Reversible inactivation of serpins at acidic pH.

The inhibitory activity of the serpins alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, alpha(2)-antiplasmin, antithrombin and C(1)-esterase inactivator is rapidly lost at pH 3 but slowly recovers at pH 7.4 with variable first-order rates (t(1/2)=1.4-19.2 min). All except alpha(1)-antichymotrypsin undergo a variation in intrinsic fluorescence intensity upon acidification (midpoint ca. 4.5) with a slow bi-exponential return to the initial intensity at pH 7.4 (mean t(1/2)=2.3-23 min). No correlation was found between the time of fluorescence recovery and that of reactivation. The acid-treated serpins are proteolyzed at neutral pH by their target proteinases. alpha(1)-Proteinase inhibitor was studied in more detail. Its acidification at pH 3 has a mild effect on its secondary structure, strongly disorders its tertiary structure, changes the microenvironment of Cys(232) and causes a very fast change in ellipticity at 225 nm (t(1/2)=1.6s). Neutralization of the acid-treated alpha(1)-proteinase inhibitor is an exothermic phenomenon. It leads to a much faster recovery of activity (t(1/2)=4+/-1 min) than of fluorescence intensity (t(1/2)=23+/-19 min), ellipticity (t(1/2)=32+/-4 min) and change in total energy, indicating that the inhibitory activity of alpha(1)-proteinase inhibitor does not require a fully native structure.

A novel type of membrane based on cholesteryl phosphocholine, cholesteryl phosphate, or sitosteryl phosphate, and dimyristoylglycerol.

Mixtures of amphiphilic cholesteryl phosphate (CP), sitosteryl phosphate (SP), or cholesteryl phosphocholine (CPC) with the nonphosphoryl diacyl lipid dimyristoylglycerol (DMG) or with cholesterol give self-organized systems (giant vesicles) in a wide range of pH, as demonstrated by fluorescence microscopy, differential scanning calorimetry, and small-angle X-ray scattering. The water permeability of a 1 : 1 molar mixture of CPC and DMG was also measured by a stopped-flow/light-scattering method. The novel self-organized systems are akin to natural eukaryotic ones, the only difference being the site of the phosphate-containing head-group, located on cholesterol instead of DMG. They might be present in some organisms not yet studied for the composition of their membranes.

Behaviour of Pluronic P84 block copolymer micelles above the gelification temperature as probed by positron annihilation lifetime spectroscopy.

The new method based on positron annihilation lifetime spectroscopy (PALS) to determine both the mean core radius, R(core), and aggregation number, N(ag), of micelles is applied to the study of aqueous solutions of the triblock Pluronic P84 copolymer as a function of temperature (T), beyond the gelification point (334 K). Two long-lived components appear in the PALS spectra, ascribed to triplet positronium in the water bulk (o-Ps(aq)) and in the organic core of the micelles (o-Ps(org)). Of the various fitting parameters, only the lifetime of the latter species, tau4, and the micellar parameters, R(core) and N(ag), disclose the occurrence of gelification by first increasing up to 334 K, then decreasing. By contrast to what is known in case of phase transition, none of the parameters shows any abrupt change at 334 K, whereas the macroscopic viscosity of the solutions suffers a drastic increase. This is attributed to the fact that positronium is sensitive to the microviscosity of the solutions. At the transition point, the properties of the polyoxipropylene aggregates forming the organic core of the P84 micelles are not greatly affected. Furthermore, the fact that the experimental N(ag) values coincide with those calculated for spheres, from the R(core) values, indicates that the shape of the P84 cores does not change significantly after gelification. The onset of gelification results from a decrease in the hydrogen bonding interactions in the solution with an ensuing relative increase in the interactions between the polyoxipropylene (PPO) groups, initially forming the corona of the P84 micelles, in an intermicellar mode. This increased solicitation of the PPO groups outside their initial location would result in depletion in the number of surfactant molecules forming the micelles, viz. a decrease in both R(core) and N(ag) above 334 K. From the data, additional information can be gained regarding the local viscosity and surface tension in the micellar cores.

A novel type of membranes based on cholesteryl phosphate.

Mixtures of the rigid amphiphile disodium cholesteryl phosphate (DCP) with the non-phosphorylated diacyl amphiphile dimyristoylglycerol (DMG) give self-organized systems in a wide range of pH, as demonstrated by differential microcalorimetry. These systems can be closed bilayer vesicles, as shown by optical microscopy (Nomarski and confocal). Neither DMG nor DCP, taken alone, give vesicles in these conditions but 10% DMG is enough to lead to the formation of vesicles from pH 5.8 to 9.3. These novel self-organized systems are akin to the classical eucaryotic ones, built on a phosphorylated diacylglycerol and free cholesterol (or analogues), the only difference being the site of the phosphate head-group.