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M F Lecompte

Publications and source records attributed to M F Lecompte.

10 recordsLinked to original sources
PubMed2005

Interaction of an amphitropic protein (factor Xa) with membrane models in a complex system.

Phosphatidylserine (PS) plays a crucial role, in the conversion of prothrombin into thrombin by the protease, factor Xa. Physiologically, the conversion occurs in the prothrombinase complex. The question of how water-soluble proteins that normally circulate in plasma bind remains to be unambiguously determined. We previously found that the amphitropic proteins (prothrombin, factors V and Va) penetrate into phospholipid layers. AC polarography has allowed the detection for the first time of insertion of factor Xa into condensed monolayers containing phosphatidylserine (PS) and phosphatidylcholine (PC) either 100% PS or 25% PS in the presence of Ca2+. This observation demonstrates that part of factor Xa can cross the phospholipid polar headgroup/hydrocarbon chain interface. In parallel experiments, radioactive surface measurements permitted measuring binding of tritium-labeled factor Xa onto a PS monolayer and calculate an association constant, 6x10(6) M(-1). Penetration of factor Xa into PS-containing vesicles was investigated also using photoactivable 5-[125I]iodonaphthalene-1-azide, which binds selectively to the lipid embedded domains of the protein. These experiments suggest that Factor Xa penetrates preferentially by its heavy chain, an alternative mode of binding to the commonly accepted binding via its Gla domain. Interaction of factor Xa with PS vesicles also changes its apparent K(m) for S 2222.

Animals↗
PubMed1998

Binding steps of apolipoprotein A-I with phospholipid monolayers: adsorption and penetration.

In whole HDL particles, the arrangement of apoA-I relative to phospholipids is of crucial interest with respect to the physiological formations of HDL. We report here new data concerning the nature of the interaction of apoA-I with condensed phospholipid (PL) monolayers (phosphatidylcholine and phosphatidylserine). The use of alternative current polarography allowed the detection for the first time of different binding steps which are dependent on apoA-I concentration. At low concentration (below 10 micrograms/mL), apoA-I adsorbs on PL polar headgroups, through electrostatic interactions. Above this threshold concentration, apoA-I penetrates within the monolayer (i.e., part of apoA-I crosses the PL polar headgroup/hydrocarbon chain interface). The process of penetration described here brings experimental evidence supporting Segrest's "snorkel" model. Penetrated helices are lying at the interface, their apolar face in contact with PL hydrocarbon chains and their polar face in contact with PL polar headgroups. In the absence of cholesterol, a second level of penetration was detected at higher apoA-I concentrations. It was facilitated in the presence of phosphatidylserine in comparison to phosphatidylcholine and disappeared in the presence of cholesterol. It is proposed that the C-terminal domain is involved in the first binding steps and that hinged domains may also be implicated. Furthermore, we propose that the apoA-I binding states stabilize the protein/phospholipid layer complex. These different binding states are discussed with respect to their roles in HDL metabolism.

Adsorption↗
PubMed1994

Electrostatic and hydrophobic interactions are involved in factor Va binding to membranes containing acidic phospholipids.

The interaction of factor Va with phospholipid monolayers was studied using alternating current polarography. For these studies a hanging mercury drop electrode is positioned in contact with the monolayer at the air-monolayer interface. Factor Va introduced into the aqueous subphase beneath the monolayer caused alterations in the capacitance of the circuit. When factor Va was introduced beneath monolayers containing 13-25% mole fraction phosphatidylserine (PS), 87-75% mole fraction phosphatidylcholine (PC), a significant capacitance increase occurred, which is interpreted to be the result of the penetration of the factor Va molecules through the monolayer. No penetration or associated capacitance changes were observed with monolayers composed of pure PC or pure PS. Polarography experiments were also conducted with the electrode placed in the aqueous subphase to measure the binding of factor Va to pure PS monolayers. This approach has allowed detection of factor Va (at nM concentrations) adsorption to a pure PS monolayer. Thus the membrane adsorption process can be distinguished from factor Va penetration through the monolayer. These results suggest that there are two types of interaction of the factor Va molecule with PS-containing monolayers. The interaction with the PS component of the monolayer is essential for binding the factor Va (at nM concentrations) that occurs by surface adsorption. For mixed PC/PS monolayers, PS adsorption is followed by penetration of the protein through the membrane.

Animals↗
PubMed1989

Interaction of human gamma-carboxyglutamic acid domainless prothrombin with phospholipids.

The conversion of prothrombin into thrombin occurs at the surface of stimulated platelets. In order to see the influence of gamma-carboxyglutamic acid in the interaction of prothrombin with phospholipid, we investigated the direct interaction of the peptide 1-41 and prothrombin (des 1-44) with phospholipid monolayers of various compositions. Adsorption of the labeled proteins was determined by surface radioactivity measurements. Penetration of the proteins into the lipid layers was inferred from capacitance variation of the monolayer, measured by a.c. polarography. Prothrombin (des 1-44) was found both to adsorb and to penetrate the lipid monolayers, in the presence and in the absence of Ca++. The effects are higher on 100% PS than on 25% PS. This protein was also found to increase the permeability of vesicles containing 25% PS to T1+ ions, in the presence and in the absence of Ca++. Comparison with prothrombin shows that Gla residues are clearly involved in the interaction at 25% PS; nevertheless, the peptide 1-41 does not penetrate. A model of interaction of prothrombin with phospholipid, including both adsorption of prothrombin by Gla residues and its penetration by another domain, is proposed.

1-Carboxyglutamic Acid↗
PubMed1987

Membrane penetration of bovine factor V and Va detected by labeling with 5-iodonaphthalene-1-azide.

The membrane-binding properties of Factor V and Factor Va were investigated using the lipophyllic, photoactivable probe 5-[125I]iodonaphthalene-1-azide. In the presence of vesicles composed of 75% phosphatidylcholine and 25% phosphatidylserine, both Factor V and Va were found to be labeled by the probe. The label was almost exclusively localized to the carboxyl-terminal-derived component E of Factor Va. The results are consistent with the interpretation that component E is the membrane binding subunit of Factor Va and that the interaction between Factor V or Factor Va and the membrane involves the penetration of the protein into the lipid bilayer.

Affinity Labels↗
PubMed1984

Membrane insertion of prothrombin.

The nature of the interaction of prothrombin with liposomes was examined by labelling with the apolar reagent 5-[125I] iodonaphthyl-1-azide (INA). When liposomes containing phosphatidylcholine were incubated with prothrombin and INA, the amount of incorporated label was greater in the absence of added calcium. Increasing the phosphatidylserine content of the liposomes, irrespective of CA++ resulted in a significant increase in the labelling of prothrombin by INA. Since INA is confined to the lipid core of the bilayer, these results strongly suggest that prothrombin binds to liposomes, and penetrates into the lipid bilayer.

Azides↗
PubMed1984

Interaction of alpha-thrombin and prethrombin 2, with phosphatidylserine-containing monolayers.

Prothrombin activation complex is located at a phospholipid surface on activated platelets. To see whether the thrombin domain of the molecule plays a role in the interaction with lipids, we investigated the direct interaction of human alpha-thrombin and its precursor prethrombin 2 with phospholipid monolayers of various compositions (PS/PC). Adsorption of the labeled proteins was determined by surface radioactivity measurements. Penetration of the proteins in the lipid layer was inferred from capacitance variation of the monolayer, measured by a.c. polarography. Disulfide bridges reduced at the electrode were determined by cyclic voltametry. In all the cases studied, although in different manners thrombin was found both to adsorb and penetrate the lipid layer, whereas prethrombin 2 did not penetrate pure phosphatidylcholine (PC). In the case of thrombin, but not of prethrombin 2, penetration is accompanied by S-S reduction which is maximum at 10 per cent of phosphatidylserine (PS). This indicates a different orientation for prethrombin 2 and thrombin in the lipid layer. This observation might be of importance for the comprehension of the architecture of the prothrombin activation complex and for the regulation of thrombin formation within the complex.

Enzyme Activation↗
PubMed1980

Interaction of prothrombin and its fragments with monolayers containing phosphatidylserine. 1. Binding of prothrombin and its fragment I to phosphatidylserine-containing monolayers.

The adsorption isotherms of prothrombin and its fragment I on phosphatidylserine monolayers and on mixed monolayers of phosphatidylcholine and phosphatidylserine were determined by measuring surface radioactivity emanating from the tritium-labeled absorbed proteins at 0.1 N NaCl and between 0 and 10 mM Ca2+. The proteins were absorbed from very dilute solutions, about 10 times more than in previous investigations on bilayer vesicles. The binding constants as obtained from the Scatchard plots were between 3 X 10(6) and 3 X 10(8) mol/L, depending on the experimental conditions. These values are between 2 and 50 times larger, respectively, than the binding constants obtained on bilayer vesicles. Prothrombin absorbs appreciably also in the absence of Ca2+. The significance of these results is discussed.

Adsorption↗