Targeting of porin to the outer membrane of Escherichia coli. Rate of trimer assembly and identification of a dimer intermediate

Porin, a transmembrane protein in the outer membrane of Escherichia coli, exists in a trimeric structure which is not dissociated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 25 degrees C. This unusual stability was utilized in the study of the conformational changes which accompany the targeting of porin to the outer membrane. A delay of 16-44 s between completion of synthesis of a monomer and its assembly into a trimer was found from the ratio of monomers to trimers found in exponentially growing cells. Pulse-chase experiments showed that rapid processing of precursor OmpF molecules was followed by assembly into sodium dodecyl sulfate-resistant oligomers with a half-time of 20 s at 30 degrees C. An intermediate in assembly was isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis below 10 degrees C and was identified as a metastable dimer.     

Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic-<Superscript>19</Superscript>F-NMR

The cause of the pregnancy condition preeclampsia (PE) is thought to be endothelial dysfunction caused by oxidative stress. As abnormal glucose tolerance has also been associated with PE, we use a fluorinated-mimic of this metabolite to establish whether any oxidative damage to lipids and proteins in the erythrocyte membrane has increased cell membrane permeability. Data were acquired using ¹⁹F Dynamic-NMR (DNMR) to measure exchange of 3-fluoro-3-deoxyglucose (3-FDG) across the membrane of erythrocytes from 10 pregnant women (5 healthy control women, and 5 from women suffering from PE). Magnetisation transfer was measured using the 1D selective inversion and 2D EXSY pulse sequences, over a range of time delays. Integrated intensities from these experiments were used in matrix diagonalisation to estimate the values of the rate constants of exchange and membrane permeability. No significant differences were observed for the rate of exchange of 3-FDG and membrane permeability between healthy pregnant women and those suffering from PE, leading us to conclude that no oxidative damage had occurred at this carrier-protein site in the membrane.     

A novel galactosyl-binding lectin from the plasma of the blood clam,Anadara granosa (L) and a study of its combining site

Control Analysis has been carried out in the first steps of a rat liver glycolytic system. Attention has been focused on the effect of several glucose concentrations on the control, particularly regarding the role of glucokinase. From kinetic studies of the whole metabolic system we have obtained information on the flux variation under different glucose concentrations. This information together with the kinetics of glucokinase has allowed us to calculate Flux Control and Elasticity Coefficients for glucokinase and the Response Coefficient of the system with respect to glucose. The changes in of the value of Flux Control Coefficients demonstrates that in conditions of low glucose concentration, glucokinase is the main enzyme in controlling the flux through the pathway, but at high glucose concentration the control moves to phosphofructokinase. Next, we have compared our results with those obtained with the shortening and titration method, previously described (Torres, N.V., Mateo, F., Mélendez-Hevia, E. and Kacser, H., (1986) Biochem. J. 234, 169–174; Torres, N.V. and Meléndez-Hevia, E. 1991. Molec. Cell. Biochem. 101, 1–10). Furthermore, from knowledge of the enzyme kinetics of the system we have been able to build a model of the pathway that allows us computer similation of its behavior and calculation of the Flux Control Coefficient profile at different glucose concentrations. By the three methods the results correlate, supporting the use of the pathway substrate as external modulator of the metabolic system as a tool for practical application of Control Analysis.     

Characterization of tetranucleotide microsatellite loci and development and validation of multiplex reactions for the study of right whale species (genus Eubalaena)

A North Atlantic right whale (Eubalaena glacialis) genomic library was developed and screened with a (GATA)₈ probe to identify tetranucleotide microsatellite loci. Sixteen characterized loci were polymorphic in North Atlantic and/or South Atlantic (Eubalaena australis) right whales, 12 being polymorphic in E. glacialis, and 15 in E. australis. Fourteen of these were combined with 21 other previously identified loci for a suite of 35 loci which can be used to increase resolution of genetic analyses of these species. Multiplex reactions were developed for genotyping samples at these loci, providing a method that is rapid, reliable and cost‐effective.