Date of Award


Degree Type


Degree Name

Doctor of Philosophy


A mutant of Escherichia coli K12 lacking pyruvate dehydrogenase and phosphoenolpyruvate synthase was used to study transport of pyruvate by whole cells. Uptake of pyruvate is maximal with mid-log phase cells and the Michaelis constant for transport is 20{dollar}\mu{dollar}M. Pretreatment of the cells with respiratory chain poisons or uncouplers, with the exception of arsenate, inhibits transport of pyruvate by up to 95%. Lactate and alanine, natural analogs of pyruvate, competitively inhibit transport only at very high concentrations. The synthetic analogues 3-bromopyruvate and pyruvic acid methyl ester are good competitive inhibitors.;In order to further minimize metabolism of pyruvate, membrane vesicles of a wild type E. coli K12 were prepared. Transport is dependent on an artificial electron donor system, phenazine methosulfate and sodium ascorbate, added to the vesicles. Pyruvate is concentrated 7-15 times in these energized vesicles and the Michaelis constant is 15{dollar}\mu{dollar}M. Uptake of pyruvate can also be energized by a phenazine methosulfate and NADH system, but not by the metabolic intermediates lactate, glucose or ATP. Energy poisons, with the exception of arsenate, inhibit the transport of pyruvate. Synthetic analogues such as 3-bromopyruvate are good competitive inhibitors of transport. Lactate initially appeared to be a good competitive inhibitor of pyruvate transport in vesicles, but under conditions in which the oxidation of lactate to pyruvate is minimized, this apparent inhibition disappears.;Transport of pyruvate in whole cells was also found to be sensitive to osmotic shock, indicating that a periplasmic binding protein was involved in the transport system. Column chromatography techniques were employed in an attempt to isolate this binding protein from osmotic shock fluid. Partial purification of pyruvate binding activity was achieved through the use of an affinity column, but purification to homogeneity was not accomplished with subsequent column steps. The binding protein has been tentatively identified as a 36,000 molecular weight moiety by SDS-polyacrylamide gel electrophoresis.;A brief summary of these results indicates that pyruvate is transported in Escherichia coli by a unique and specific active transport system. This transport system utilizes the respiratory chain to provide the driving force for uptake and appears to have a periplasmic component capable of binding pyruvate.



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