cytochrome-c-t and 1-2-oleoylphosphatidylcholine

Topics: Cardiolipins; Cytochromes c; Humans; Lipid Bilayers; Mitochondrial Membranes; Phosphatidylcholines; Secretory Vesicles; Unilamellar Liposomes

Experiments investigating the adsorption and desorption of cytochrome c onto and from liposomes containing 50 mol% 1,2-diacylphosphatidylglycerol lipids [10:0, 12:0, 14:0, 16:0, 18:1(Δ9 cis)] with 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) in pH 7.4 buffered solutions of low to moderate ionic strength are reported. Fluorescence experiments show that cytochrome c has a similar adsorption affinity for the five labeled 50 mol% PG liposome systems investigated. Fluorescence recovery experiments reveal the extent of cytochrome c desorption upon the addition of >10× excess of unlabeled 100% 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG) liposomes is dependent on the lipid's acyl chain length. The extent of desorption is also shown to be independent of temperature, albeit over a narrow range. The differences in the extent of cytochrome c desorption from liposomes containing PG lipids with different acyl chain lengths is attributed to the varying contribution of the binding motif involving the extended lipid anchorage in response to lipid packing stress.

Topics: Adsorption; Cytochromes c; Diglycerides; Glycosylphosphatidylinositols; Liposomes; Membrane Lipids; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Osmolar Concentration; Phosphatidylcholines; Phosphatidylglycerols

The Met80-heme iron bond of cytochrome c (cyt c) is cleaved by the interaction of cyt c with cardiolipin (CL) in membranes. The Met80 dissociation enhances the peroxidase activity of cyt c and triggers cyt c release from mitochondrion to the cytosol at the early stage of apoptosis. This paper demonstrates the selective oxidation of Met80 for the reaction of ferric cyt c with a peroxide, meta-chloroperbenzoic acid (mCPBA), in the presence of CL-containing liposomes by formation of a ferryl species (Compound I). After the reaction of cyt c with mCPBA in the presence of 1,2-dioloeyl-sn-glycero-3-phosphocholine (DOPC) liposomes containing CL, the electrospray ionization mass spectrum of the peptide fragments, obtained by digestion of cyt c with lysyl endopeptidase, exhibited a peak at m/z = 795.45; whereas, this peak was not observed for the peptide fragments obtained after the reaction in the presence of DOPC liposomes not containing CL. According to the tandem mass spectrum of the m/z = 795.45 peptide fragment, Met80 was modified with a 16 Da mass increase. The purified Met80-modified cyt c exhibited a peroxidase activity more than 5-fold higher than that of the unmodified protein. Transient absorption bands around 650 nm were generated by the reactions with mCPBA for ferric wild-type cyt c in the presence of CL-containing DOPC liposomes and ferric Y67F cyt c in the absence of liposomes. The formation and decomposition rates of the 650-nm absorption species increased and decreased, respectively, by increasing the mCPBA concentration in the reaction, indicating transient formation of Compound I.

Topics: Cardiolipins; Cytochromes c; Liposomes; Methionine; Oxidation-Reduction; Peroxidase; Peroxides; Phosphatidylcholines; Tandem Mass Spectrometry

Molecular understanding of the impact of nanomaterials on cell membranes is critical for the prediction of effects that span environmental exposures to nanoenabled therapies. Experimental and computational studies employing phospholipid bilayers as model systems for membranes have yielded important insights but lack the biomolecular complexity of actual membranes. Here, we increase model membrane complexity by incorporating the peripheral membrane protein cytochrome c and studying the interactions of the resulting membrane systems with two types of anionic nanoparticles. Experimental and computational studies reveal that the extent of cytochrome c binding to supported lipid bilayers depends on anionic phospholipid number density and headgroup chemistry. Gold nanoparticles functionalized with short, anionic ligands or wrapped with an anionic polymer do not interact with silica-supported bilayers composed solely of phospholipids. Strikingly, when cytochrome c was bound to these bilayers, nanoparticles functionalized with short anionic ligands attached to model biomembranes in amounts proportional to the number of bound cytochrome c molecules. In contrast, anionic polymer-wrapped gold nanoparticles appeared to remove cytochrome c from supported lipid bilayers in a manner inversely proportional to the strength of cytochrome c binding to the bilayer; this reflects the removal of a weakly bound pool of cytochrome c, as suggested by molecular dynamics simulations. These results highlight the importance of the surface chemistry of both the nanoparticle and the membrane in predicting nano-bio interactions.

Topics: Animals; Binding Sites; Cardiolipins; Cattle; Cytochromes c; Gold; Lipid Bilayers; Membrane Proteins; Metal Nanoparticles; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylinositols; Protein Binding; Static Electricity

Non-fluidic bio-layer interferometry (BLI) has rapidly become a standard tool for monitoring almost all biomolecular interactions in a label-free, real-time and high-throughput manner. High-efficiency screening methods which measure the kinetics of liposomes with a variety of compounds require the immobilization of liposomes. In this work, a method is described for immobilizing liposomes for interaction studies, based on the biophysical principles of this biosensor platform. The immobilization approach includes the loading of DSPE-PEG

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adsorption; Biosensing Techniques; Biotin; Cardiolipins; Cytochromes c; Drosophila Proteins; Fluoresceins; Fluorescent Dyes; High-Throughput Screening Assays; Hydrophobic and Hydrophilic Interactions; Interferometry; Kinetics; Liposomes; Micelles; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Polyethylene Glycols; Protein Phosphatase 1; Reproducibility of Results

Ferricytochrome c binding to cardiolipin-containing liposomes produces a heterogeneous distribution of conformations comprising native-like and non-native misfolded proteins. We utilized the photoreduction of native ferricytochrome c in the presence of potassium ferrocyanide and resonance Raman spectroscopy to probe the population of native and misfolded cytochrome c on liposomes with 20 % tetraoleylcardiolipin (TOCL)/80 % dioleylphosphocholine (DOPC) and with 100 % TOCL as a function of TOCL concentration. Our data provided strong support for an earlier model, which predicts that the equilibrium between native and non-native conformations is shifted to the latter with decreasing protein occupation of liposomes.

Topics: Cardiolipins; Cytochromes c; Ferrocyanides; Light; Liposomes; Oxidation-Reduction; Phosphatidylcholines; Spectrum Analysis, Raman

The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly (13)C,(15)N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.

Topics: Apoptosis; Carbon-13 Magnetic Resonance Spectroscopy; Cardiolipins; Cytochromes c; Escherichia coli; Lipid Bilayers; Mitochondria; Nuclear Magnetic Resonance, Biomolecular; Peroxidase; Phosphatidylcholines; Protein Conformation; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis

Cytochrome c binding to cardiolipin receptors on the surface of TOCL/DOPC(20% : 80%) liposomes induces a conformational change, which is not reversible after the protein's dissociation at low ionic strength. Addition of 100 mM NaCl switches the protein back to its native state.

Topics: Cardiolipins; Catalysis; Cell Membrane; Cytochromes c; Liposomes; Mitochondria; Phosphatidylcholines; Protein Folding; Sodium Chloride

Mitochondria have emerged as the major regulatory platform responsible for the coordination of numerous metabolic reactions as well as cell death processes, whereby the execution of intrinsic apoptosis includes the production of reactive oxygen species fueling oxidation of cardiolipin (CL) catalyzed by cytochrome (Cyt) c. As this oxidation occurs within the peroxidase complex of Cyt c with CL, the latter represents a promising target for the discovery and design of drugs with antiapoptotic mechanisms of action. In this work, we designed and synthesized a new group of mitochondria-targeted imidazole-substituted analogs of stearic acid TPP-n-ISAs with various positions of the attached imidazole group on the fatty acid (n = 6, 8, 10, 13, and 14). By using a combination of absorption spectroscopy and EPR protocols (continuous wave electron paramagnetic resonance and electron spin echo envelope modulation) we demonstrated that TPP-n-ISAs indeed were able to potently suppress CL-induced structural rearrangements in Cyt c, paving the way to its peroxidase competence. TPP-n-ISA analogs preserved the low-spin hexa-coordinated heme-iron state in Cyt c/CL complexes whereby TPP-6-ISA displayed a significantly more effective preservation pattern than TPP-14-ISA. Elucidation of these intermolecular stabilization mechanisms of Cyt c identified TPP-6-ISA as an effective inhibitor of the peroxidase function of Cyt c/CL complexes with a significant antiapoptotic potential realized in mouse embryonic cells exposed to ionizing irradiation. These experimental findings were detailed and supported by all-atom molecular dynamics simulations. Based on the experimental data and computation predictions, we identified TPP-6-ISA as a candidate drug with optimized antiapoptotic potency.

Topics: Animals; Apoptosis; Cardiolipins; Cytochromes c; Drug Design; Embryonic Stem Cells; Enzyme Inhibitors; Gamma Rays; Horses; Imidazoles; Mice; Mitochondria, Heart; Molecular Dynamics Simulation; Organophosphorus Compounds; Peroxidase; Phosphatidylcholines; Ricinoleic Acids; Stearic Acids; Structure-Activity Relationship

Interaction of cytochrome c with cardiolipin in the presence of hydrogen peroxide induces peroxidase activity in cytochrome c and the ability to oxidize membrane lipids. These cytochrome c properties play a substantial role in the cytochrome c-mediated apoptotic reactions. In the present study the electric properties (specific capacitance and integral conductance) of the cardiolipin-containing asolectin planar bilayer lipid membranes (pBLM) in the presence of cytochrome c and hydrogen peroxide were studied. Cytochrome c interaction with cardiolipin-containing pBLM in the presence of hydrogen peroxide resulted in the dramatic increase of the conductance, pore production, their growth up to 3.5 nm diameter and subsequent membrane destruction. In the absence of hydrogen peroxide cytochrome c demonstrated almost no effect on the membrane capacitance and conductance. The data obtained prove the pivotal role of cytochrome c and membrane lipids in the permeabilization of pBLM. Correlation of apoptotic reactions and cytochrome c-mediated membrane permeability is discussed.

Topics: Apoptosis; Cardiolipins; Cell Membrane; Cell Membrane Permeability; Cytochromes c; Hydrogen Peroxide; Kinetics; Lipid Bilayers; Membrane Lipids; Permeability; Peroxidase; Phosphatidylcholines; Time Factors

Cytochrome c (cyt c) is a small soluble protein from the intermembrane space of mitochondria. This protein is essential because it transfers electrons between two membrane complexes of the respiratory chain. In fact, during this transfer, the positively charged amino-acid residues surrounding the heme in the protein structure allow the cyt c to interact properly with the anionic part of other molecules: mainly the cardiolipin-rich membrane of mitochondria and respiratory complexes. We have previously shown that besides its interaction with anionic lipids, the cyt c is also able to cross neutral lipid membranes. In this work, with the help of AFM and punch-through experiments, we have measured the force required to penetrate the membrane in the fluid and in the gel phases with or without cyt c molecules. In the presence of cyt c molecules, the structures generated by the interaction with the protein were considerably weakened, which led to the desorption of the fluid bilayer and to a considerable loss of cohesion of the gel phase. These results show the usefulness of punch-through experiments in determining the changes of membrane properties in the presence of external agents.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Biomechanical Phenomena; Cytochromes c; Gels; Horses; Ions; Lipid Bilayers; Membrane Fluidity; Membranes, Artificial; Microscopy, Atomic Force; Models, Biological; Nanoparticles; Phosphatidylcholines; Spectrum Analysis

Induction of the peroxidase activity of cytochrome c (cyt c) by cardiolipin (CL) and H(2)O(2) in mitochondria is suggested to be a key event in early apoptosis. Although electrostatic interaction between the positively charged cyt c and negatively charged CL is a predominant force behind the formation of a specific cyt c-CL complex and sequential induction of the peroxidase activity, molecular mechanisms of hydrophobic interactions involving the fatty acyl chains of CL remain to be investigated. To elucidate the function of the acyl chains, particularly the role of the double bond, we synthesized a variety of CL analogues and examined their peroxidase inducing activity. Irrespective of the number of double bonds in the acyl chains, the peroxidase activity of cyt c induced by liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and a different CL (9:1 molar ratio) was similar, except for that of 1,1',2,2'-tetrastearoylcardiolipin (TSCL, C18:0)-containing liposomes. The peroxidase inducing activity of TSCL-containing liposomes was 3-4-fold greater than that of other CL-containing liposomes. The peroxidase activity induced by all CL-containing liposomes was much lower at high ionic strengths than that at low ionic strengths because of diminution of the electrostatic interaction. The peroxidase inducing effects of various CL-containing liposomes were related well to their ability to associate with cyt c. Thus, our results revealed that at low CL levels, the saturated acyl chain of CL is favorable for the activation of peroxidase activity of CL-bound cyt c and the proposed critical role of the double bond is not a general feature of the cyt c-CL interaction. The polarity of the membrane surface of TSCL-containing liposomes was slightly, but significantly, lower than that of other CL-containing liposomes, suggesting that the higher activating ability of TSCL-containing liposomes may be due to a reduced level of hydration of the polar head region reflecting tighter packing of the fully saturated acyl chains. Moreover, using CL analogues in which a central glycerol head moiety was modified, we revealed that the natural structure of the head moiety is not critical for the formation of the active cyt c-CL complex. The effects of the CL content of the liposomal membrane on the cyt c-CL interaction are discussed.

Topics: Cardiolipins; Cytochromes c; Enzyme Induction; Hydrogen Peroxide; Liposomes; Mitochondria; Osmolar Concentration; Peroxidases; Phosphatidylcholines

The interaction of cytochrome c (cyt c) with fluid/gel neutral supported lipid membranes was investigated by time-lapse atomic force microscopy (AFM). AFM revealed the random formation of depressed areas in fluid membranes promoted by cyt c. These depressions corresponded to the desorption of fluid bilayer patches induced by cyt c. By contrast, the gel domains were never desorbed but they were progressively thickened in the presence of the protein. These results suggest that cyt c molecules might intercalate between the mica and the lipid bilayer. Although the interaction of cyt c with the mica surface is likely to be an artifact, this work is the first direct observation of cyt c ability to cross membranes. Furthermore, our data show that the net positive charge of cyt c molecules plays a pivotal role but it is not the sole factor responsible for cyt c insertion in the membrane.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Chickens; Cytochromes c; Egg Yolk; Horses; Lipid Bilayers; Membranes, Artificial; Models, Molecular; Muramidase; Phosphatidylcholines; Static Electricity; Surface Properties

Specular neutron reflectivity was used to study the time course and nature of the interaction of the positively charged, peripheral membrane protein cytochrome c with supported bilayers of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) containing the anionic lipid 1-palmitoyl-2-oleoyl-glycero-3-phosphatidylserine (POPS). The supported bilayers were prepared by deposition on silicon blocks of two monolayers of DOPC, the second of which contained either 10 or 20 mol % POPS at surface pressures of either 15 or 20 mN/m using a combination of Langmuir-Blodgett and Schaefer deposition techniques. Each supported bilayer was initially characterized by specular neutron reflectivity using subphases of 10 mM NaCl aqueous solutions. Regardless of POPS content and bilayer deposition pressure, the molecular architecture of the bilayers was similar. The addition of cytochrome c resulted in an almost immediate change in reflectivity, which was well modeled by assuming that an additional layer was present next to the outer leaflet of the bilayer. The thickness of this layer, which contained the volume fraction of approximately 15% protein, was approximately 30 A (consistent with the cross-section of a single cytochrome c molecule). The addition of cytochrome c to the subphase also resulted in a change in the structure of the phospholipid bilayer, suggesting some penetration of cytochrome c into the bilayer. Specular neutron reflectivity studies after careful washing with solvent showed that although most of the protein was washed off by flushing 10 mM NaCl D2O through the cell a small amount remained both within the bilayer and bound to the membrane surface.

Topics: Cell Membrane; Cytochromes c; Lipid Bilayers; Neutron Diffraction; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Silicon; Sodium Chloride

Small increases in physiological nitrite concentrations have now been shown to mediate a number of biological responses, including hypoxic vasodilation, cytoprotection after ischemia/reperfusion, and regulation of gene and protein expression. Thus, while nitrite was until recently believed to be biologically inert, it is now recognized as a potentially important hypoxic signaling molecule and therapeutic agent. Nitrite mediates signaling through its reduction to nitric oxide, via reactions with several heme-containing proteins. In this report, we show for the first time that the mitochondrial electron carrier cytochrome c can also effectively reduce nitrite to NO. This nitrite reductase activity is highly regulated as it is dependent on pentacoordination of the heme iron in the protein and occurs under anoxic and acidic conditions. Further, we demonstrate that in the presence of nitrite, pentacoordinate cytochrome c generates bioavailable NO that is able to inhibit mitochondrial respiration. These data suggest an additional role for cytochrome c as a nitrite reductase that may play an important role in regulating mitochondrial function and contributing to hypoxic, redox, and apoptotic signaling within the cell.

Topics: Animals; Apoptosis; Cattle; Cytochromes c; Hydrogen-Ion Concentration; Hypoxia; Kinetics; Mitochondria; Nitric Oxide; Nitrite Reductases; Oxygen; Oxygen Consumption; Phosphatidylcholines; Signal Transduction; Spectrophotometry

The interaction of cytochrome c (cyt c) with supported lipid membranes was investigated on the nanoscale by real-time atomic force microscopy. Cyt c promoted the formation and the expansion of depressed areas in the fluid parts of the bilayer. When the depressions reached the gel domains, they induced the thickening of their edges. According to the step-height differences, cyt c was able to remove neutral lipids in the fluid phase and then to reside on the mica surface. Concerning gel phases, cyt c might insert between the two lipid leaflets, or it might intercalate between the mica and the bilayer.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Aluminum Silicates; Cytochromes c; Gels; Lipid Bilayers; Microfluidics; Microscopy, Atomic Force; Nanotechnology; Phosphatidylcholines; Surface Properties

Activation of peroxidase catalytic function of cytochrome c (cyt c) by anionic lipids is associated with destabilization of its tertiary structure. We studied effects of several anionic phospholipids on the protein structure by monitoring (1) Trp59 fluorescence, (2) Fe-S(Met80) absorbance at 695 nm, and (3) EPR of heme nitrosylation. Peroxidase activity was probed using several substrates and protein-derived radicals. Peroxidase activation of cyt c did not require complete protein unfolding or breakage of the Fe-S(Met80) bond. The activation energy of cyt c peroxidase changed in parallel with stability energies of structural regions of the protein probed spectroscopically. Cardiolipin (CL) and phosphatidic acid (PA) were most effective in inducing cyt c peroxidase activity. Phosphatidylserine (PS) and phosphatidylinositol bisphosphate (PIP2) displayed a significant but much weaker capacity to destabilize the protein and induce peroxidase activity. Phosphatidylinositol trisphosphate (PIP3) appeared to be a stronger inducer of cyt c structural changes than PIP2, indicating a role for the negatively charged extra phosphate group. Comparison of cyt c-deficient HeLa cells and mouse embryonic cells with those expressing a full complement of cyt c demonstrated the involvement of cyt c peroxidase activity in selective catalysis of peroxidation of CL, PS, and PI, which corresponded to the potency of these lipids in inducing cyt c's structural destabilization.

Topics: Animals; Apoptosis; Cardiolipins; Cytochromes c; Electron Spin Resonance Spectroscopy; Enzyme Activation; Etoposide; Fluorescence; Heme; Humans; Mice; Peroxidase; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylserines; Phospholipids; Protein Structure, Tertiary; Tryptophan

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.

Topics: Acridine Orange; Animals; Binding, Competitive; Cardiolipins; Cell Membrane; Cytochromes c; Electrophoresis; Enzyme Activation; Etoposide; Fluoresceins; Horses; Hydrophobic and Hydrophilic Interactions; Lipids; Liposomes; Osmolar Concentration; Oxidation-Reduction; Peroxidase; Phosphatidylcholines; Spectrometry, Fluorescence; Structure-Activity Relationship; Time Factors; Tryptophan

The interaction of phenethyl alcohol with model membranes and its effect on translocation of the chemically prepared mitochondrial precursor protein apocytochrome c across a lipid bilayer was studied. Phenethyl alcohol efficiently penetrates into monolayers and causes acyl chain disordering judged from deuterium nuclear magnetic resonance measurements with specific acyl chain-deuterated phospholipids. Translocation of apocytochrome c across a phospholipid bilayer was stimulated on addition of phenethyl alcohol indicating that the efficiency of translocation of this precursor protein is enhanced due to a disorder of the acyl chain region of the bilayer.

Topics: Apoproteins; Biological Transport; Cytochrome c Group; Cytochromes c; Ethanol; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Lipids; Mitochondria; Phenylethyl Alcohol; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Protein Precursors; Trypsin

(1) The interaction of apocytochrome c with different molecular species of phosphatidylserine was studied using monolayers at constant surface area or constant surface pressure. The protein inserted readily into dioleoylphosphatidylserine monolayers up to a limiting pressure of 50 mN/m, whereas the interaction decreased with increasing molecular packing of the phosphatidylserine species, indicating the importance of the hydrophobic core of the lipid layer for the interaction. (2) The high affinity of apocytochrome c for dioleoylphosphatidylserine is indicated by the low Kd of 0.017 microM. There is little or no interaction with phosphatidylcholines. The importance of charge interactions is underlined by its ionic strength and pH dependency. (3) Experiments using 14C-labelled apocytochrome c indicate that cholesterol can enhance the protein binding. (4) It was demonstrated that apocytochrome c monomers penetrate the monolayer whereas oligomers can be formed in an adsorbed layer and washed off without changing the surface pressure. Preincubation of apocytochrome c in 3 M guanidine, to obtain the monomeric form, was essential to measure the full effect of interfacial interaction. (5) The molecular area of apocytochrome c changed from 1200-1300 A2/molecule in the absence of lipid to 700-900 A2/molecule after penetration of dioleoylphosphatidylserine monolayers. (6) Apocytochrome c-dioleoylphosphatidylserine interactions are only possible when the monolayer is approached from the subphase. It is concluded that the charge interactions are required for binding and penetration of the protein.

Topics: Apoproteins; Cholesterol; Cytochrome c Group; Cytochromes c; Hydrogen-Ion Concentration; Membrane Lipids; Mitochondria; Phosphatidylcholines; Phosphatidylserines; Temperature