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C Kahle

Publications and source records attributed to C Kahle.

6 recordsLinked to original sources
PubMed1999

Thermodynamics and Structural Studies of the Interaction of Polymyxin B with Deep Rough Mutant Lipopolysaccharides.

Deep rough mutant lipopolysaccharide (ReLPS) dissolved in aqueous solution spontaneously forms supramolecular structures which mainly consist of vesicles. Addition of Polymyxin B (PmB) to these ReLPS vesicles influence the shape of these structures as demonstrated here by electronmicroscopy and dynamic light scattering techniques. The main phase transition of the ReLPS is found at 21.3 +/- 0.1 degrees C for ReLPS from Escherichia coli and at 24.0 +/- 0.5 degrees C for ReLPS from Salmonella minnesota by differential scanning calorimetry (DSC). Using isothermal differential titration calorimetry (ITC), the thermodynamic behavior of the interaction of PmB with ReLPS vesicles has been studied. The stoichiometric ratio for the binding of PmB to ReLPS was found to lie between 0.6 and 1, as determined from ITC and monolayer experiments. No phase transition was observed for ReLPS monolayers saturated with PmB. The results indicate specific interaction of PmB with ReLPS. We propose a two-step mechanism for this interaction, which involves electrostatic attraction between charged parts of the molecules and, in the second step, hydrophobic interactions between the nonpolar parts of both compounds. Copyright 1999 Academic Press.

Journal Article↗
PubMed1992

Regulation of hemicholinium binding sites in isolated nerve terminals.

High-affinity uptake of choline, the rate-limiting, regulatory step for the synthesis of acetylcholine is regulated via presynaptic auto- and heteroreceptors. Binding studies using tritiated hemicholinium-3 ([3H]HCh-3) as the specific ligand for the choline carrier revealed that the number of hemicholinium binding sites in nerve terminals isolated from insect brain changes corresponding to the activity of synaptosomal kinase A and kinase C. Activation of kinase A apparently increases the total number of hemicholinium binding sites by recruiting additional occult carriers, whereas the effect of kinase C activity is most appropriately explained by preventing a down-regulation of carrier proteins. The kinase-mediated regulation of choline transporters is obviously due to a phosphorylation of the carrier protein itself.

Animals↗
PubMed1992

Pentitol metabolism of Rhodobacter sphaeroides Si4: purification and characterization of a ribitol dehydrogenase.

The phototrophic bacterium Rhodobacter sphaeroides strain Si4 induced ribitol dehydrogenase (EC 1.1.1.56) when grown on ribitol- or xylitol-containing medium. This ribitol dehydrogenase was purified to apparent homogeneity by ammonium sulphate precipitation, affinity chromatography on Procion red, and chromatography on Q-Sepharose. For the native enzyme an isoelectric point of pH 6.1 and an apparent M(r) of 50,000 was determined. SDS-PAGE yielded a single peptide band of M(r) 25,000 suggesting a dimeric enzyme structure. The ribitol dehydrogenase was specific for NAD+ but unspecific as to its polyol substrate. In order of decreasing activity ribitol, xylitol, erythritol, D-glucitol and D-arabitol were oxidized. The pH optimum of substrate oxidation was 10, and that of substrate reduction was 6.5. The equilibrium constant of the interconversion of ribitol to D-ribulose was determined to be 0.33 nM at pH 7.0 and 25 degrees C. The Km-values determined for ribitol, ribulose, xylitol and NAD+ (in the presence of ribitol) were 6.3, 12.5, 77 and 0.077 mM, respectively. Because of the favourable Km for ribitol, a method for quantitative ribitol determination was elaborated.

Enzyme Induction↗
PubMed1991

Purification and reconstitution of the high affinity choline transporter.

The high-affinity choline transporter has been solubilized from synaptosomal membranes by various detergents. The solubilized carrier protein has been incorporated into liposomes after removal of the detergent by dialysis. Using the reconstitution of choline transport activity as an assay, the components catalyzing choline translocation were purified from the detergent extract by ion-exchange chromatography on a Mono-Q column followed by immunoaffinity chromatography. Monitoring the active fractions by sodium dodecylsulfate polyacrylamide gel electrophoresis and isoelectrofocussing gave one major protein with an apparent molecular weight of about 90,000 and an isoelectric point of pH 4.7. The isolated protein appeared to be heavily glycosylated as shown by lectin binding; upon treatment with endoglycosidase F the polypeptide was degraded to an apparent molecular weight of about 65,000. Accumulation of choline into liposomes reconstituted with the purified protein was driven by artificially imposed sodium gradients and inhibited by hemicholinium-3.

Animals↗
PubMed1990

Functional renaturation of receptor polypeptides eluted from SDS polyacrylamide gels.

In order to gain further support for the concept that a homo-oligomeric protein-complex may be sufficient to form a functional ligand-activated ion channel and to explore additional possibilities for the reconstitution of channel activity, a single polypeptide band of the purified neuronal AChR from insects has been electroeluted from SDS-polyacrylamide gels, the SDS removed and the polypeptides incorporated into liposomes. Liposomes were fused into planar lipid bilayers which were subsequently analysed for channel activity. Fluctuations of cation-channels were detected after addition of agonists (carbamylcholine); channel activity was blocked by antagonists (d-tubocurarine). The channels formed by electroeluted polypeptides gave conductance values, as well as kinetic data, quite similar to channels formed by the native receptor protein. Sedimentation experiments using sucrose density gradient centrifugation revealed that a considerable portion of the electroeluted polypeptides assembled during the reconstitution process to form oligomeric complexes with a sedimentation coefficient of about 10 S; thus resembling the native receptor complex.

Animals↗