Measurement of net proton-hydroxyl permeability of large unilamellar liposomes with the fluorescent pH probe, 9-aminoacridine

The fluorescent probe 9-aminoacridine was used to measure the rate of decay of experimentally established pH gradients across liposome membranes. From the rate of decay, separate permeability coefficients for protons (PH) and hydroxyls (POH) were calculated and summed to yield the net proton-hydroxyl permeability (Pnet). The net permeability of protons and hydroxyls was found to be approximately 10(-4) cm/s, six orders of magnitude greater than that measured for sodium and pyrophosphate ions under similar conditions. This suggests that protons and/or hydroxyls cross lipid bilayers by a different mechanism than do other monovalent cations and anions. In addition, the measurements provide a standard for net proton-hydroxyl permeability in pure phospholipid bilayers for comparison with biological membranes.     

Interaction of the voltage-sensing fluorescent probe diS-C3-(5) with dipalmitoylphosphatidylcholine liposomes

The interaction of the probe diS-C3-(5) with dipalmitoylphosphatidylcholine (DPPC) liposomes has been studied using fluorescence and differential scanning calorimetry (DSC). The partition coefficients (K) of the probe for the lipid and the aqueous phase (in terms of molar part units) were (1.20 +/- 0.4) X 10(6) at 45 degrees C and (0.50 +/- 0.07) X 10(6) at 23 and 36 degrees C. In terms of volume concentration units, these values correspond to Kp = (2.88 +/- 0.10) X 10(4) and Kp = (1.20 +/- 0.17) X 10(4), respectively. DSC thermograms were practically identical both for large unilamellar and multilamellar liposomes. The main transition peak remained practically unchanged over the entire range of the probe concentrations used. The pretransition could be observed up to maximal probe concentrations applied and it widened and shifted from 35.4 degrees C in pure DPPC to approximately 32 degrees C at a probe/lipid ratio of 0.027. These results suggest that in both quasicrystalline and liquid crystalline lipid bilayers the probe molecules are included in "defects" between structurally ordered microregions (microdomains or clusters). The dependence of the fluorescence response on the transmembrane potential in a suspension of unilamellar DPPC vesicles suggest that the equilibrium thermodynamic model is valid for liquid crystalline bilayers.     

Interaction of cytochrome c with cytochrome c oxidase studied by monoclonal antibodies and a protein modifying reagent.

C/57 black mice were immunized with beef heart cytochrome c oxidase, generating 48 hybrid cell lines that secrete antibodies against the different subunits of the enzyme. Immunoblot analysis showed reactions with 7 of the 13 subunits. Among the monoclonal antibodies produced, only those to subunit II gave significant inhibition; these inhibited the enzyme activity completely and prevented cytochrome c binding to the enzyme. Epitope mapping studies indicate that a peptide including residues 200-227 reacts with the antibody, suggesting that the C-terminus of the protein is essential for the binding of this antibody. The carboxyl modifying reagent 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) was chosen to investigate further the relationship between antibody and cytochrome c binding domains. ETC caused 50% inhibition of the enzyme activity with a first-order time during the first 20 min; a slower reaction over 3 h resulted in 90% inhibition. Cytochrome c binding to the oxidase was inhibited to a similar extent as cytochrome c oxidation, and protection against both effects was afforded by the presence of cytochrome c during ETC modification. Anion-exchange of FPLC of the modified forms of cytochrome oxidase revealed extensive inhomogeneity, indicating random derivatization of a number of different carboxyls even during the first-order reaction, and precluding identification of carboxyl residues related to a specific phase of the reaction. Cytochrome c and the subunit II-specific antibody protected against radioactive labeling of subunit II by ETC in the presence of [14C]glycine ethyl ester, demonstrating that the antibody and cytochrome c occupy significant and overlapping areas on the subunit II surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of L-arginine with dihexadecylphosphate unilamellar liposomes: the effect of the lipid phase organization

The interaction of L-arginine with unilamellar liposomes of dihexadecylphosphate sodium salt (DHP-Na) has been investigated using calorimetric, light scattering, fluorescence spectroscopy and zeta-potential techniques. Heating from room temperature, the bilayer exhibits a phase transition from a subgel (L(c)) to the gel (L(beta')) phase as well as a pre-transition (L(beta')-P(beta')), which is followed by the main lipid phase transition (P(beta')-L(alpha)). Direct studies of the interaction of L-arginine with the DHP-Na bilayers via isothermal titration calorimetry at 27 degrees C depict significant differences between samples in the L(c) and the L(beta') phases reflecting the effect of molecular organization of the lipids upon the interaction. While L-arginine has only a small impact upon the L(c) to L(beta') phase transition, it affects more significantly the transition temperature as well as the shape of the DSC peaks of the main lipid phase transition. Based on fluorescence and zeta-potential studies, the permeability of L-arginine through the liposomal membrane is higher within the temperature range of the main lipid phase transition. Encapsulated l-arginine obstructs the formation of the subgel phase.     

Investigation of spectrin binding to phospholipid vesicles using isoindole fluorescent probe. Thermal properties of the bound and unbound protein

Fluorescence of isoindole probe covalently bound to spectrin from pig erythrocytes, and fluorescence of tryptophanyl residues were used to study spectrin interaction with phospholipid bilayers. Evidence would be provided for conformational changes of spectrin occurring upon its binding to lipid bilayers. Fluorescence quenching experiments allowed to determine thermal stability of the protein in bound and unbound state. Spectrin binding to lipids was shown to protect the protein against thermal denaturation.     

The interaction of covalently bound heme with the cytochrome c maturation protein CcmE

The heme chaperone CcmE is a novel protein that binds heme covalently via a histidine residue as part of its essential function in the process of cytochrome c biogenesis in many bacteria as well as plant mitochondria. In the continued absence of a structure of the holoform of CcmE, identification of the heme ligands is an important step in understanding the molecular function of this protein and the role of covalent heme binding to CcmE during the maturation of c-type cytochromes. In this work, we present spectroscopic data that provide insight into the ligation of the heme iron in the soluble domain of CcmE from Escherichia coli. Resonance Raman spectra demonstrated that one of the heme axial ligands is a histidine residue and that the other is likely to be Tyr134. In addition, the properties of the heme resonances of the holo-protein as compared with those of a form of CcmE with non-covalently bound heme provide evidence for the modification of one of the heme vinyl side chains by the protein, most likely the 2-vinyl group.     

Structure of the cytochrome c oxidase complex: labeling by hydrophilic and hydrophobic protein modifying reagents

Beef heart cytochrome c oxidase has been reacted with [35S]diazobenzenesulfonate ([35S]DABS), [35S]-N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate ([35S]NAP-taurine), and two different radioactive arylazidophospholipids. The labeling of the seven different subunits of the enzyme with these protein modifying reagents has been examined. DABS, a water-soluble, lipid-insoluble reagent, reacted with subunits II, III, IV, V, and VII but labeled I or VI only poorly. The arylazidophospholipids, probes for the bilayer-intercalated portion of cytochrome c oxidase, labeled I, III, and VII heavily and II and IV lightly but did not react with V or VI. NAP-taurine labeled all of the subunits of cytochrome c oxidase. Evidence is presented that this latter reagent reacts with the enzyme from outside the bilayer, and the pattern of labeling with the different hydrophilic and hydrophobic labeling reagents is used to derive a model for the arrangement of subunits in cytochrome c oxidase.     

Raft-like domain formation in large unilamellar vesicles probed by the fluorescent phospholipid analogue, C12NBD-PC

The liquid-ordered/disordered-phase domain co-existence in large unilamellar vesicle membranes consisting of phosphatidylcholine:sphingomyelin (2:1) with different amounts of cholesterol has been examined using a concentration-dependent self-quenching of a single reporter molecule, C12NBD-PC. A temperature-dependent decrease of fluorescence intensity was associated with the expected formation and increase of l_o-phase membrane fraction in the vesicles. The result is consistent with exclusion of the fluorescent probe from the liquid-ordered phase which partitions preferentially into the liquid-disordered phase membrane domains. This leads to an increase of the local concentration of fluorophore in the liquid-disordered phase and a decrease of the quantum yield. This effect was used to obtain a quantitative estimation of the fraction of the vesicle membrane occupied by the liquid-ordered phase, Φ_o, as a function of temperature and cholesterol content between 0 and 45 mol%. The value of Φ_o was related to the assumed partition coefficient k_p of probe between liquid-ordered/disordered phases. For large unilamellar vesicles containing 20 and 4 mol% cholesterol and probe, respectively, with k_p = 0 (probe completely excluded from liquid-ordered phase), Φ_o = 0.16 and with k_p = 0.2, Φ_o = 0.2. The results are relevant to the action of detergent in the fractionation of detergent-resistant membrane from living cells.     

Interaction of a peripheral protein of the erythrocyte membrane, band 4.1, with phosphatidylserine-containing liposomes and erythrocyte inside-out vesicles

Interactions of band 4.1 with mixed phospholipid membranes [phosphatidylserine (PtdSer), phosphatidylethanolamine, phosphatidylcholine, etc.] and erythrocyte inside-out vesicles were studied. Band 4.1 showed a higher affinity to PtdSer-containing membranes. The amount of binding to PtdSer-containing liposomes was larger than that to PtdSer-lacking liposomes. The amount of binding to inside-out vesicles did not change significantly on a protease treatment of the vesicles. The amount of band 4.1 bound on inside-out vesicles decreased on PtdSer-decarboxylase treatment of the vesicles. Ca2+ acted inhibitory to the binding of band 4.1. Band 4.1 together with PtdSer-containing vesicles but not with PtdSer-lacking vesicles induced gelation of spectrin-actin copolymer solution. Ca2+ inhibited the gelation. Fluorescence energy transfer from PtdSer-containing vesicles to band 4.1 was larger than that from PtdSer-lacking vesicles. Band 4.1 caused a marked release of tempocholine from preloaded PtdSer-containing liposomes but not from PtdSer-lacking liposomes. The release was larger from liposomes containing more PtdSer. Ca2+ was inhibitory to the tempocholine release. We suggest from these results that band 4.1 provides another anchoring site for the cytoskeletal spectrin-actin network to PtdSer domains in the inner layer of erythrocyte membrane. This anchoring may be involved in functional regulation since the interaction causes the membrane permeability change that is dependent on Ca2+.     

Interaction of cytochrome c with cytochrome c oxidase: an understanding of the high- to low-affinity transition

The steady-state kinetics of high- and low-affinity electron transfer reactions between various cytochromes c and cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) preparations were studied spectrophotometrically and polarographically. The dissociation constants for the binding of the first and second molecules of horse cytochrome c (I = 15 mM) are 5.10(-8) M and 1.10(-5) M, respectively, close to the spectrophotometric Km values and consistent with the controlled binding model for the interaction between cytochrome c and cytochrome oxidase (Speck, S.H., Dye, D. and Margoliash, E. (1984) Proc. Natl. Acad. Sci. USA 81, 346-351) which postulates that the binding of a second molecule of cytochrome c weakens that of the first, resulting in low-affinity kinetics. While the Km of the polarographically assayed high-affinity reaction is comparable to that observed spectrophotometrically, the low-affinity Km is over an order of magnitude smaller and cannot be attributed to the binding of a second molecule of cytochrome c. Increasing the viscosity has no effect on the Vmax of the low-affinity reaction assayed polarographically, but increases the Km. Thus, the transition from high- to low-affinity kinetics is dependent on the frequency of productive collisions, as expected for a hysteresis model ascribing the transition to the trapping of the oxidase in a primed state for turnover. At ionic strengths above 150 mM, the rate of cytochrome c oxidation decreases without any correlation to the calculated net charge of the cytochrome c, indicating rate-limiting rearrangement of the two proteins in proximity to each other.     

Interaction of the polyene antibiotic etruscomycin with large unilamellar lipid vesicles: binding and proton permeability inducement

The effect of the polyene antibiotic etruscomycin on the permeability of large unilamellar lipid vesicles was investigated. Proton leakage was induced in egg-yolk phosphatidylcholine (EPC) vesicles only when sterol was present in the membrane; the extent of leakage was limited. High etruscomycin/lipid ratios (R) were necessary (R greater than 0.1). Higher percentages of sterol increased the permeability, slightly more strongly for ergosterol than for cholesterol. Dipalmitoylphosphatidylcholine (DPPC) vesicles were more sensitive to permeability inducement, even in the absence of sterol in the bilayer (inducement for R greater than 0.06). The interactions of etruscomycin with the vesicles were examined by circular dichroism, fluorescence and 31P-NMR. In the range of antibiotic concentration where permeability was induced, R greater than 0.1 for EPC vesicles, R greater than 0.06 for DPPC vesicles, etruscomycin exhibited characteristic circular dichroism spectra independent of the presence of sterol. Under the same conditions, 31P-NMR and fluorescence studies indicated a destruction or a fusion of the vesicle bilayer. At lower etruscomycin concentrations (R less than 0.03), the etruscomycin circular dichroism spectra were different, indicating that the interaction with membranes containing ergosterol differed from that with membranes containing cholesterol. From correlating the increase in fluorescence intensity with this interaction, as well as from exchange experiments, it was inferred that etruscomycin at a low antibiotic/lipid ratio is more strongly bound to ergosterol-containing vesicles than to cholesterol-containing vesicles. These results and their comparison with the results obtained with other polyene antibiotics indicate that at low R etruscomycin resembles amphotericin rather than filipin in its preferential binding to ergosterol-containing vesicles. At higher R, that is in conditions where permeability is induced, the selectivity is different. The corresponding mechanism seems not to involve the formation of an etruscomycin-sterol channel, since the hydrophobic chain of the complex would be too short to form a channel.     

H+/e- stoichiometry of mitochondrial cytochrome complexes reconstituted in liposomes. Rate-dependent changes of the stoichiometry in the cytochrome c oxidase vesicles.

The H+/e- stoichiometry of protonmotive cytochrome c oxidase, isolated from bovine heart mitochondria and reconstituted in liposomes, has been determined by making use of direct spectrophotometric measurements of the initial rates of e- flow and H+ translocation. It is shown that the ----H+/e- ratio for redox-linked proton ejection by the oxidase varies from around 0 to a maximum of 1 as a function of the rate of overall electron flow in the complex.     

Subzero temperature study of the inner mitochondrial membrane and related phospholipid membrane systems with the fluorescent probe, trans-parinaric acid.

The fluorescence intensity of trans-parinaric acid as a function of the temperature indicates a phase transition in bovine heart mitochondrial inner membranes below 0 degrees C. The comparison of the dye fluorescence intensity in intact inner mitochondrial membranes and in vesicles from extracted phospho lipids of mitochondria revealed a similar intensity increase with decreasing temperature. A synthetic phospholipid system of dioleoyl phosphatidylcholine was investigated because of its low phase transition temperature and showed a very definite intensity change at -25 degrees C. trans-Parinaric acid in membrane systems probes an environment of intermediate polarity; this was found from the excitation and emission spectra and from fluorescence decay.     

Interaction of negatively charged liposomes with nuclear membranes: adsorption, lipid mixing and lysis of the vesicles

Fluorescence energy transfer studies reveal that negatively charged lipid vesicles interact with nuclei from mouse liver cells. This interaction was observed with charged lipid vesicles composed of PA or PS but not with the uncharged PC or PE:PC vesicles. The vesicles were prepared by bath sonication and contained either a fluorescent marker in the lipid bilayer or in the vesicular interior. The negatively charged vesicles showed an adsorption to the nuclear membrane visible by fluorescence microscopy. The results obtained by resonance energy transfer experiments are interpreted in terms of a mixing of the lipids from the vesicles with the nuclear membrane. Encapsulation studies documented a staining of the nuclei only if the dye molecules of high or low molecular weight were encapsulated inside negatively charged vesicles. As consequence of the vesicle-nuclei interaction morphological changes on the nuclear surface became visible.     

Interaction of the polyene antibiotic amphotericin B with model membranes: differences between small and large unilamellar vesicles

The interaction of the polyene antibiotic amphotericin B (AmB) (Fig. 1) with large unilamellar vesicles (LUV) was monitored by circular dichroism (CD) and carboxyfluorescein (CF) release. LUV afford a far better model for biological membranes than small unilamellar vesicles (SUV) which have been used until now. With dimyristoyl phosphatidyl choline (DMPC) LUV (i.e., containing saturated acyl chains), a strong and not saturable binding for AmB/lipid ratios up to 0.5 was observed both above and below the phase transition temperature. Incorporation of cholesterol into the vesicles did not significantly change the interaction. With egg PC (EPC) LUV (i.e., containing unsaturated acyl chains), quite a different picture emerged: the binding reached saturation for AmB/lipid ratios of about 5 x 10(-3), a result not observed with EPC SUV. When sterols were introduced into membranes, the CD spectral features obtained in the presence of ergosterol were different from those obtained in the presence of cholesterol. Such a different behavior was not observed with SUV. We suggest that species whose CD spectrum was observed after 15 min in the presence of ergosterol-containing EPC LUV is the particular one which forms wide channels and induces a Ca2+ release. (H. Ramos, A. Attias, B.E. Cohen and J. Bolard, submitted for publication). The CF release from EPC LUV induced by AmB was very low, even at very high concentrations of the antibiotic (3 x 10(-4)M). In contrast, an important release of the fluorescent dye was observed with DMPC LUV at concentrations of approximately 10(-5)M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incorporation of the cytochrome P-450 monooxygenase system into large unilamellar liposomes using octylglucoside,especially for measurements of protein diffusion in membranes

Cytochrome P-450 and NADPH cytochrome P-450 reductase were incorporated into large unilamellar lipid vesicles (200–300 nm in diameter) removing octylglucoside from mixed micelles by dialysis. The large size of the protein-containing liposomes guarantees a negligibly small vesicle tumbling. Such large vesicles are better suited for studies of protein rotation in reconstituted membranes than vesicles prepared by use of bile salts. At present the octylglucoside reconstituted monooxygenase system seems to be the most appropriate model for studying protein-protein and protein-lipid interactions in liver microsomes due to the similarity with respect to the main structural and functional properties, including size.     

Subzero temperature study of the inner mitochondrial membrane and related phospholipid membrane systems with the fluorescent probe, trans-parinaric acid

The fluorescence intensity of trans-parinaric acid as a function of the temperature indicates a phase transition in bovine heart mitochondrial inner membranes below 0°C. The comparison of the dye fluorescence intensity in intact inner mitochondrial membranes and in vesicles from extracted phospholipids of mitochondria revealed a similar intensity increase with decreasing temperature. A synthetic phospholipid system of dioleoyl phosphatidylcholine was investigated because of its low phase transition temperature and showed a very definite intensity change at ?25°C. trans-Parinaric acid in membrane systems probes an environment of intermediate polarity; this was found from the excitation and emission spectra and from fluorescence decay.     

Interaction of the 4 S polycyclic aromatic hydrocarbon-binding protein with the cytochrome P-450c gene

The induction of cytochrome P-450c, the isozyme most closely associated with aryl hydrocarbon hydroxylase activity in the rat, is mediated through a cytosolic polycyclic aromatic hydrocarbon (PAH)-binding protein(s). We have reported on the purification and characterization of a 4 S protein that interacts in a specific and saturable manner with [3H]benzo[a]pyrene and other PAHs. (W. H. Houser et al. (1985) Biochemistry 24, 7839-7845). We have also reported on the specific and saturable interaction of the 4 S protein with a plasmid containing 1.9 kbp of cloned rat P-450c sequence including exon 1, the 5' half of intron 1, and approximately 882 bp upstream information. Our investigations now show that incubation of this protein with a portion of the rat P-450c gene, followed by digestion with either lambda exonuclease or exonuclease III, tentatively identified two protected regions at -225 and -455 bp 5' from exon 1. To further study the significance of these protected regions, a 3.4-kbp fragment containing cytochrome P-450c promoter and 5'-upstream sequences (-882 to +2545) was fused to the chloramphenicol acetyl transferase (CAT) reporter gene and transfected into either rat epithelial RL-PR-C cells or rat hepatoma H-4-II-E cells. Both cell lines expressed CAT activity in response to induction by 3-methylcholanthrene (3MC), indicating that important regulatory regions responsive to 3MC are present in these constructs. However, neither cell line expressed CAT activity in response to 3MC when transfected with plasmids containing deletions (-95 to -724 or -240 to -720) in the regions shown to be protected by our footprinting studies. These results corroborate previous studies which indicated that the 4 S PAH-binding protein interacts in a specific manner with regions of the rat cytochrome P-450c gene. We conclude that the 4 S protein may play an important role in the regulation of expression of cytochrome P-450c in the rat.     

Proton NMR and electrophoretic studies of the covalent complex formed by cross-linking yeast cytochrome c peroxidase and horse cytochrome c with a water-soluble carbodiimide

The 1:1 covalently cross-linked complex between horse cytochrome c and yeast cytochrome c peroxidase (ccp) has been formed by a slight modification of the method of Waldmeyer and Bosshard [Waldmeyer, B., & Bosshard, H. R. (1985) J. Biol. Chem. 260, 5184-5190]. This earlier study has been extended to show that efficient cross-linking of the two proteins can occur in a variety of buffers over a broad ionic strength range. The substitution of ferrocytochrome c for ferricytochrome c in the cross-linking studies resulted in an increased yield of 1:1 complex (approximately 10-20%) under the conditions studied. An improved method for purifying the covalent complex in relatively large quantities is presented here as are the results of electrophoresis and proton NMR studies of the complex. Both electrophoresis and NMR studies indicate modification of some surface acidic amino acids in the covalent complex by the carbodiimide. The proton hyperfine-shifted resonances of cytochrome c are broadened in the covalent complex relative to free cytochrome c, and the resonances corresponding to the cytochrome c heme 3-CH3 and 8-CH3 groups are shifted closer together in the complex. Integration of NMR resonances confirms a 1:1 complex as the primary cross-linking reaction product. However, we also demonstrate that the covalent complex can be further coupled to ccp and to cytochrome c to form higher molecular weight aggregates.     

Proton NMR detection of porphyrins and cytochrome C in small unilamellar vesicles: role of the dissociation kinetic constant

The molecular tumbling of small unilamellar vesicles is not fast enough to enable the detection of (1)H NMR signals of molecules associated with phospholipids. We show that relatively fast kinetic exchange of the interacting molecules is able to induce a strong decrease of the residual homonuclear dipolar coupling, allowing the acquisition of sharp signals. At low molecule/lipids molecular ratio, this can be lead to signal broadening due to exchange at intermediate rates on the NMR chemical timescale. However, proton resonances can be easily detected when sufficient lipids are added to prevent the occurrence of any free compounds in solution. This is demonstrated, using lipid signal suppression, in the case of paramagnetic porphyrin derivatives as well as diamagnetic hematoporphyrin. Since several peptides and proteins are expected to be associated with lipids having relatively fast dynamics, this study addresses, as a first example, the interaction of cytochrome c.