No abstract

No abstract

The interaction between alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein (AP-GPI), and phospholipids was monitored using Langmuir isotherms and PM-IRRAS spectroscopy. AP-GPI was injected under C16 phospholipid monolayers with either a neutral polar head (1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC)) or an anionic polar head (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS)). The increase in molecular area due to the injection of protein depended on the surface pressure and the type of phospholipid. At all surface pressures, it was highest in the case of DPPS monolayers. The surface elasticity coefficient E, determined from the pi-A diagrams, allowed to deduct that the AP-GPI-phospholipid mixtures presented a molecular arrangement less condensed than the corresponding pure phospholipid films. PM-IRRAS spectra suggested different protein-lipid interactions as a function of the nature of the lipids. AP-GPI modified the organization of the DPPS deuterated chains whereas AP-GPI affected only the polar group of DPPC at low surface pressure (8 mN/m). Different protein hydration layers between the DPPC and DPPS monolayers were suggested to explain these results. PM-IRRAS spectra of AP-GPI in the presence of lipids showed a shape similar to those collected for pure AP-GPI, indicating a similar orientation of AP-GPI in the presence or absence of phospholipids, where the active sites of the enzyme are turned outside of the membrane.

The monolayer organization of intestinal alkaline phosphatase (AP), a glycosylphosphatidyl inositol (GPI) anchored dimeric protein (AP-GPI), was monitored using polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and Brewster angle microscopy (BAM). The behavior of the monolayer was reversible during the compression−expansion cycles as shown by BAM. At high surface pressure, we observed formation of condensed protein domains. Upon decompression, the protein clusters remained stable over a period of several tenths of a minute, and then the condensed domain disappeared. These results suggest strong interactions between proteins forming clusters, which could be involved in the formation of membrane microdomains. The protein did not unfold as evidenced by PM-IRRAS, whatever the initial spreading surface pressure used. The PM-IRRAS spectra obtained during compression of a native AP-GPI monolayer induced slight reversible modifications on PM-IRRAS spectra. From the amide I/amide II ratio and the α-helix band position, it can be deduced that the protein was anchored at the interface with the GPI standing up and the great axis of the ellipsoid model of AP-GPI remained parallel to the interface. The PM-IRRAS spectra obtained during compression and their simulated spectra were consistent with the existence of small movements of intramolecular protein domains. Such movements may be related to the allosteric properties of mammalian alkaline phosphatases.

Chlorophyll a, chlorophyll b, and bacteriochlorophyll a in aqueous micellar solutions of Triton X 100 (2%) are readily oxidized by pulse-radiolytically generated N3*, Brz-., and (f4CN)z-m radicals at nearly diffusion-controlled rates. The kinetic study suggests that pigment molecules occupy multiple sites in the micelle. Pheophytin a is only oxidized by NB* and Brz--radicals. The absolute spectra and the molar extinction coefficients of chlorophyll a, bacteriochlorophyll a, chlorophyll b, and pheophytin a cations have been determined. The chlorophyll a cation has been observed in the presence of pigment aggregates.