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Details about the parameters of kinetic systems are crucial for progress in both medical and industrial research, including drug development, clinical diagnosis and biotechnology applications. Such details must be collected by a series of kinetic experiments and investigations. The correct design of the experiment is essential to collecting data suitable for analysis, modelling and deriving the correct information. We have developed a systematic and iterative Bayesian method and sets of rules for the design of enzyme kinetic experiments. Our method selects the optimum design to collect data suitable for accurate modelling and analysis and minimises the error in the parameters estimated. The rules select features of the design such as the substrate range and the number of measurements. We show here that this method can be directly applied to the study of other important kinetic systems, including drug transport, receptor binding, microbial culture and cell transport kinetics. It is possible to reduce the errors in the estimated parameters and, most importantly, increase the efficiency and cost-effectiveness by reducing the necessary amount of experiments and data points measured. [Copyright &y& Elsevier]

Wild-type rat lens main intrinsic protein (MIP) was heterologously expressed in the membrane of Spodoptera frugiperda (Sf21) cells using the baculovirus expression system and in mouse erythroid leukaemia cells (MEL C88). Both MEL and Sf21 cell lines expressing wild-type MIP were investigated for the conductance of ions using a whole cell patch clamp technique. An increase in conductance was seen in both expression systems, particularly on lowering the pH to 6.3. In Sf21 cells, addition of antibodies to the NPA1 box resulted in a reduction of current flow. These results suggest that MIP has pH-dependent ion channel activity, which involves the NPA1 box domain. [Copyright &y& Elsevier]

Wild type rat lens main intrinsic protein (MIP) and MIP mutated (F73I, F75L) to resemble the glycerol facilitator of Escherichia coli in the region of the NPA1 box were used to investigate the topology of MIP in the membrane of Spodoptera frugiperda (Sf21) cells using the baculovirus expression system and expression in mouse erythroid leukaemia cells (MEL C88). Differential fixation for staining was used, with paraformaldehyde for externally exposed antigenic sites, and acetone for both externally and internally exposed protein antigenic sites. Immunofluorescence using antibodies to synthetic MIP peptides showed that wild type MIP had a six transmembrane topography. The N- and C-termini were intracellular in both expression systems, and both NPA boxes were found to be extracellular. These results show that residues around the NPA1 box can influence the folding of the MIP in the membrane, and provide structural evidence for the poor water transport properties of MIP, as the NPA boxes lie outside the plane of the membrane. [Copyright &y& Elsevier]