Microcalorimetric study of the inhibition of butyrylcholinesterase by paraoxon.

The inhibition of horse serum butyrylcholinesterase (EC 3.1.1.8) by the organophosphorus compound paraoxon (diethyl 4-nitrophenyl phosphate) was studied by flow microcalorimetry at 37 degrees C in Tris buffer (pH 7.5) using a modification of the kinetic model described by Stojan and coworkers [J. Stojan, V. Marcel, S. Estrada-Mondaca, A. Klaebe, P. Masson, D. Fournier, A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase, FEBS Lett. 440 (1998) 85-88]. The reversible steps of the inhibition were studied in the mixing cell of the calorimeter, whereas the irreversible step was studied in the flow-through cell. A new pseudo-first-order approximation was developed to allow the kinetic analysis of inhibition progress curves in the presence of substrate when a significant amount of substrate is transformed. This approximation also allowed one to compute an analytical expression of the calorimetric curves using a gamma distribution to describe the impulse response of the calorimeter. Fitting models to data by nonlinear regression, with simulated annealing as a stochastic optimization method, allowed the determination of all kinetic parameters. It was found that paraoxon binds to both the enzyme and acyl-enzyme, but with weak affinities (K(i) = 0.123 mM and K'(i) = 5.5 mM). A slight activation was observed at the lowest paraoxon concentrations and was attributed to the binding of the substrate to the enzyme-inhibitor complex. The bimolecular inhibition rate constant k(i) = 2.8 x 10(4) M(-1) s(-1) was in agreement with previous studies. It is hoped that the methods developed in this work will contribute to extending the application range of microcalorimetry in the field of irreversible inhibitors.

Flow microcalorimetric study of butyrylcholinesterase kinetics and inhibition.

The enzymatic hydrolysis of butyrylcholine, catalyzed by horse serum butyrylcholinesterase (EC 3.1.1.8), was studied at 37 degrees C in Tris buffer (pH 7.5) by flow microcalorimetry. A convolution procedure, using the Gamma distribution to represent the impulse response of the calorimeter, was developed to analyze the microcalorimetric curves. After correction for buffer protonation, the hydrolysis reaction was found to be slightly endothermic, with Delta H=+9.8 kJ mol(-1). Enzyme kinetics was studied with both the differential and integrated forms of the Michaelis equation with equivalent results: Michaelis constant K(m)=3.3mM, catalytic constant k(cat)=1.7 x 10(3)s(-1), bimolecular rate constant k(s)=5.1 x 10(5)M(-1)s(-1). The reaction product, choline, was found to be a competitive inhibitor with a dissociation constant K(i)=9.1mM. Betaine had a slightly higher affinity for the enzyme, but the inhibition was only partial. This study confirms the usefulness of microcalorimetry for the kinetic study of enzymes and their inhibitors.

New photoantimicrobial films composed of porphyrinated lipophilic cellulose esters.

Porphyrinated cellulose laurate esters have been prepared in homogeneous DMA/LiCl medium by "one-pot, two-step" reactions starting from cellulose, protoporphyrin IX, and lauric acid and using a TsCl/Pyridine system. The plastic films obtained after casting were shown to display photobactericidal activity against Gram positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. This new photobactericidal polymer has potential for industrial, medical, or household applications.

Synthesis and properties of new bolaform and macrocyclic galactose-based surfactants obtained by olefin metathesis.

A series of galactose-based surfactants with various structures likely to display new interesting properties were synthesized. Four monocatenary surfactants were elaborated by microwave-assisted galactosylation of undecanol or 10-undecenol. These compounds were slightly soluble in water. Their tensioactive properties were determined at 45 degrees C. Olefin metathesis was used to synthesize the two single-chain bolaforms from undec-10-enyl galactopyranosides; two pseudomacrocyclic bolaforms were prepared by grafting two carbamates at O-4 and O-4' sugar positions of the single-chain bolaforms. These four surfactants are insoluble in water and undergo monolayer compression. Cyclization of these bolaforms by olefin metathesis led to macrocyclic surfactant analogues of archaeobacterial membrane components.

Amicoumacin antibiotic production and genetic diversity of Bacillus subtilis strains isolated from different habitats.

One of the most interesting groups of phenolic compounds is comprised of the low molecular weight phenylpropanol derivative substances named isocoumarins, which possess important biological activities. In this study, the isocoumarin production and genetic diversity of 51 Bacillus strains isolated from different geographical and ecological niches were studied. Using molecular identification techniques, 47 strains were identified as B. subtilis, three as B. licheniformis and one as B. pumilus. When these strains were screened for isocumarin production, 11 belonging to the species B. subtilis produced amicoumacins, antibiotics of the isocoumarin group. RAPD analysis demonstrated that these strains fell into two groups which contained only these amicoumacin producers. No association was detected between RAPD profiles and the geographic origin or habitat of the strains tested. In conclusion, production of amicoumacin antibiotics by B. subtilis is a common characteristic of individual strains that presented genetic and physiological homogeneity.