Methionine recycling in fruit studies on the metabolism and function of selected intermediates Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/wp988n777

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  • This research has focussed upon both the in vitro and in vivo characterization of the recycling pathway leading from 5'-deoxy-5'-methylthioadenosine to methionine in fruit. In addition to contributing towards polyamine biosynthesis and other important cellular functions, in plant tissues these events also serve to generate the hormone ethylene. A broad range of physiological functions governing plant growth and development, including fruit ripening, are regulated by this compound. Whereas most of the enzymatic steps leading to the synthesis of ethylene from methionine have received considerable attention in efforts to suppress ethylene evolution and thereby control its effects, much less is known concerning the relative contribution of methionine recycling. Furthermore, the complete methionine cycle and the impact of its constituent reactions upon ethylene have yet to be elucidated. To address these issues in vitro, cell-free extracts of two varieties of avocado and four varieties of pear fruit were utilized. Several methods of tissue extraction, protein quantification, and enzyme purification were investigated and optimized with respect to enzyme activity. Two of the enzymes (5'- deoxy-5'-methylthioadenosine nucleosidase and 5-deoxy-5-methylthioribose kinase) involved in the recycling pathway were partially purified and biochemically characterized in each system. Of those compounds examined for their effect on the individual activities of these two enzymes, several were found to have significant influences. For the in vivo work, one variety of suspension-cultured pear cells previously utilized for in vitro studies was employed. The cells were found to take up both radiolabelled preparations of methionine and two of its recycling intermediates and convert these compounds into radiolabelled ethylene and metabolites known to participate in methionine recycling. The effect of various compounds upon cellular growth was examined as well. The data suggest both that the recycling pathway is necessary for normal cellular growth and also that these cells serve well to study these events. Additionally, the analyses of radiolabelled investigations (i.e., enzyme assays and metabolic uptake studies) were modified to utilize a radioisotope detector coupled to a high performance liquid chromatograph (HPLC). This application was found to be rapid, sensitive, cost-effective, and in some cases, provided more information than previously documented methods.
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