RESUMEN

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.

Asunto(s)

Glicoproteínas Hemaglutininas del Virus de la Influenza/farmacología , Fusión de Membrana/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Polietilenglicoles/farmacología , Proteínas Virales de Fusión/farmacología , Liposomas , Péptidos , Fosfatidilcolinas

RESUMEN

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the posttranslational modification of select glutamate residues of its vitamin K-dependent substrates to gamma-carboxyglutamate. In this report, we describe a new fluorescence assay that is sensitive and specific for the propeptide binding site of active carboxylase. We employed the assay to make three important observations: (1) A tight binding fluorescein-labeled consensus propeptide can be used to quantify the active fraction of the enzyme. (2) The off-rate for a fluorescein-labeled factor IX propeptide was 3000-fold slower than the rate of carboxylation, a difference that may explain how carboxylase can carry out multiple carboxylations of a substrate during the same binding event. (3) We show evidence that substrate binding to the active site modifies the propeptide binding site of carboxylase. The significant (9-fold) differences in off-rates for the propeptide in the presence and absence of its co-substrates may represent a release mechanism for macromolecular substrates from the enzyme. Additionally, sedimentation velocity and equilibrium experiments indicate a monomeric association of enzyme with propeptide. Furthermore, the carboxylase preparation is monodisperse in the buffer used for our studies.

Asunto(s)

Ligasas de Carbono-Carbono/metabolismo , Péptidos/metabolismo , Unión Competitiva , Fluoresceína , Espectrometría de Fluorescencia , Especificidad por Sustrato

RESUMEN

Factor V(a) (FV(a)) is a cofactor for the serine protease factor X(a) that activates prothrombin to thrombin in the presence of Ca(2+) and a membrane surface. FV(a) is a heterodimer composed of one heavy chain (A1 and A2 domains) and one light chain (A3, C1, and C2 domains). We use fluorescence, circular dichroism, and equilibrium dialysis to demonstrate that (1) the FV C2 domain expressed in Sf9 cells binds one molecule of C6PS with a k(d) of approximately 2 microM, (2) stabilizing changes occur in the FV C2 domain upon C6PS binding, (3) the C6PS binding site in the FV C2 domain is located near residue Cys(2113), which reacts with DTNB, and (4) binding to a PS-containing membrane is an order of magnitude tighter than that to soluble C6PS. Coupled with a recently published crystal structure of the C2 domain, these results support a model for the mechanism of C2-membrane interaction.

Asunto(s)

Factor Va/metabolismo , Fragmentos de Péptidos/metabolismo , Fosfatidilserinas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Rastreo Diferencial de Calorimetría , Factor Va/genética , Vectores Genéticos/síntesis química , Calor , Humanos , Micelas , Datos de Secuencia Molecular , Concentración Osmolar , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Fosfatidilcolinas/metabolismo , Fosfatidilgliceroles/metabolismo , Unión Proteica/genética , Desnaturalización Proteica , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Solubilidad , Spodoptera/genética , Transfección

RESUMEN

A number of fluorescent probes have been used to follow membrane fusion events, particularly intermixing of lipids. None of them is ideal. The most popular pair of probes is NBD-PE and Rh-PE, in which the fluorescent groups are attached to the lipid headgroups, making them sensitive to changes in the surrounding medium. Here we present a new assay for monitoring lipid transfer during membrane fusion using the acyl chain tagged fluorescent probes BODIPY500-PC and BODIPY530-PE. Like the NBD-PE/Rh-PE assay, this assay is based on fluorescence resonance energy transfer (FRET) between the donor, BODIPY500, and the acceptor, BODIPY530. The magnitude of FRET is sensitive to the probe surface concentration, allowing one to detect movement of probes from labeled to unlabeled vesicles during fusion. The high quantum yield of fluorescence, high efficiency of FRET (R(o) is estimated to be approximately 60 A), photostability, and localization in the central hydrophobic region of a bilayer all make this pair of probes quite promising for detecting fusion. We have compared this and two other lipid mixing assays for their abilities to detect the initial events of poly(ethylene glycol) (PEG)-mediated fusion of small unilamellar vesicles (SUVs). We found that the BODIPY500/530 assay showed lipid transfer rates consistent with those obtained using the DPHpPC self-quenching assay, while lipid mixing rates measured with the NBD-PE/Rh-PE RET assay were significantly slower. We speculate that the bulky labeled headgroups of NBD-PE and especially Rh-PE molecules hamper movement of probes through the stalk between fusing vesicles, and thus reduce the apparent rate of lipid mixing.

Asunto(s)

Compuestos de Boro/química , Colorantes Fluorescentes/química , Fusión de Membrana , Lípidos de la Membrana/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Calibración , Transferencia de Energía , Glicerofosfolípidos/química , Liposomas/química , Polietilenglicoles/química , Rodaminas/química , Espectrometría de Fluorescencia/métodos

RESUMEN

Poly(ethylene glycol) (PEG)-mediated fusion of phosphatidylcholine model membranes has been shown to mimic the protein-mediated biomembrane process [Lee, J., and Lentz, B. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9274-9279]. Unlike the simple model membranes used in this earlier study, the lipid composition of fusogenic biomembranes is quite complex. The purpose of this paper was to examine PEG-mediated fusion of highly curved (SUV) and largely uncurved (LUV) membrane vesicles composed of different lipids in order to identify lipid compositions that produce highly fusogenic membranes. Starting with liposomes composed of five lipids with different physical properties, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), bovine brain sphingomyelin (SM), and cholesterol (CH), we systematically varied the composition and tested for the extent of PEG-mediated fusion after 5 min of treatment. We found that a vesicle system composed of four lipids, DOPC/DOPE/SM/CH, fused optimally at a 35/30/15/20 molar ratio. Each lipid seemed to play a part in optimizing the membrane for fusion. PE disrupted outer leaflet packing as demonstrated with TMA-DPH lifetime, C(6)-NBD-PC partitioning, and DPH anisotropy measurements, and thus significantly enhanced fusion and rupture, without significantly altering interbilayer approach (X-ray diffraction). An optimal ratio of PC/PE (35/30) produced a balance between fusion and rupture. CH and SM, when present at an optimal ratio of 3/4 in vesicles containing the optimal PC/PE ratio, reduced rupture without significantly reducing fusion. This optimal CH/SM ratio also enhanced outer leaflet packing, suggesting that fusion is dependent not only on outer leaflet packing but also on the properties of the inner leaflet. Addition of CH without SM enhanced rupture relative to fusion, while SM alone reduced both rupture and fusion. The optimal lipid composition is very close to the natural synaptic vesicle composition, suggesting that the synaptic vesicle composition is optimized with respect to fusogenicity.

Asunto(s)

Membrana Dobles de Lípidos/química , Liposomas/química , Fusión de Membrana/efectos de los fármacos , Lípidos de la Membrana/química , Polietilenglicoles/farmacología , Colesterol/química , Difenilhexatrieno/análogos & derivados , Difenilhexatrieno/química , Polarización de Fluorescencia , Colorantes Fluorescentes/química , Semivida , Lípidos de la Membrana/análisis , Filtros Microporos , Modelos Químicos , Presión Osmótica , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Sonicación , Espectrometría de Fluorescencia , Esfingomielinas/química , Propiedades de Superficie , Ultracentrifugación , Difracción de Rayos X

RESUMEN

Protein machines and lipid bilayers both play central roles in cell membrane fusion, a process crucial to life. Recent results provide clues to how both components function in fusion. Recent observations suggest a common mechanism by which very different fusion machines (from lipid-enveloped viruses and synaptic vesicles) may function to produce compartment-joining pores. This mechanism presumes that fusion proteins act as machines that use stored conformational energy to assemble closely juxtaposed lipid bilayers, bend these to form fusion-competent structures, stabilize unfavorable lipid structures and destabilize a committed intermediate to drive fusion pore formation.

Asunto(s)

Membrana Dobles de Lípidos , Fusión de Membrana , Proteínas de la Membrana/fisiología

RESUMEN

Thrombin-activated factor Va exists as two isoforms, factor Va(1) and factor Va(2), which differ in the size of their light chains and their affinity for biological membranes. The heterogeneity of the light chain remained following incubation of factor Va with N-glycanase. However, we found that the factor V C2 domain, which contains a single potential glycosylation site at Asn-2181, was partially glycosylated when expressed in COS cells. To confirm the structural basis for factor Va(1) and factor Va(2), we mutated Asn-2181 to glutamine (N2181Q) and expressed this mutant using a B domain deletion construct (rHFV des B) in COS cells. Thrombin activation of N2181Q released a light chain with mobility identical to that of factor Va(2) on SDS-PAGE. The functional properties of purified N2181Q were similar to those of factor Va(2) in prothrombinase assays carried out in the presence of limiting concentrations of phosphatidylserine. The binding of human factor Va(1) and factor Va(2) to 75:25 POPC/POPS vesicles was also investigated in equilibrium binding assays using proteins containing a fluorescein-labeled heavy chain. The affinity of human factor Va(2) binding to POPC/POPS vesicles was approximately 3-fold higher than that of factor Va(1). These results indicate that partial glycosylation of factor V at asparagine-2181 is the structural basis of the light chain doublet and that the presence of this oligosaccharide reduces the affinity of factor Va for biological membranes.

Asunto(s)

Asparagina/metabolismo , Factor V/metabolismo , Fragmentos de Péptidos/metabolismo , Tromboplastina/metabolismo , Animales , Asparagina/genética , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Factor V/biosíntesis , Factor V/genética , Factor V/aislamiento & purificación , Factor Va/química , Factor Va/genética , Factor Va/aislamiento & purificación , Factor Va/metabolismo , Glutamina/genética , Glicósido Hidrolasas/metabolismo , Glicosilación , Humanos , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Unión Proteica/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transfección

RESUMEN

Membrane fusion is fundamental to the life of eukaryotic cells. Cellular trafficking and compartmentalization, import of food stuffs and export of waste, inter-cellular communication, sexual reproduction, and cell division are all dependent on this basic process. Yet, little is known about the molecular mechanism(s) by which fusion occurs. It is known that fusing membranes must somehow be docked and brought into close contact. Specific proteins, many of which have been identified within the past decade, accomplish this. An electrical connection or 'fusion pore' is established between compartments surrounded by the fusing membranes. Three primary views of the mechanism of pore formation during secretory and viral fusion have been proposed within the past decade. In one view, a protein ring forms an initial transient connection that expands slowly by recruiting lipid so as to form a lipidic junction. In another view, the initial fusion pore consists of a protein-lipid complex that transforms slowly until the fusion proteins dissociate from the complex to form an irreversible lipidic pore. In a third view, the initial pore is a transient lipid pore that fluctuates between open and closed states before either expanding irreversibly or closing. Recent work has helped define the mechanism by which poly(ethylene glycol) (PEG) mediates fusion of highly curved model membranes composed only of synthetic phospholipids. PEG is a highly hydrated polymer that can bring vesicle membranes to near molecular contact by making water between them thermodynamically unfavourable. Disrupted packing in the contacting monolayers of these vesicle membranes is necessary to induce fusion. The time course and sequence of molecular events of the ensuing fusion process have also been defined. This sequence of events involves the formation of an initial, transient intermediate in which outer leaflet lipids have mixed and small transient pores join fusing compartments ('stalk'). The transient intermediate transforms in 1-3 min to a fusion-committed, second intermediate ('septum') that then 'pops' to form the fusion pore. Inner leaflet mixing, which is shown to be distinct from outer leaflet mixing, accompanies contents mixing that marks formation of the fusion pore. Both the sequence of events and the activation energies of these events correspond well to those observed in viral membrane fusion and secretory granule fusion. These results strongly support the contention that both viral and secretory fusion events occur by lipid molecule rearrangements that can be studied and defined through the use of PEG-mediated vesicle fusion as a model system. A possible mechanism by which fusion proteins might mediate this lipidic process is described.

Asunto(s)

Membrana Dobles de Lípidos/química , Fusión de Membrana , Polietilenglicoles/química , Fenómenos Fisiológicos de los Virus , Animales , Membrana Celular/química , Membrana Celular/fisiología , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/fisiología , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/fisiología , Liposomas/química , Liposomas/fisiología

RESUMEN

The plasma protein beta2-glycoprotein I (beta2-GPI) is a major target of autoantibodies in patients with the antiphospholipid syndrome. To understand the physiological function of beta2-GPI and its potential role in the pathophysiology of the antiphospholipid syndrome, the binding of beta2-GPI to phospholipid membranes was characterized. The interaction of beta2-GPI with unilamellar vesicles containing varying amounts of acidic phospholipids with phosphatidylcholine (PC) was measured at equilibrium via relative light scattering. Analysis of binding isotherms gave apparent Kd values ranging from approximately 5.0 to 0.5 microM over a range of 5-20 mol % anionic phospholipid. Inhibition of binding by increasing ionic strength and Ca2+ ions suggests that binding is primarily electrostatic. These data indicate that beta2-GPI binding to membranes with physiological anionic phospholipid content is relatively weak in comparison to plasma coagulation proteins, suggesting that beta2-GPI does not function as a physiological anticoagulant based on its phospholipid-binding properties.

Asunto(s)

Glicoproteínas/química , Fosfolípidos/química , Humanos , Membranas Artificiales , Unión Proteica , beta 2 Glicoproteína I

RESUMEN

Activation energies for the individual steps of secretory and viral fusion are reported to be large [Oberhauser, A. F., Monck, J. R. & Fernandez, J. M. (1992) Biophys. J. 61, 800-809; Clague, M. J., Schoch, C., Zech, L. & Blumenthal, R. (1990) Biochemistry 29, 1303-1308]. Understanding the cause for these large activation energies is crucial to defining the mechanisms of these two types of biological membrane fusion. We showed recently that the fusion of protein-free model lipid bilayers mimics the sequence of steps observed during secretory and viral fusion, suggesting that these processes may involve common lipid, rather than protein, rearrangements. To test for this possibility, we determined the activation energies for the three steps that we were able to distinguish as contributing to the fusion of protein-free model lipid bilayers. Activation energies for lipid rearrangements associated with formation of the reversible first intermediate, with conversion of this to a semi-stable second intermediate, and with irreversible fusion pore formation were 37 kcal/mol, 27 kcal/mol, and 22 kcal/mol, respectively. The first and last of these were comparable to the activation energies observed for membrane lipid exchange (42 kcal/mol) during viral fusion and for the rate of fusion pore opening during secretory granule release (23 kcal/mol). This striking similarity suggests strongly that the basic molecular processes involved in secretory and viral fusion involve a set of lipid molecule rearrangements that also are involved in model membrane fusion.

Asunto(s)

Membrana Dobles de Lípidos , Fusión de Membrana , Cinética , Modelos Químicos

RESUMEN

Factor Va is an essential protein cofactor of the enzyme factor Xa, which activates prothrombin to thrombin during blood coagulation. Peptides with an apparent Mr of approximately 94,000 (heavy chain; HC) and approximately 74,000 or 72,000 (light chain; LC) interact in the presence of Ca2+ to form active Va. The two forms of Va-LC differ in their carboxyl-terminal C2 domain. Using Va reconstituted with either LC form, we examined the effects of the two LC species on membrane binding and on the activity of membrane-bound Va. We found that 1) Va composed of the 72,000 LC bound only slightly more tightly to membranes composed of a mixture of neutral and acidic lipids, the Kd being reduced by a factor of approximately 3 at 5 mM and by a factor of 6 at 2 mM Ca2+. 2) The two forms of Va seemed to undergo different conformational changes when bound to a membrane. 3) The activity of bovine Va varied somewhat with LC species, the difference being greatest at limiting Xa concentration. We have also addressed the role of the two Va peptides in membrane lipid rearrangements and binding: 1) Va binding increased lateral packing density in mixed neutral/acidic lipid membranes. In the solid phase, Va-HC had no effect, whereas Va-LC and whole Va had similar but small effects. In the fluid phase, Va-HC and whole Va both altered membrane packing, with Va-HC having the largest effect. 2) Va-HC bound reversibly and in a Ca2+-independent fashion to membranes composed of neutral phospholipid (Kd, approximately 0.3 microM; stoichiometry approximately 91). High ionic strength had little effect on binding. 3) The substantial effect of Va on packing within neutral phospholipid membranes was mimicked by Va-HC. 4) Based on measurements of membrane phase behavior, binding of Va or its peptide components did not induce thermodynamically discernible lateral membrane domains. These results suggest that the membrane association of factor Va is a complex process involving both chains of Va, changes in lipid packing, and changes in protein structure.

Asunto(s)

Membrana Celular/metabolismo , Factor Va/química , Factor Va/metabolismo , Membrana Dobles de Lípidos/metabolismo , Metabolismo de los Lípidos , Animales , Arginina/análogos & derivados , Arginina/farmacología , Bovinos , Membrana Celular/química , Compuestos de Dansilo/farmacología , Electroforesis en Gel de Poliacrilamida , Factor Xa/metabolismo , Polarización de Fluorescencia , Colorantes Fluorescentes , Cinética , Membrana Dobles de Lípidos/química , Lípidos/química , Liposomas/química , Liposomas/metabolismo , Modelos Biológicos , Fosfolípidos/química , Fosfolípidos/metabolismo , Fosfolípidos/farmacología , Unión Proteica , Conformación Proteica , Protrombina/metabolismo , Temperatura

RESUMEN

The poly(ethylene glycol) (PEG)-induced fusion of sonicated, unilamellar vesicles (SUV) and large, unilamellar vesicles (LUV) composed of a variety of phosphatidylcholine species was compared using two assays for the mixing and leakage of internal vesicle contents. In the first [Lentz et al. (1992) Biochemistry, 31, 2643], disodium 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) fluorescence is quenched by co-encapsulated N,N'-p-xylylenebis(pyridinium bromide) (DPX). For this assay, interference by the fluorescence of impurities in PEG demands that the PEG content of the sample be reduced by dilution before measurements are taken. The second assay [Viguera et al. (1993) Biochemistry, 32, 3708] monitors the fluorescence of Tb3+ complexed with dipicolinic acid (DPA) directly in concentrated PEG solutions. The two assays gave identical fusion profiles for egg PC SUVs treated with increasing concentrations of PEG, demonstrating that fusion occurs in the dehydrated state in the presence of PEG and does not require dilution. Comparison of results obtained with lipid species of varying degrees of unsaturation incorporated into either SUV or LUV suggested that acyl chain unsaturation and high membrane curvature combine to favor fusion of pure phosphatidylcholine membranes. There was a clear correlation between the fluorescence lifetime or the order parameter of the membrane probe 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) in different membranes and the concentration of PEG needed to induce fusion of these membranes. However, the ratios of TMA-DPH lifetimes measured in D2O versus H2O buffers were the same for different lipid species, indicating that probe penetration was not very different for different lipid species. The results suggest that the combined effect of high membrane curvature and extensive chain unsaturation is an enhanced rate of lipid motion in the upper region of the bilayer, reflective of decreased packing density in the outer leaflet of unsaturated SUV bilayers, probably allowing for enhanced water penetration leading to an enhanced probability of fusion.

Asunto(s)

Ácidos Grasos Insaturados/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fusión de Membrana/efectos de los fármacos , Polietilenglicoles/farmacología , Difenilhexatrieno/análogos & derivados , Ácidos Grasos Insaturados/química , Colorantes Fluorescentes , Membrana Dobles de Lípidos/química , Fosfatidilcolinas , Ácidos Picolínicos , Terbio

RESUMEN

The sequence of events involved in poly(ethylene glycol)-mediated fusion of small unilamellar vesicles (SUVs) has been studied. Fusion events were monitored using light scattering for vesicle aggregation, the fluorescence lifetime of membrane probe lipids (DPHpPC and NBD-PS) for membrane mixing, the aqueous fluorescent marker (Tb3+/DPA and H+/HPTS) for contents mixing; and quasi-elastic light scattering for the change in the size of vesicles. Poly(ethylene glycol) is a highly hydrated polymer that can bring vesicle membranes to near molecular contact but is unable to induce vesicle fusion without manipulations that reduce packing density and encourage molecular motions in the backbone regions of both contacting membrane leaflets. Once this condition is achieved, the sequence of events involved in vesicle fusion is shown here to be (1) outer leaflet mixing accompanied by (2) transient pore formation, both occurring on a time scale of approximately 10 s and leading to an initial, reversible intermediate; (3) a 1-3 min delay leading to formation of a fusion-committed second intermediate; (4) inner leaflet mixing on a time scale of ca. 150 s; and (5) contents mixing on a time scale of 150-300 s. Inner leaflet mixing, which has never before been shown to be distinct from outer leaflet mixing, begins simultaneously with, but is completed before, contents mixing. Fusion products, which seem to be large vesicles, are estimated to be formed from four to six SUVs. The fusion intermediates are shown to have merged outer leaflets and distinct inner leaflets prior to formation of fusion pores. Using quasi-elastic light scattering, the initial intermediate was shown to revert to SUVs upon removal of PEG, while the second intermediate irreversibly continued to a fusion pore in the presence or absence of PEG. The sequence of events for this pure lipid bilayer fusion process shows remarkable homology to what is known about the sequence of protein-mediated cell membrane fusion events, suggesting a commonality between these two processes.

Asunto(s)

Membrana Dobles de Lípidos/química , Fusión de Membrana , Lípidos de la Membrana/química , Biopolímeros/química , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Fusión de Membrana/efectos de los fármacos , Modelos Moleculares , Fosfolípidos/química , Polietilenglicoles/farmacología , Protones , Dispersión de Radiación

RESUMEN

Prothrombin activation to thrombin is a key control reaction in blood coagulation. During the process, prothrombin is sequentially cleaved at two peptide bonds (Arg323-Ile and Arg274-Thr) by factor X(a) to generate meizothrombin and then thrombin. Phosphatidylserine (PS)-containing membranes from platelets are believed to facilitate this two-step process. Using fluorescence energy transfer (FRET), we determined the distances of closest approach between a specifically located C-terminal fluorescein of a double mutant bovine prothrombin (P(S528A, G581C)-FM) or meizothrombin (M(S528A, G581C)-FM) and phosphatidylethanolamine-N-rhodamine B (PE-Rh; 0-8.7 mol %) contained in membranes composed of PS (25 mol %) and phosphatidylcholine (66.3-75 mol %). Plots of the energy transfer efficiency as a function of membrane concentration, at six PE-Rh surface densities, were analyzed globally to obtain dissociation constants and binding stoichiometries as global parameters and saturating energy transfer efficiencies characteristic of each surface density. From the global analysis, the dissociation constants were estimated to be 0.32 +/- 0.10 and 0.28 +/- 0.12 microM with stoichiometries of 42 +/- 12 and 44 +/- 9 lipid/protein for prothrombin and meizothrombin, respectively. The distance of closest approach was obtained from the dependence of the saturating energy transfer efficiency on the acceptor (PE-Rh) surface density. With the assumptions of kappa2 = 2/3 and n = 1.4, the distances were 94 +/- 3 A for prothrombin and 114 +/- 2 A for meizothrombin. Since both prothrombin and meizothrombin behave in solution as oblate ellipsoids of revolution with a long axis of 120 A, our FRET measurements suggest that binding to PS-containing membranes induced tighter folding of the prothrombin molecule but not of the meizothrombin intermediate. This observation is consistent with our hypothesis that membrane binding plays an essential role in the sequential alignment of the bond Arg323-Ile in prothrombin and Arg274-Thr in meizothrombin with the active site of the membrane-bound prothrombinase in the two-step thrombin-generating process.

Asunto(s)

Transferencia de Energía , Precursores Enzimáticos/química , Lípidos de la Membrana/química , Protrombina/química , Trombina/química , Secuencia de Aminoácidos , Animales , Bovinos , Precursores Enzimáticos/metabolismo , Fluoresceína , Fluoresceínas , Protrombina/metabolismo , Rodaminas , Espectrometría de Fluorescencia/métodos , Trombina/metabolismo

RESUMEN

Partially purified snake venom of Echis carinatus (ECV) is a well-known enzyme used to generate meizothrombin from prothrombin. In this reaction, meizothrombin is slowly converted to two smaller fragments. It is not certain if this slow reaction is catalyzed by meizothrombin or by ECV. Using previously constructed active site mutant bovine prothrombin, we eliminated meizothrombin autolysis and examined ECV reactions. ECV did slowly generate two fragments during activation of the active site mutant prothrombin. This result indicates that ECV cleaved bovine prothrombin at two sites: at Arg323-Ile to expose the active site, at another bond at or near the known meizothrombin autolysis site. Because the former cleavage is faster than the latter one, ECV is still a reasonable tool to generate meizothrombin under proper conditions.

Asunto(s)

Protrombina/metabolismo , Venenos de Víboras/farmacología , Animales , Arginina , Sitios de Unión , Bovinos , Compuestos Cromogénicos/metabolismo , Dipéptidos/metabolismo , Precursores Enzimáticos/metabolismo , Esterasas/metabolismo , Mutagénesis Sitio-Dirigida , Protrombina/química , Protrombina/genética , Trombina/metabolismo

RESUMEN

Small, unilamellar vesicles (SUV) or large, unilamellar vesicles (LUV) containing a small amount of N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) or the corresponding phosphatidylserine (NBD-PS) were made asymmetric in labeled lipid by reduction of outer leaflet probe with externally added sodium dithionite. Following removal of dithionite, transbilayer lipid redistribution (presumably due to lipid flip-flop) was indicated by a loss of fluorescence intensity upon readdition of dithionite. Vesicle rupture and fusion in the presence of PEG were measured by changes in the fluorescence of trapped Tb3+ complexed with dipicolinic acid (DPA) or by the increase of fluorescence from 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) coencapsulated with a quenching agent. NBD-PE redistributed slowly (approximately 2%/h) in all symmetrically labeled vesicles examined, while NBD-PS did not. NBD-PE redistribution was not accelerated by treatment of vesicles with PEG below concentrations that induced vesicle rupture or fusion, but was enhanced at or above these PEG concentrations. SUV prepared from hen egg yolk phosphatidylcholine (egg PC) or from dioleoylphosphatidylcholine (DOPC)/dilinolenoylphosphatidylcholine (diC(18:3)PC) (85/15) mixtures were shown to fuse without rupturing in the presence of appropriate concentrations of PEG. Matching the osmolalities inside and outside the vesicle mitigated against rupture but did not prevent fusion. Under these conditions, NBD-PE flip-flop was proportional to the amount of fusion, but with different proportionality constants for the two lipid systems, while NBD-PS flip-flop did not occur. Redistribution of total mass from the outer to the inner leaflet during the fusion process could be detected in terms of a change in the ratio of dithionite-reducible probe to total probe. Both probes detected inwardly directed redistribution of lipid mass under conditions that induced fusion of SUV. We conclude that inward mass redistribution must accompany PEG-mediated SUV fusion, but that lipid flip-flop is not mechanistically related to the fusion process.

Asunto(s)

Membrana Dobles de Lípidos/metabolismo , Fusión de Membrana , Polietilenglicoles

RESUMEN

Poly(ethylene glycol)-induced fusion of two different vesicle systems has been examined: dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles (LUV) and cardiolipin (CL)/dioleoylphosphatidylcholine (DOPC) (1:10) LUVs. A slight perturbation was established in the outer leaflets of DPPC LUVs by hydrolyzing 0.8% of the outer leaflet lipid with phospholipase A2 to produce lysophosphatidylcholine and palmitate which were then removed by bovine serum albumin. Similarly, 5 mM Ca2+ was added to the external compartment of CL/DOPC LUVs to alter the shape of the CL molecule and thereby create a perturbation in the outer leaflet packing of these vesicles. Contents mixing assays showed that both vesicle systems fused only when the outer leaflets of both contacting vesicles were perturbed as described. Two fluorescent probes (C6-NBD-PC and TMA-DPH) were used to detect changes in outer leaflet molecular packing between nonfusing and fusing systems. The steady-state fluorescence intensity of C6-NBD-PC added externally to either fusing system was enhanced relative to that of nonfusing vesicles. Phase-resolved measurements of probe lifetime showed that this was due mainly to enhanced partitioning of probe from a micellar state into fusing versus nonfusing membranes. Similarly, TMA-DPH was found to undergo more rapid motion when incorporated into fusing as opposed to nonfusing vesicles. The effects of deuterium exchange on probe lifetime also indicated that C6-NBD-PC and TMA-DPH penetrated more deeply into fusing than into nonfusing membranes. These results suggest that the fusogenic perturbations produced in these two very different lipid systems took the form of altered outer leaflet packing. We conclude that, for the two model lipid bilayers examined, small perturbations in lipid packing within contacting bilayer leaflets are necessary and probably sufficient to promote membrane fusion.

Asunto(s)

1,2-Dipalmitoilfosfatidilcolina , Cardiolipinas , Membrana Dobles de Lípidos , Fusión de Membrana , Fosfatidilcolinas , Polietilenglicoles , Calcio , Deuterio , Hidrólisis , Cinética , Modelos Biológicos , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Albúmina Sérica Bovina , Espectrometría de Fluorescencia

RESUMEN

Several structural methods were used to probe the influence of three fusogenic and four nonfusogenic amphipaths on large, unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles. For four of these structural measurements there was a correlation observed between the ability of an amphipath to favor poly(ethylene glycol) (PEG)-induced fusion and the structural perturbation reported by each method. First, the fluorescence anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenyhexa-1,3,5-triene (TMA-DPH), which probes the upper region of the bilayer, decreased in the range of PEG concentrations previously found to cause fusion of membranes containing fusogenic amphipaths. For nonfusogenic amphipaths, the anisotropy increased monotonically with PEG concentration. The properties of similar probes that locate in the hydrophobic core of the bilayer showed no correlation with fusogenicity, nor did the properties of probes purported to sense the aqueous surface of the membrane. Second, the frequency of the C=O stretch increased and then decreased dramatically as fusogenic but not nonfusogenic membranes were heated through their phase transition. Third, there was a dramatic increase in the frequency of the C-O-C ester stretch at the membrane order/disorder phase transition for membranes containing fusogenic amphipaths, twice the increase observed for nonfusogenic amphipaths. The spectral characteristics of phosphate, choline, and acyl chain motions showed no such correlation with fusogenicity. Finally, calorimetric measurements showed that low levels of fusogenic amphipaths eliminated the "pretransition" (L beta-->P beta) in DPPC membranes, whereas other amphipaths shifted but did not eliminate this transition. Taken together, these results indicate that fusogenic amphipaths perturb the interface or "backbone" region of the bilayer rather than the hydrophobic core, the headgroup, or the water interface regions of DPPC bilayers.

Asunto(s)

Diglicéridos , Dimiristoilfosfatidilcolina/química , Membrana Dobles de Lípidos/química , Fusión de Membrana , Calorimetría , Difenilhexatrieno/análogos & derivados , Electroquímica , Polarización de Fluorescencia , Colorantes Fluorescentes , Cinética , Modelos Biológicos , Polietilenglicoles , Espectroscopía Infrarroja por Transformada de Fourier , Relación Estructura-Actividad

RESUMEN

Large, unilamellar vesicles (LUV) composed of dipalmitoylphosphatidylcholine (DPPC) were made asymmetric in L-alpha-lysopalmitoylphosphatidylcholine (LPC) either by adding a small amount (total mole fraction = 0.003) of LPC to their outer leaflets (LUV-LPCout) or by extracting a small amount from outer leaflets which already contained 0.0015 mol fraction LPC (LUV-LPCin). The slow rate of the transbilayer redistribution of LPC allowed the asymmetric vesicles to be characterized with regard both to their physical properties and to their ability to fuse in the presence of poly(ethylene glycol) (PEG). The fraction of LPC extractable with bovine serum albumin was taken as a measure of LPC transbilayer asymmetry. The ratio of LPC available to that unavailable to BSA extraction was 6 for LUV-LPCout and 0.3 for LUV-LPCin. These asymmetries could not be enhanced significantly by increasing the vesicle LPC content. Measurements of the mixing and leakage of vesicle contents showed that LUV-LPCin fused in the presence of 15% (w/w) PEG without loss of contents but that LUV-LPCout did not fuse in the presence of up to 35% PEG. Vesicles prepared from palmitoyloleoylphosphatidylcholine could also be made asymmetric in LPC, but did not fuse even in the presence of 30% PEG. Quasi-elastic light scattering revealed that LUV-LPCin aggregated at 25 degrees C except when swollen by an osmotic gradient while LUV-LPCout were much less likely to aggregate. Trapped volume determinations suggested that neither type of vesicle was perfectly spherical in shape, but no correlation was found between fusogenicity and vesicle shape. Measurements of the fluorescence properties of TMA-DPH and of C6-NBD-PC suggested that the interface region of the outer leaflet of LUV-LPCin was slightly less ordered and less well packed than that of LUV-LPCout. This slight perturbation of the external vesicle surface correlated with the ability of juxtaposed vesicle bilayers to fuse.

Asunto(s)

1,2-Dipalmitoilfosfatidilcolina/química , Membrana Dobles de Lípidos/química , Liposomas/química , Lisofosfatidilcolinas/química , Fusión de Membrana/fisiología , Polietilenglicoles/farmacología , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , Cromatografía en Gel , Difenilhexatrieno/análogos & derivados , Polarización de Fluorescencia , Colorantes Fluorescentes/metabolismo , Presión Osmótica , Fosfatidilcolinas/química , Albúmina Sérica Bovina , Propiedades de Superficie , Temperatura

RESUMEN

Acidic phospholipids play an important but incompletely understood role in prothrombin activation. Here we report the effect of short-chain phosphatidylserine (dicaproylphosphatidylserine, C6PS) and the corresponding phosphatidylglycerol (C6PG) and phosphatidylcholine (C6PC) derivatives on the rate of prothrombin activation by factor Xa. The critical micellar concentrations of these short-chained phospholipids have been determined under a variety of conditions that we used for kinetic and structural studies. Under conditions for which these lipids exist in a soluble form, the results demonstrate that: (i) the rate of human prothrombin activation by human factor Xa was enhanced in a calcium-dependent fashion up to 60-fold by addition of C6PS, roughly 20% of the optimal enhancement seen with bovine phosphatidylserine/palmitoyloleoylphosphatidylcholine (25/75 PS/POPC) membranes; (ii) C6PS inhibited the rate of hydrolysis of synthetic factor Xa substrate (S-2765), an effect that was mimicked, but at much lower lipid concentrations, by PS/POPC membranes; (iii) there was no enhancement of prothrombin activation and much less inhibition of hydrolysis of S-2765 by factor Xa in the presence of C6PG or C6PC; and (iv) the thermal denaturation of prothrombin was altered in a calcium-independent but dose-dependent fashion by either C6PS or C6PG. These results have been interpreted in terms of the existence of (a) specific PS binding site(s) on factor Xa (Kd approximately 73 microM) that regulate(s) the activity of this serine protease. Our results do not rule out the possibility that the rate of prothrombin activation is also influenced by a weaker, calcium-independent, and less specific acidic lipid binding site on prothrombin, the occupancy of which results in conformational changes in this protein. The results clearly suggest that PS binding regulates the rate of prothrombin activation.

Asunto(s)

Factor Xa/metabolismo , Fosfolípidos/farmacología , Protrombina/metabolismo , Animales , Sitios de Unión , Calcio/farmacología , Rastreo Diferencial de Calorimetría , Bovinos , Calor , Humanos , Cinética , Micelas , Desnaturalización de Ácido Nucleico , Protrombina/química , Protrombina/aislamiento & purificación , Soluciones , Relación Estructura-Actividad