No description available.

No description available.

No description available.

The theoretical description of multicomponent transport across nonporous polymeric membranes was investigated using two alternative frameworks; the phenomenological approach of irreversible thermodynamics and the mechanistic Stefan-Maxwell formulation. The transport models developed account for potential equilibrium and/or kinetic coupling of fluxes and the contribution of diffusion induced non-selective flow within the polymer. Transient measurements coupled with transient models enable a more detailed evaluation of the complex multicomponent transport phenomena occurring within the nonporous polymer. The models developed in this study were validated against transient dialysis and pervaporation data for (ethanol-water)/silicone rubber system. A critical assessment was obtained by recovering the model parameters from the dialysis data and using the same parameters to predict the transient pervaporation performance. Separate evaluation of the equilibrium and kinetic contributions requires a thermodynamic model to describe the non-ideality of the polymer solution. The uptake of small polar solutes by hydrophobic polymers is not well described by the classical Flory-Huggins model. An empirical modification was developed which retains the basic form of the Flory-Huggins model but allows the interaction parameters to be a simple function of activity. This modification provided a physically realistic description of the sorption equilibria for the {ethanol- water}/silicone rubber system over conditions ranging from a low pressure vapour to a saturated liquid. The phenomenological approach of irreversible thermodynamics was used to develop transient models of dialysis and pervaporation. The numerical solution of the model equations, which constitute a set of coupled partial differential equations, was accomplished by the application of the method of lines. Average phenomenological diffusion coefficients recovered from dialysis data can give a good qualitative prediction of pervaporation performance provided the diffusion coefficients satisfy the Onsagar reciprocal relationships. However, a quantitative prediction requires the explicit inclusion of the concentration dependence of the diffusivities, which is best achieved within the mechanistic Stefan-Maxwell formulation. A generic model of membrane transport was formulated using the mechanistic Stephan-Maxwell approach and generalised driving forces, which included the contribution from the various internal and external driving forces. Transient models of dialysis and pervaporation were developed which used exactly the same generic model to describe the transport through the membrane. A notable advantage of the generic model lies in the fact that the Stephan-Maxwell diffusivities retain their physical significance irrespective of the number of components present. This offers the opportunity of recovering many of the model parameters from relatively simple binary experiments. The results obtained indicate that the generic model is capable of describing the transient dialysis and pervaporation of the {ethanol-water}/silicone rubber system with an identical set of concentration dependent equilibrium and diffusive parameters. The generic model provides a solid framework for the theoretical description of diverse processes employing a nonporous polymer as the selective separation barrier.

No description available.

No description available.

Aspects of the molecular basis of potassium transport via the KefC potassium efflux system in <I>E. coli</I> were examined. Separate cloning of the proposed membrane-spanning and cytoplasmic domains of KefC was carried out. A KefC construct was created that contained a amber stop codon at residue 382, between the two domains. When cloned into a prokaryotic expression vector, transformed strains grew poorly. No activity was detected in either an amber suppressor strain, or a strain lacking an amber suppressor, in the presence or absence of IPTG. A series of strains possessing mutant <I>kefC</I> alleles that cause spontaneous potassium loss via KefC were studied. The mutations in the mutant <I>kefC</I> strains were identified as missense mutations, which mapped to two regions of KefC; a sequence of amino acids in the N-terminal domain, (HALESDIE), and the Rossman fold within the C-terminal, cytoplasmic, domain. Recently, it has been discovered that an upstream gene, <I>yabF</I>, which overlaps <I>kefC</I> by eight base pairs, is involved in KefC activity. The sequence of <I>yabF</I>, from Frag5 and the mutant strains was examined and was not mutated in any of the strains, but the sequence of <I>yabF</I> was different from that published by the <I>E. coli</I> genome sequencing project. Studies on <I>kefB</I> mutant strains were initiated. <I>In vitro</I> site-directed mutagenesis of cloned <I>kefC</I> was used to analyse the importance of two residues in the Rossman fold, R416 and S420. The KefCR416S mutations results in spontaneous potassium loss, whereas the KefCS420A mutation does not. The interaction of KefC subunits was confirmed. The R416S mutation, (in the Rossman fold), and the D264A mutation, (in the HALESDIE sequence), were co-dominant. Spontaneous potassium loss via KefCR416S was found to require YabF and glutathione.

No description available.

No description available.

Inorganic phosphate is an essential nutrient for all organisms. It is required for many cellular components as nucleic acids and phospholipids, and as energy-carrying compounds such as ATP. Thus, a regulated uptake of this pivotal nutrient is of outermost importance. Depending of the availability of phosphate in the surroundings the yeast Saccharomyces cerevisiae make use of two different systems for transporting phosphate into the interior of the cell: a low-affinity system that is active during surplus phosphate conditions and a high-affinity system that is active when the availability becomes limited. This thesis focuses on the high-affinity system, which is comprised of the Pho84 and Pho89 transporters. Of the two transporters, Pho84 is the predominant one, responsible for almost all phosphate uptake during low phosphate conditions, and the contribution of Pho89 is of minor importance. Hence Pho84 is by far the most well characterized phosphate transporter. Even though much is known about phosphate transporters in yeast little in known about how phosphate is transported. The work in this thesis aims to broaden the knowledge about the transport mechanism by the means of site-directed mutagenesis and functional characterization. Also the similarity of Pho84 to glucose sensors and the potential role of conserved residues in phosphate signaling are investigated. By the use of a high-affinity system deletion strain (∆Pho84 ∆Pho89), we also managed to investigate the functional importance of well conserved residues in Pho89. In summary: the work presented in this thesis has contributed to increase the knowledge about transport mechanisms in phosphate transporters.