Graduate Thesis Or Dissertation
 

An investigation of the metabolism of poly-β-hydroxybutyrate and its possible role in nitrogen fixation in soybean root nodules

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  • A major objective of this investigation was to determine the possible roles, if any, of poly-β-hydroxybutyrate and products of its metabolic breakdown in symbiotic nitrogen fixation by nodule bacteroids. Changes in poly -β- hydroxybutyrate content and in activities of nitrogenase, β-hydroxybutyrate dehydrogenase, and isocitrate lyase were studied under various conditions where the supply of carbohydrate from the soybean plants to the nodule bacteroids was interrupted. Experiments of this type were conducted with detached root nodules, nodules from plants incubated in the dark, and nodules from plants of different ages. The results indicated that nodule bacteroids did not utilize poly- β-hydroxybutyrate until the carbohydrate supply from the host plants was limited by nodule exicison or incubation in the dark, or by senescence of the host plant. Isocitrate lyase activity was not detected until poly-β-hydroxybutyrate utilization began. The results of these experiments suggest that poly-β-hydroxybutyrate was not capable of fulfilling the requirement for maintenance of high activity of nitrogenase under conditions where the supply of carbohydrate from the host plant was limited. β-Hydroxybutyrate dehydrogenase from nodule bacteroids was purified, and its properties were investigated. Kinetic studies revealed that the mechanism of the β-hydroxybutyrate dehydrogenase reaction was a compulsory order type involving the formation of a ternary complex between the dehydrogenase, NAD⁺, and β-hydroxybutyrate. Three different systems for the transport of electrons to bacteroid nitrogenase were studied. Effective systems included the following: (a) hydrogen in the presence of Clostridium pasteurianum hydrogenase and ferredoxin, (b) NADH generated by the p-hydroxybutyrate dehydrogenase reaction in the presence of an NADH dehydrogenase and either FMN or FAD, (c) NADPH generated by the glucose-6-phosphate dehydrogenase reaction, provided that bacteroid non-heme iron protein, azotoflavin from Azotobacter and ferredoxin- NADP reductase from spinach were added, It is suggested that the NADPH donor system is physiologically more important than the other systems investigated.
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