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Title: Energy coupling in bacterial transport : studies with isolated cytoplasmic membrane vesicles
Author: Nichols, W. W.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1975
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1. The research literature is reviewed concerning the application of the bacterial membrane vesicle, as an experimental tool, to the problem of energy coupling in active transport. The conclusion drawn is that the experimental data from studies with bacterial membrane vesicles either positively support, or are compatible with, the chemiosmotic hypothesis of energy coupling. Furthermore, the experimental data which gave rise to the redox carrier hypothesis of energy coupling in bacterial transport are explained in terms of the membrane orientation of individual vesicles and in terms of the permeability properties of individual vesicles. 2. Membrane vesicles from Micrococcus denitrificans are described and chosen as the tool for examining the above interpretation experimentally. 3. A description is made of variations in the basic membrane-preparation procedure which were carried out in an effort to obtain homogeneous populations of right-side-out membrane vesicles (ghosts) and homogeneous populations of inside-out membrane vesicles (sub-bacterial particles; SBP) from M. denitrificans. In all cases, heterogenerous preparations were obtained which contained both ghosts and SBP. 4. The following techniques, which were examined in order to test their applicability to resolving heterogeneous vesicle populations, are discussed: sedimentation in ficoll and in sucrose density gradients, affinity chromatography, and electrophoresis. Data is presented showing that, in my hands, none of these methods showed extensive separation of ghosts from SBP. However, a useful technical finding is described: sedimentation of a membrane vesicle preparation over a pad of (5% w/v) ficoll solution removes unsealed vesicles from the total preparation. 5. Of the amino acids tested, only glycine is accumulated by membrane vesicles from M. denitrificans. The accumulation is uncoupled-sensitive and is dependent upon the addition of an appropriate physiological electron donor, such as; D-lactate, L-lactate or succinate, or the artificial electron-donating systems ascorbate plus phenazine methosulphate (PMS) or ascorbate plus N:N:N':N' - tetramethyl-p-phenylene-diamine dihydrochloride (TMPD). Reduced nicotinamide adenine dinucleotide is found not to act as an electron donor for stimulating active transport of glycine. 6. The variation between D-lactate, L-lactate and succinate in extent of stimulation of active transport in membrane vesicle preparations from M. denitrificans grown on different media is found to be most likely due to variations in the respective complements of the particular respiratory dehydrogenases. 7. A scheme is proposed which explains the lack of NADH-stimulated glycine transport in membrane vesicles from M. denitrificans as being due to the localization of the respiratory dehydrogenases inside whole cells (and therefore ghosts). In addition, it is proposed that externally added NADH cannot cross the vesicle membrane to reach its dehydrogenase site. Experiments are described which show that the above scheme is correct. 8. A further part of the scheme also proposes that D-lactate is an effective energy donor for active transport in certain bacterial membrane vesicles (i.e. those which possess adequate lactate dehydrogenase activity) because it does cross the membrane, via a specific transport system, to reach its dehydrogenase site in ghosts. Evidence is presented showing that this is the case for membrane vesicles from Escherichia coli (studied, for technical reasons, as a parallel to the M. denitrificans vesicle system) and for membrane vesicles from M. denitrificans (studied after these technical difficulties were resolved). 9. Finally, as a pointer to a possible direction of future work, the following test of the above propositions is evaluated. I submit that D-lactate transport is required for D-lactate-driven glycine transport in membrane vesicles from M. denitrificans. Therefore, inhibition of D-lactate transport (without affecting D-lactate oxidation per se or glycine transport per se) should inhibit D-lactate-powered glycine transport. As preliminary observations, I tested the D-lactate transport inhibitors N-ethylmaleimide (NEM) and α-cvano, 4-hydroxycinnamic (αCHCA) acid for this purpose. These compounds may not be used for the above critical test due to their interference, in control experiments, withh the D-lactate dehydrogenase (αCHCA) or glycine transport (NEM).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available