-Burk plot of cytokinin nucleosidase activity in peak fraction of i6Ado NS III against cytokinin concentration................................................................. 59 19. Lineweaver-Burk plot of adenosine nucleosidase activity in peak fraction of Ado NS III against adenosine concentration............................................................... 61 20. SDS-polyacrylamide electrophoregram of partially purified nucleosidase attained from i6Ado-Agarose affinity chromatography column in Fig. 10 and ......11 65 viii 21. The calibration curve of the molecular weight versus relative mobility f Rvalue on a semi-logarithmic scale.................................................................. 66 22. Effect of calcium lactate and calcium gluconate vacuum infiltration treatments on the firmness of tomato slices at day zero.............................................................
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