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A study of oxidative phosphorylation in Pseudomonas aeruginosa Strasdine, George Alfred


The earlier failure to demonstrate substrate-dependent oxidative phosphorylation in cell free extracts of Pseudomonas aeruginosa led to an investigation of the conditions affecting the incorporation of radioactive phosphorus into resting cell suspensions of this organism. Incorporation of radioactive phosphorus was shown to be dependent on the substrate concentration, the presence of magnesium ions, a source of available nitrogen and to be associated with the oxidative enzymes of the cell. The more common methods of cell breakage employed for the preparation of bacterial cell free extracts were considered detrimental to the mechanisms of oxidative phosphorylation and were abandoned in favor of a method involving the osmotic lysis of spheroplasts with versene and lysozyme. These preparations were shown to be easily separated into membranes, cytoplasm, and ribosomes by differential centrifugation and had the advantage of not having been subjected to severe physical treatments. Previous studies with cell free extracts had demonstrated the formation of ATP³² in the presence of ADP and P³², presumably through a coupled oxidative phosphorylation process. The formation of ATP³² was shown however to be the result of a coupled enzyme reaction involving polynucleotide phosphorylase and adenylate kinase (equations 1, 2 and 3), and although influenced by a concurrent oxidative phosphorylation process was itself not a measure of oxidative phosphorylation. [Equations omitted] The enzyme mediating the exchange reaction (equation 1) was shown to be polynucleotide phosphorylase and that at least in this organism this enzyme is associated with the ribosomal fraction of the cell. Oxidative phosphorylation was demonstrated in crude cell extracts prepared from succinate-grown cultures by the lysozyme-versene treatment. Maximum P:0 ratios of 2.0 with succinate as substrate and 4.3 for -keto-glutarate were obtained thus presenting further evidence for the similarity of this fundamental process in bacterial and animal tissue

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