Graduate Thesis Or Dissertation
 

The estimation of glucose pathways in Saccharomyces cerevisiae by the catabolic rate method

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  • Existing methods for the estimation of relative participation of glucose catabolic pathways make use of yield data of respiratory ¹⁴CO₂ or intermediary ¹⁴C products derived from glucose substrates specifically labeled with ¹⁴C. Cumulative yields of respiratory ¹⁴CO₂ observed at the end of time course of complete substrate utilization are analyzed to provide direct information on the participation of the pentose phosphate pathway only. The other glucose catabolic pathways such as glycolysis and the Entner-Doudoroff pathway are obtained by difference. In the present work a novel method for pathway estimation has been developed which uses data of the catabolic rate of individual glucose pathways. This method relies on newly designed and constructed apparatus which employs an ion-chamber-electrometer assembly to determine the amount of respiratory ¹⁴CO₂ produced by biological systems metabolizing substrates labeled with ¹⁴C. Kinetic information on the production of respiratory ¹⁴CO₂ is obtained with excellent resolution. Hence, the new method has been designated as the catabolic rate method based on high resolution radiorespirometry. With the high resolution system, the catabolic rate, derived by following the ¹⁴CO yield data over short time intervals (one or two minutes), of not only the PP pathway, but also the glycolytic pathway can be determined directly. Information on the total rate of glucose catabolism permits one to estimate the participation of anabolic pathways if the overall rate of glucose assimilation is known. Thus, insight on the total metabolism of glucose in biological systems is provided by the new method. Saccharomyces cerevisiae has been employed as the test organism in the development of the catabolic rate method by high resolution radiorespirometry. Since the catabolic rate method uses data collected over a short period of time, it is possible to examine the metabolic mechanisms of glucose metabolism in yeast cells at different growth phases. Such information is of great importance in the understanding of developmental physiology in microorganisms and possibly other biological systems. The individual catabolic and anabolic rates of glucose metabolism in Saccharomyces cerevisiae have been assessed as a function of growth physiology with the new method. The catabolic rate of the pentose phosphate pathway decreases as cells develop from middle logarithmic to early stationary phase (0.27 μmole of glucose per minute per 10⁹ middle log cells compared to 0.17 μmole of glucose per minute per 10⁹ early stationary cells). Since the glucose assimilation rate also decreases during the same growth period, the relative participation of pentose phosphate pathway in overall glucose metabolism remains essentially constant. However, the glycolysis-Krebs cycle pathway assumes a more dominant role in glucose metabolism in yeast growth physiology through the middle log to the early stationary phase. The role played by anabolic pathways resembles the case of the pentose phosphate pathway, i.e. , decreasing as growth continues in the cell population. It appears that the increased resolving power of high resolution radiorespirometry can be successfully employed in the study of other physiological phenomena such as substrate transport and oscillatory mechanisms occurring in metabolic sequences.
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