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Y Engelborghs

Publications and source records attributed to Y Engelborghs.

At least 91 records · Page 5Linked to original sources
PubMed1983

Interactions of clathrin coat with model lipid membranes.

The interaction of coat proteins from coated vesicles with model lipid membranes was examined using small unilamellar vesicles of dipalmitoylglycerophosphocholine as model membranes. Changes in membrane permeability were measured by the leakage of entrapped fluorescent dye, carboxyfluorescein. Both clathrin and the 55000-Da protein were found to be active. Density gradient centrifugation showed the formation of an irreversible protein-lipid complex. Dynamic light-scattering measurements showed that this complex is significantly larger than the original vesicles, suggesting that fusion is induced. The effects of pH, urea, Tris and ionic strength were studied and the possible biological relevance of the results is discussed.

Animals↗
PubMed1983

A quantitative description of microtubule formation in the presence of tubulin-colchicine.

The overall polymerization of microtubule protein in the presence of tubulin-colchicine is described by competition between an intrinsically unaltered nucleation process and the process of propagation inhibited by the binding of tubulin-colchicine to the microtubule ends. The inhibition of propagation can be quantified with the binding constant previously determined [Lambeir and Engelborghs (1980) Eur. J. Biochem. 109, 619-624]. A quantitative description of the competition between nucleation and propagation follows from the kinetic theory of Oosawa. Comparison of several subsequent cycles of polymerization/depolymerization shows that a fraction of cold-stable complexes are formed. An equilibrium derivation is presented which shows the enhanced nucleation upon binding of inhibiting proteins, by the increase of the nucleation parameter A [Oosawa and Asakura (1975) Thermodynamics of the Polymerization of Protein, Academic Press, London, New York]. The kinetic and equilibrium derivations presented here are generally applicable to all capping factors, e.g. some of the actin-binding proteins.

Animals↗
PubMed1982

Tubulin polymerization in dimethyl sulfoxide.

The self-assembly of tubulin devoid of microtubule-associated proteins (MAPs) has been studied using a MES buffer containing dimethyl sulfoxide (Me2SO). Between 6% and 12% v/v Me2SO, the tubulin forms polymers which resemble microtubules in their morphology and chemical properties. These Me2SO microtubules, like normal microtubules, require GTP for assembly and are sensitive to cold, calcium ions, colchicine, and hydrostatic pressure. The polymerization shows a critical concentration which is dependent on the concentration of Me2SO. 8% Me2SO was found to be the optimum concentration for microtubule assembly. In these conditions, a linear Van t'Hoff plot is obtained, with delta H0/kJ . mol-1 = 26.5 over the range of 10-35 degrees C, and delta S0/J . K-1 . mol-1 = 186, in contrast to the assembly with MAPS or glycerol. The kinetics of polymerization shows that the apparent stoichiometry coefficient of nucleation has the value of 2. Ultracentrifugation analysis shows that there are no oligomers present at low temperatures in the absence of free nucleotide, while in identical conditions, tubulin with MAPs does form oligomers. Although the solvent conditions used supported propagation of assembly, nucleation was found to be very dependent on the transiently locally high Me2SO concentrations formed when Me2SO was added to initiate assembly. It is concluded that Me2SO preferentially stabilizes the lateral interactions.

Animals↗
PubMed1982

Fluorimetric analysis of the binding of warfarin to human serum albumin. Equilibrium and kinetic study.

Binding of warfarin to human serum albumin results in a red shift of the UV absorption maximum, suggesting that the binding site is a hydrophobic area of the protein. The enhancement of the fluorescence of warfarin upon binding to human serum albumin was used to study the binding equilibrium and the kinetics of this drug-protein interaction. From equilibrium fluorescence measurements, contributions from free and bound warfarin could be evaluated. From the resulting Scatchard plots, equilibrium constants ranging from 4.2 X 10(5) to 3.5 X 10(5) M-1 for temperatures from 8 degrees to 37 degrees were calculated. The reaction is slightly exothermic (delta H = -1.2 kcal m mole-1) and strongly entropy-driven (delta S = +21 cal . mole-1 . K-1). The reaction rate constants of the warfarin-albumin interaction were determined by the stopped-flow technique. The association rate constant varies from 2.2 X 10(5) to 7.7 X 10(5) M-1 sec-1 from 10 degrees to 32 degrees. The corresponding activation enthalpy is 9.0 kcal . mole-1. These values are not consistent with a diffusion-controlled reaction. The dissociation of the complex was studied by making use of the direct competition between warfarin and phenylbutazone for the same binding site. The dissociation rate constant varies from 2.5 to 10.8 sec-1 in the same temperature range. Activation parameters obtained in the kinetic experiments correspond very well with the thermodynamic parameters calculated from the equilibrium study, validating the fluorescence approach to the equilibrium studies.

Biotransformation↗
PubMed1981

A thermodynamic study of colchicine and colcemid dimerization.

The dimerization of colchicine is demonstrated using the technique of concentration difference spectra. The difference spectra are characterized by an isosbestic point at 372 nm, a positive peak at 387 nm, and two negative peaks at 360 and 330 nm. The study of the concentration dependence and the effect of temperature allowed the determination of the molar extinction change and the dimerization equilibrium constant at four temperatures. The van't Hoff plot is linear, and the following thermodynamic parameters are calculated: standard enthalpy change, delta Ho/kJ . mol-1 = -31.0; and standard entropy change, delta So/J . mol-1 . K-1 = -69.5. For colcemid, similar results are obtained. The difference spectra show an isosbestic point at 385 nm and a positive peak at 397 nm. The thermodynamic parameters obtained are delta Ho/kJ . mol-1 = -26.8, and delta So/J . mol-1 . K-1 = -61.1. These thermodynamic parameters are comparable to the values obtained for the dimerization of polar dyes and the stacking of nucleotide bases.

Chemical Phenomena↗
PubMed1981

A fluorescence stopped flow study of colchicine binding to tubulin.

The kinetics of colchicine binding to tubulin has been studied, using a fluorescence stopped flow. The measurements of Garland (Garland, D. L. (1978) Biochemistry 17, 4266-4272) have been extended to high colchicine concentrations and different temperatures. The appearance of fluorescence is biphasic. Both phases depend in a nonlinear way on colchicine concentration. The presence of colchicine dimers at these concentrations has been taken into account. The fast phase is analyzed as a two-step mechanism. The thermodynamic parameters of the fast initial binding, and the activation energy of the slow conformational change, have been determined. The relative magnitude of the slow phase depends on temperature. It is interpreted as a slow preequilibrium between two tubulin conformers. The effect of the microtubule-associated proteins on the different processes is studied. The binding of colchicine to tubulin ring-like oligomers is discussed.

Animals↗
PubMed1981

Temperature jump relaxation study of microtubule elongation in the presence of GTP/GDP mixtures.

GDP was added to microtubules at steady state. The amount of dissociation obtained was dependent on the GDP/GTP ratio and a method was developed to extrapolate to pure GDP conditions. From this extrapolation it was concluded that in the absence of GTP no elongation events occur. It was shown that at 35 degrees C nucleotide exchange is very fast, but at 25 degrees C, it is rate limiting for GDP-induced dissociation. Relaxation experiments, using temperature jumps before and after the addition of GDP, show that the nucleotide composition of the ends has to be taken into account. The model accepted so far cannot explain the observations. Several model mechanisms are described and their implications for equilibrium and relaxation data are analysed. All the acceptable models predict an increase in treadmilling efficiency at high GDP concentrations.

Animals↗
PubMed1980

A quantitative analysis of tubulin-colchicine binding to microtubules.

The binding of the tubulin-colchicine complex to microtubules has been studied in conditions where copolymerisation, as observed by Sternlicht and Ringel [J. Biol. Chem. 254, 10540-10550 (1979)], is negligible. The binding is shown to be rapid and reversible, in contrast with assumptions found in the literature. The binding constant was determined by a quantitative analysis of the concentration dependence of growth inhibition. The binding constant is of the same order of magnitude as the equilibrium constant of growth for tubulin. The temperature dependence was also studied and the Van't Hoff plot was found to be biphasic with an exothermic part at temperatures lower than 30 degrees C. A molecular model is presented for the interpretation of the copolymerisation observed by Strenlicht and Ringel. When a large excess of colchicine was added to microtubules, only a small phase of dissociation was observed. The new end state was metastable, as further dissociation could not be reversed. This is in conflict with a pure head-to-tail polymerisation mechanism, but in agreement with the recent observations of Bergen and Borisy [J. Cell Biol. 84, 141-150 (1980)], that head-to-tail polymerisation contributes only a small fraction of the total polymerisation.

Animals↗
PubMed1976

Differential hydrogen ion titrations of the histidine residues in Helix pomatia haemocyanin.

The properties of the histidine residues in Helix pomatia haemocyanin have been studied by differential hydrogen ion titrations. In oxy-and deoxyhaemocyanin 31 X 10(-5) histidine residues per g protein are titrated in contrast to 35 X 10(-5) residues in apohaemocyanin. The difference corresponds to a stoichiometry of one histidine residue per copper atom bound. Even in apohaemocyanin about 6 X 10(-5) histidine residues per g protein are not titrated in their normal pH region. In the presence of sufficient calcium to displace the dissociation completely out of the titration region, the titration curve of apohaemocyanin could be linarized according to the model of Linderstrom--Lang. In oxy-and deoxyhaemocyanin, however, a distinct deviation from linearity was found under the same conditions. In the absence of calcium the effect of the dissociation adds up to this deviation. The electrostatic interaction factors were determined for the protein at 0.1 M KC1 and for the dissociation products: halves and tenths at 1.0 M KC1. The electrostatic interaction factor for the wholes and the halves are much smaller than the values calculated from the Linderstrom--Lang equation, using the radius of the equivalent sphere either obtained from electron microscopy or from the partial specific volume. This probably due to solvent penetration. For the tenths at 1.0 M KC1, this effect is small.

Animals↗
PubMed1975

A quenched-flow study of the reaction catalysed by creatine kinase.

The reaction catalysed by creatine kinase was studied in both directions by quenched-flow techniques to follow the initial product formation in the millisecond range. In both directions the amount of product formed increases linearly with time, and the turnover number corresponds to the steady-state value. Extrapolation to zero time indicates the absence of either a large transient phase or a large lag phase in both directions. This indicates that the actual chemical reaction is rate-limiting, and that all possible isomerizations before or after the chemical step must be much more rapid.

Adenosine Diphosphate↗