Tonoplast of Beta vulgaris L. contains detergent-resistant membrane microdomains.

The experiments conducted on tonoplast of Beta vulgaris L. roots were performed to identify detergent-resistant lipid-protein microdomains (DRMs, interpreted as lipid rafts).The presence of DRMs can be found when dynamic clustering of sphingolipids, sterols, saturated fatty acids is registered, and the insolubility of these microdomains in nonionic detergents at low temperatures is proven. The elucidation of tonoplast microdomains has been based on results obtained with the aid of high-speed centrifuging in the sucrose gradient. The experiments have shown that tonoplast microdomains are rich in sphingolipids, free sterols and saturated fatty acids (such a lipid content is also typical of lipid-protein microdomains of other membranes), while only few phospholipids are present in tonoplast microdomains. The presence of microdomains has been confirmed by fluorescence and confocal microscopy using filipin and Laurdan as fluorescent probes. The experiments with Laurdan have shown that tonoplast microdomains are characterized by a high order compared to characteristics of the rest of the tonoplast. Thus, the presence of detergent-resistant lipid-protein microdomains in the tonoplast has been demonstrated.

Fluorescence labels in kinases: a high-throughput kinase binding assay for the identification of DFG-out binding ligands.

Despite the hundreds of kinase inhibitors currently in discovery and pre-clinical phases, the number of kinase inhibitors which have been approved and are on the market remains low by comparison. This discrepancy reflects the challenges which accompany the development of kinase inhibitors which are relatively specific and less toxic. Targeting protein kinases with ATP-competitive inhibitors has been the classical approach to inhibiting kinase activity, but the highly conserved nature of the ATP-binding site contributes to poor inhibitor selectivity, issues which have particularly hampered the development of novel kinase inhibitors. We developed a high-throughput screening technology that can discriminate for inhibitors which stabilize the inactive "DFG-out" kinase conformation by binding within an allosteric pocket adjacent to the ATP-binding site. Here, we describe how to use this approach to measure the K (d) of ligands, as well as how to kinetically characterize the binding and dissociation of ligands to the kinase. We also describe how this technology can be used to rapidly screen small molecule libraries at high throughput.

The use of solvent relaxation technique to investigate headgroup hydration and protein binding of simple and mixed phosphatidylcholine/surfactant bilayer membranes.

The subject of this report was to investigate headgroup hydration and mobility of two types of mixed lipid vesicles, containing nonionic surfactants; straight chain Brij 98, and polysorbat Tween 80, with the same number of oxyethylene units as Brij, but attached via a sorbitan ring to oleic acid. We used the fluorescence solvent relaxation (SR) approach for the purpose and revealed differences between the two systems. Fluorescent solvent relaxation probes (Prodan, Laurdan, Patman) were found to be localized in mixed lipid vesicles similarly as in pure phospholipid bilayers. The SR parameters (i.e. dynamic Stokes shift, Deltanu, and the time course of the correlation function, C(t)) of such labels are in the same range in both kinds of systems. Each type of the tested surfactants has its own impact on water organization in the bilayer headgroup region probed by Patman. Brij 98 does not modify the solvation characteristics of the dye. In contrast, Tween 80 apparently dehydrates the headgroup and decreases its mobility. The SR data measured in lipid bilayers in presence of Interferon alfa-2b reveal that this protein, a candidate for non-invasive delivery, affects the bilayer in a different way than the peptide melittin. Interferon alfa-2b binds to mixed lipid bilayers peripherally, whereas melittin is deeply inserted into lipid membranes and affects their headgroup hydration and mobility measurably.

Distribution of selenium-containing proteins in human serum.

Selenium-containing proteins in human serum of four volunteers in Beijing were separated and purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Selenium contents in the proteins were quantified by high-performance liquid chromatography (HPLC) coupled with a fluorescence detector (FLD) after pretreatment with a microwave digestion system and derivatization with 2,3-diaminonaphthalene (DAN). Five selenium-containing proteins with apparent molecular weights (MWs) of 68+/-3, 57.5+/-2.5, 47+/-2, 41+/-1, and 21+/-1 kDa, respectively, were detected. By comparison with known data on serum selenium-containing proteins, the 68+/-3 kDa protein should belong to albumin, which took 6.3-9.8% of the total serum Se. The 57.5+/-2.5 kDa protein should be selenoprotein P and the 47+/-2 kDa protein was believed to be an isoform of selenoprotein P. The sum of Se in selenoprotein P and its isomer took about 41.1-69.3% and was the major form of human serum selenium. The 21+/-1 kDa protein should be plasma glutathione peroxidase (p-GPx) and its Se content was about 21.1-24.3%. Also, protein of 41+/-1 kDa should be a selenium-containing protein that, to our best knowledge, was reported for the first time. The Se percentage in this protein corresponded to 12.6-20.4% of total human serum Se.

Disclosure of discrete sites for phospholipid and sterols at the protein-lipid interface in native acetylcholine receptor-rich membrane.

There is an increasing body of evidence to support the notion that the function of the nicotinic acetylcholine receptor (AChR) is influenced by its lipid microenvironment [see Barrantes, F. J. (1993) FASEB J. 7, 1460-1467]. We have recently made use of the so-called generalized polarization (GP) of the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino)naphthalene) to learn about the physical state of the lipids in Torpedo marmorata AChR native membrane [Antollini, S. S., Soto, M. A., Bonini de Romanelli, I., Gutiérrez Merino, C., Sotomayor, P., and Barrantes, F. J. (1996) Biophys. J. 70, 1275-1284] and cells expressing endogenous or heterologous AChR [Zanello, L. P., Aztiria, E., Antollini, S., and Barrantes, F. J. (1996) Biophys. J. 70, 2155-2164]. In the present work, Laurdan GP was measured in T. marmorata native AChR membrane by direct excitation or under energy transfer conditions in the presence of exogenous lipids. GP was found to diminish in these two regions upon addition of oleic acid and dioleoylphosphatidylcholine and not to vary significantly upon addition of cholesterol hemisuccinate, indicating an increase in the polarity of the single, ordered-liquid lipid phase in the two former cases. Complementary information about the bulk lipid order was obtained from measurements of fluorescence anisotropy of DPH and two of its derivatives. The membrane order diminished in the presence of oleic acid and dioleoylphosphatidylcholine. The location of Laurdan was determined using the parallax method. Laurdan lies at approximately 10 A from the center of the bilayer, i.e., at depth of approximately 5 A from the lipid-water interface. Exogenous lipids modified the energy transfer efficiency from the intrinsic fluorescence to Laurdan. This strategy is introduced as a new analytic tool that discloses for the first time the occurrence of discrete and independent sites for phospholipids and sterols, respectively, both accessible to fatty acids, and presumably located at a shallow depth close to the phospholipid polar head region in the native AChR membrane.