Plasma membrane coating with cationic silica particles and osmotic shock alters the morphology of bovine aortic endothelial cells.

We have used a published method of membrane preparation based on the precoating of the apical membrane of aortic endothelial cells with cationic silica microbeads (with or without polyacrylic acid) in combination with an osmotic shock and mechanical shearing to isolate the apical from the basal plasma membranes of these cells, in vitro. After labeling of the plasma membrane of adherent endothelial cells with a fluorescent derivative of phosphatidylcholine and by using laser confocal fluorescence scanning microscopy, we found that this method of membrane isolation rapidly induced invaginations of the basal plasma membrane to an extent which makes this method unsuitable for further membrane lipid analysis. Morphological analysis of the cells and fluorescence recovery after photobleaching experiments on the plasma membranes were performed at each step of the purification procedure and showed that only hypotonic shock and mechanical shearing of the cells enabled the basal plasma membranes to be purified without significant morphological changes.

Plasma membrane coating with cationic silica particles and osmotic shock alters the morphology of bovine aortic endothelial cells.

We have used a published method of membrane preparation based on the precoating of the apical membrane of aortic endothelial cells with cationic silica microbeads (with or without polyacrylic acid) in combination with an osmotic shock and mechanical shearing to isolate the apical from the basal plasma membranes of these cells, in vitro. After labeling of the plasma membrane of adherent endothelial cells with a fluorescent derivative of phosphatidylcholine and by using laser confocal fluorescence scanning microscopy, we found that this method of membrane isolation rapidly induced invaginations of the basal plasma membrane to an extent which makes this method unsuitable for further membrane lipid analysis. Morphological analysis of the cells and fluorescence recovery after photobleaching experiments on the plasma membranes were performed at each step of the purification procedure and showed that only hypotonic shock and mechanical shearing of the cells enabled the basal plasma membranes to be purified without significant morphological changes.

Mechanism of porcine pancreatic alpha-amylase inhibition of amylose and maltopentaose hydrolysis by kidney bean (Phaseolus vulgaris) inhibitor and comparison with that by acarbose.

The effects of Phaseolus vulgaris inhibitor (alpha-AI) on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic alpha-amylase (PPA) were investigated. Based on a statistical analysis of the kinetic data and using the general velocity equation, which is valid at equilibrium for all types of inhibition in a single-substrate reaction, it was concluded that the inhibitory mode is of the mixed noncompetitive type involving two molecules of inhibitor. In line with this conclusion, the Lineweaver-Burk primary plots intersect in the second quadrant and the secondary plots of the slopes and the intercepts versus the inhibitor concentrations are parabolic curves, whether the substrate used was amylose or maltopentaose. A specific inhibition model of the mixed noncompetitive type applies here. This model differs from those previously proposed for acarbose [Al Kazaz, M., Desseaux, V., Marchis-Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787-796 and Al Kazaz, M., Desseaux, V., Marchis-Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100-107]. In particular, with alpha-AI, the inhibition takes place only when PPA and alpha-AI are preincubated together before the substrate is added. This shows that the inhibitory PPA-alphaAI complex is formed during the preincubation period. Secondly, other inhibitory complexes are formed, in which two molecules of inhibitor are bound to either the free enzyme or the enzyme-substrate complex. The catalytic efficiency was determined both with and without inhibitor. Using the same molar concentration of inhibitor, alpha-AI was found to be a much stronger inhibitor than acarbose. However, when the inhibitor amount is expressed on a weight basis (mg x L-1), the opposite conclusion is drawn. In addition, limited proteolysis was performed on PPA alone and on the alpha-AI-PPA complex. The results show that, in the complex, PPA is more sensitive to subtilisin attack, and shorter fragments are obtained. These data reflect the conformational changes undergone by PPA as the result of the protein inhibitor binding, which differ from those previously observed with acarbose.

A plant-seed inhibitor of two classes of alpha-amylases: X-ray analysis of Tenebrio molitor larvae alpha-amylase in complex with the bean Phaseolus vulgaris inhibitor.

The alpha-amylase from Tenebrio molitor larvae (TMA) has been crystallized in complex with the alpha-amylase inhibitor (alpha-AI) from the bean Phaseolus vulgaris. A molecular-replacement solution of the structure was obtained using the refined pig pancreatic alpha-amylase (PPA) and alpha-AI atomic coordinates as starting models. The structural analysis showed that although TMA has the typical structure common to alpha-amylases, large deviations from the mammalian alpha-amylase models occur in the loops. Despite these differences in the interacting loops, the bean inhibitor is still able to inhibit both the insect and mammalian alpha-amylase.

Characterization and functional properties of the alpha-amylase inhibitor (alpha-AI) from kidney bean (Phaseolus vulgaris) seeds.

Alpha-amylase inhibitor (alpha-AI) from kidney bean (Phaseolus vulgaris L. cv Tendergreen) seeds has been purified to homogeneity by heat treatment in acidic medium, ammonium sulphate fractionation, chromatofocusing and gel filtration. Two isoforms, alpha-AI1 and alpha-AI1', of 43 kDa have been isolated which differ from each other by their isoelectric points and neutral sugar contents. The major isoform alpha-AI1 inhibited human and porcine pancreatic alpha-amylases (PPA) but was devoid of activity on alpha-amylases of bacterial or fungal origins. As shown on the Lineweaver-Burk plots, the nature of the inhibition is explained by a mixed non-competitive inhibition mechanism. Alpha-AI1 formed a 1:2 stoichiometric complex with PPA which showed an optimum pH of 4.5 at 30 degrees C. Owing to the low optimum pH found for alpha-AI activity, inhibitor-containing diets such as beans or transgenic plants expressing alpha-AI should be devoid of any harmful effect on human health.