UBC Theses and Dissertations
Mechanism of energization of transhydrogenase in Escherichia coli membranes Chang, David Yeun Bin
Low concentrations of the group IIA metals Mg²⁺, Ca²⁺, and Ba²⁺ stimulated energy-independent transhydrogenase activity. High concentrations of Mg²⁺ inhibited this activity. Transhydrogenase requires Mg²⁺-complexed NADP(H) rather than free NADP(H) as its substrate. High concentrations of Mg2+, however, may change the conformation of the enzyme to inhibit its enzymatic reaction by binding directly to the NADP(H) site. Upon transhydrogenation between NADPH and 3-acetylpyridine dinucleotide, E. coli pyridine nucleotide transhydrogenase can establish a proton gradient across the cell membrane. The primary component of the proton gradient for energization of transhydrogenase was found to be the pH gradient and not the membrane potential. A similar conclusion was drawn for the ATP-driven transhydrogenase reactions. In strains of E. coli that harbored plasmids to give the cells elevated levels of transhydrogenase, it was found that uncouplers stimulated the aerobic-driven transhydrogenase reaction. This is a chemiosmotic anomaly and is in contrast to the non-plasmid containing parent strains where uncouplers inhibited the activity. Further investigation revealed that the plasmid strains contained a much lower NADH oxidase activity than the non-plasmid strains and that neither KCN nor QNO can inhibit the aerobic-dependent activity in both types of strains even though they were effective in blocking the respiratory chain. These effects prompted us to inquire whether the anomaly was due to differences in the respiratory chain, but no differences were found between the NADH dehydrogenase activities, quinone and cytochrome contents of the plasmid and non-plasmid strains. The bacterial cells with amplified transhydrogenases induce extra intracellular tubular membrane structures to accomodate the extra proteins (Clark, D.M., Pyridine Nucleotide Transhydrogenase, PhD thesis, University of British Columbia, 1986). Separation of the E. coli membrane vesicles on a shallow sucrose gradient, however, did not reveal any differences between the vesicles of the plasmid and non-plasmid strains. Therefore, it seems unlikely that the anomaly is due to the plasmid strains performing a unique form of energization on these induced structures. Finally, it was established by SDS-PAGE and Western blot using anti-transhydrogenase antisera that the plasmid strains express a much higher level of transhydrogenase enzymes in their cell membranes than do the non-plasmid strains.
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