Date of Award
Doctor of Philosophy
The C(,4)-dicarboxylate transport system of Escherichia coli K12 is responsible for the active uptake of succinate, fumarate and malate. Genetic and biochemical evidence has indicated that at least three distinct components appear to be involved in this transport process. Two of these transport components are the cytoplasmic membrane-bound succinate binding proteins, SBP1 and SBP2, which are coded for by the dctB (16 minutes) and dctA (78 minutes) genes respectively. Early work on these two transport proteins involved their isolation via aspartate-Sepharose chromatography from detergent-solubilized cytoplasmic membrane vesicles. The impurity of the isolated dicarboxylate transport proteins necessitated the development of techniques for their identification and purification. Three affinity techniques were developed that utilized the recognition of the transport components' substrate binding sites in order to identify and enrich the proteins prior to further purification steps. These techniques included aspartate-Sepharose affinity chromatography, immunoblotting of cytoplasmic membrane proteins with succinate-specific antiidiotypic antibodies and photoaffinity labelling of proteins in isolated membranes and whole cells. All three techniques involved a comparison of a wild-type strain (CBT43) with various dicarboxylate transport-mutant strains. The original aspartate-Sepharose protocol was optimized in terms of the isolation, solubilization and affinity chromatography of the cytoplasmic membrane proteins. The results of ('14)C -succinate binding studies with affinity-purified proteins from strain CBT43 and affinity chromatography of transport-mutant cytoplasmic membrane proteins suggests that a 53K protein was a good candidate for the SBP2 transport component. The immunoblotting studies employed antiidiotypic antibodies raised against the antigen binding site of succinate-specific IgG molecules and again implicated a 53K protein in the transport process. Finally, photoaffinity labelling of whole cells with N-(4-azido-2-nitrophenyl)aspartic acid suggested that a 47K protein might be involved in dicarboxylate transport. Further studies with dctA and dctB transformants are necessary to confirm the identity of the 53K protein as a succinate transport component and to establish the relationship, if any, between the 47K and 53K proteins.
Walker, Katherine Elizabeth, "The Cytoplasmic Membrane Carbon(4)-transport Components In Escherichia Coli K12: Identification And Purification Studies" (1986). Digitized Theses. 1564.