Biomedical subjects
Publications and source records attributed to M De Cuyper.
A fast, simple two-step purification scheme is presented for the isolation of lysozyme at a high yield from equine milk. In the first step, fluidized bed technology, using the Streamline system, was exploited. In the following step, advantage was taken of Ca(2+)-induced conformational changes to obtain a pure, high specific activity, enzyme fraction by hydrophobic interaction chromatography.
We have studied the capacity of different iodinated derivatives of phenylalanine and tyrosine to inhibit the incorporation of [3H]tyrosine into tubulin catalyzed by tubulin:tyrosine ligase. In contrast to thyronine and its iodinated derivatives, iodotyrosines were efficient inhibitors. That they also functioned as substrates of the enzyme was shown by the effective incorporation of [125I]mono- and diiodotyrosine into tubulin. The label was shown to be located at the carboxy terminus. Labeling by this method conserves the polymerization capacity of tubulin in contrast with classical radioiodination methods involving oxidation.
The adsorption of different types of phosphatidylglycerols onto magnetizable solid particles is studied. The super-paramagnetic magnetite spheres used have an average diameter of only 14 nm and are stabilized by lauric acid to keep them in solution. During incubation and dialysis of this water-based magnetic fluid in the presence of preformed sonicated phospholipid vesicles, magnetoliposomes are formed which are captured from solution with high efficiency by high-gradient magnetophoresis. Support for the bilayer character of the phospholipid coat is derived from both theoretical calculations and experimental data. Phospholipids which form the inner monolayer are adsorbed very quickly with their charged head-group orientated towards the iron oxide surface. The high-affinity character of the binding is reflected in the adsorption isotherms and is further illustrated by their non-extractability with high concentrations of Tween 20. The outer layer assembles through interaction with the exposed hydrocarbon chains. As compared to the inner layer, the phospholipids adsorb at a much slower rate and are displaced by Tween 20 concentrations which usually disrupt conventional membranes. The adsorption isotherms for this layer obey the Langmuir expression. The affinity constants, derived from them, progressively increase as the hydrophobic nature of the phosphatidylglycerols is more pronounced.
Basal-lateral and brush border membranes from pig kidney cortex were prepared by differential centrifugation followed by free-flow electrophoresis. In each type of membrane, azide-insensitive, low-affinity Ca2+-ATPase and Mg2+-ATPase activities are demonstrated. A comparative study for both membranes further reveals the following analogies between these ATPases: (a) they show maximal activity between pH 8 and 8.5; (b) they exhibit Km values for Ca-ATP or Mg-ATP in the millimolar range and have a comparable low substrate specificity; (c) they are insensitive to 10 microM of vanadate, N,N'-dicyclohexylcarbodiimide, e diethylstilbestrol, quercetin, harmaline and amiloride. The partial inhibition by 1 mM of the various compounds is rather aspecific. In view of these similarities it is concluded that only one enzyme entity is responsible for the activity which is measured in both membrane types. The HCO3-stimulated Mg2+-ATPase activity in pig kidney cortex was also studied. This enzyme, however, is clearly of mitochondrial origin since the HCO3-stimulation coincides with the distribution profile of succinate dehydrogenase, a mitochondrial marker; and since it is inhibited by azide.
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The nonconjugated bichromophoric molecule 1,3-di(1-pyrenyl)propane shows, besides the pyrene monomer fluorescence, a structureless emission due to an intramolecular excited dimer (excimer). In the case of intramolecular excimer forming systems, the ratio of the emission intensities of excimer vs. monomer (IE/IM) is sensitive to changes in membrane structure as will be illustrated here. The present molecule is a useful probe to report on lipid-protein interactions, at least in our model system. We have introduced it into the hydrocarbon layer of dimyristoylphosphatidylcholine vesicles in order to study their interaction with alpha-lactalbumin (alpha-LA) as a function of pH and temperature. On the basis of steady-state fluorescence, kinetic, and energy transfer studies, we have found that, at pH 4, alpha-LA strongly interacts with the lipid bilayer. In steady-state fluorescence experiments, changes of the ratio IE/IM and shifts of the transition temperature have been observed, reflecting changes of membrane structure caused by interaction with alpha-LA. Kinetic studies of the rate of interaction of alpha-lactalbumin and measurements of energy transfer from excited tryptophan(s) to the fluorescent probe confirm the steady-state experiments. Our results agree with previously reported microcalorimetric, gel chromatographic, and fluorescence polarization studies.
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1. Phospholipid-depleted cytochrome c oxidase is incorporated in vesicles, built up of phospholipids of known polar headgroup and fatty-acyl side chains. 2. Maximal reactivation is obtained only when the fatty-acyl side chains provide a fluid environment. 3. Fluid zwitterionic phospholipids are found to be more efficient reactivators than fluid anionic ones. 4. Irrespective of the polar headgroup type, two narrow ranges of activation energies for the enzymatic reaction are calculated from the Arrhenius plots: 81--92 kJ/mol in solid and 51--61 kJ/mol in fluid conditions. 5. Cytochrome c oxidase is also incorporated in a series of vesicles, each built up of an equimolar amount of two phospholipids which differ in their polar headgroup type and/or their fatty-acyl side chain characteristics. From the localization of the enzyme activity profiles, obtained with these mixtures, tentative deductions are made about the preference of cytochrome c oxidase for different phospholipid molecules.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.