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W2002088627 abstract "Binding of cytochrome c (cytc) to fatty acids and acidic phospholipid membranes produces pronounced and essentially identical changes in the spectral properties of cyt c, revealing conformational changes in the protein. The exact mechanism of the interaction of fatty acids and acidic phospholipids with cyt c is unknown. Binding of cytc to liposomes with high contents (mole fractionX > 0.7) of acidic phospholipids caused spectral changes identical to those due to binding of oleic acid. Fluorescence spectroscopy of a cyt c analog containing a Zn2+ substituted heme moiety and brominated lipid derivatives (9,10)-dibromostearate and 1-palmitoyl-2-(9,10)-dibromo-sn-glycero-3-phospho-rac-glycerol demonstrated a direct contact between the fluorescent [Zn2+-heme] group and the brominated acyl chain. These data constitute direct evidence for interaction between an acyl chain of a membrane phospholipid and the inside of the protein containing the heme moiety and provide direct evidence for the so-called extended-lipid anchorage of cyt c to phospholipid membranes. In this mechanism, one of the phospholipid acyl chains protrudes out of the membrane and intercalates into a hydrophobic channel in cyt c while the other chain remains in the bilayer. Binding of cytochrome c (cytc) to fatty acids and acidic phospholipid membranes produces pronounced and essentially identical changes in the spectral properties of cyt c, revealing conformational changes in the protein. The exact mechanism of the interaction of fatty acids and acidic phospholipids with cyt c is unknown. Binding of cytc to liposomes with high contents (mole fractionX > 0.7) of acidic phospholipids caused spectral changes identical to those due to binding of oleic acid. Fluorescence spectroscopy of a cyt c analog containing a Zn2+ substituted heme moiety and brominated lipid derivatives (9,10)-dibromostearate and 1-palmitoyl-2-(9,10)-dibromo-sn-glycero-3-phospho-rac-glycerol demonstrated a direct contact between the fluorescent [Zn2+-heme] group and the brominated acyl chain. These data constitute direct evidence for interaction between an acyl chain of a membrane phospholipid and the inside of the protein containing the heme moiety and provide direct evidence for the so-called extended-lipid anchorage of cyt c to phospholipid membranes. In this mechanism, one of the phospholipid acyl chains protrudes out of the membrane and intercalates into a hydrophobic channel in cyt c while the other chain remains in the bilayer. cytochrome c large unilamellar vesicle phosphatidylcholine phosphatidylglycerol 1-palmitoyl-2-(9,10)-dibromo-sn-glycero-3-phospho-rac-glycerol relative fluorescence intensity mole fraction of PG Zn-substituted cytochrome circular dichroism magnetic circular dichroism electron paramagnetic resonance 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleyl-sn-glycero-phosphoglycerol Cytochrome c (cytc)1 is a mitochondrial peripheral membrane protein functioning in the respiratory chain in the inner mitochondrial membrane. With its heme moiety switching between ferro and ferri forms, this water-soluble 13-kDa protein shuttles electrons from cyt c reductase to cyt c oxidase. (1Moore G.R. Pettigrew G.W. Cytochromes c: Evolutionary, Structural and Physicochemical Aspects. Springer-Verlag, Berlin1990Crossref Google Scholar). In addition to its role in mitochondrial respiration, a novel function has been discovered for cyt cin apoptosis (2Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4405) Google Scholar, 3Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4362) Google Scholar, 4Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4241) Google Scholar). In the early phase of apoptosis, cyt cis released from mitochondria. In the cytoplasm, cyt c forms a complex with a protein called Apaf-1 and caspase 9, the formation of this “apoptosome” complex leading to the activation of the cascade of proteases executing apoptosis in cells (5Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6157) Google Scholar). The apoptosis inducing form of cyt c has been suggested to be membrane-bound (6Jemmerson R. Liu J. Hausauer D. Lam K.P. Mondino A. Nelson R.D. Biochemistry. 1999; 38: 3599-3609Crossref PubMed Scopus (113) Google Scholar). An acidic phospholipid, cardiolipin, either by itself or complexed with cyt c oxidase, provides the membrane binding site for cyt c in mitochondria (7Vik S.B. Georgevich G. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1456-1460Crossref PubMed Scopus (183) Google Scholar, 8Speck S.H. Neu C.A. Swanson M.S. Margoliash E. FEBS Lett. 1983; 164: 379-382Crossref PubMed Scopus (28) Google Scholar). Cyt c bears a net positive charge of +8 and binds avidly to model membranes containing acidic phospholipids (9Rytömaa M. Mustonen P. Kinnunen P.K.J. J. Biol. Chem. 1992; 267: 22243-22248Abstract Full Text PDF PubMed Google Scholar). Yet, the interaction of cytc with acidic phospholipids involves more than electrostatic attraction. Two different types of interaction have been characterized for the membrane binding of cyt c and have been shown to be due to distinct sites, named the A-site and the C-site, respectively (10Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1995; 270: 3197-3202Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). More specifically, negative surface charge density of the liposomes, pH, and ionic strength together determine whether cytc is bound electrostatically via its A-site or by hydrogen bonding and hydrophobic interaction via its C-site (9Rytömaa M. Mustonen P. Kinnunen P.K.J. J. Biol. Chem. 1992; 267: 22243-22248Abstract Full Text PDF PubMed Google Scholar, 10Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1995; 270: 3197-3202Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The electrostatic A-site interaction can be reversed by ATP and does not involve pronounced changes in the protein conformation as revealed by circular dichroism measurements on native cyt c and fluorescence spectroscopy studies using a derivative of cytc, in which the heme iron has been substituted by zinc (11Tuominen E.K.J. Zhu K. Wallace C.J.A. Clark-Lewis I. Craig D.B. Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 2001; 276: 19356-19362Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Studies of the C-site lipid-bound cyt c have demonstrated this type of interaction not to be reversed by ATP (10Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1995; 270: 3197-3202Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Conformational studies revealed C-site binding to membranes to involve significant changes in the Soret region CD-spectrum thus suggesting changes in the heme environment and thus perturbations in the interior of the protein (11Tuominen E.K.J. Zhu K. Wallace C.J.A. Clark-Lewis I. Craig D.B. Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 2001; 276: 19356-19362Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). ATP is able to interact with the C-site membrane-bound cytc, leading to further changes in its conformation (11Tuominen E.K.J. Zhu K. Wallace C.J.A. Clark-Lewis I. Craig D.B. Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 2001; 276: 19356-19362Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The hydrophobic component of C-site-mediated interaction of cytc with an acidic phospholipid has been suggested to be due to the so-called extended lipid anchorage (10Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1995; 270: 3197-3202Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 12Kinnunen P.K.J. Kõiv A. Lehtonen J.Y.A. Rytömaa M. Mustonen P. Chem. Phys. Lipids. 1994; 73: 181-207Crossref PubMed Scopus (137) Google Scholar). In this mechanism one of the acyl chains of acidic phospholipid would be accommodated within a hydrophobic channel in cyt c, whereas the other chain of the glycerophospholipid remains in the lipid bilayer. In this study we provide further evidence for the extended lipid anchorage of cyt c binding to liposomes. The extended lipid anchorage has been proposed to represent a general mechanism for peripheral membrane protein-lipid interaction (12Kinnunen P.K.J. Kõiv A. Lehtonen J.Y.A. Rytömaa M. Mustonen P. Chem. Phys. Lipids. 1994; 73: 181-207Crossref PubMed Scopus (137) Google Scholar, 13Kinnunen P.K.J. Chem. Phys. Lipids. 1996; 81: 151-166Crossref Scopus (91) Google Scholar). Horse heart cyt c (type VI, oxidized form), POPG, POPC, and oleic acid were from Sigma Chemical Co. No impurities were detected in the above lipids upon thin layer chromatography on silicic acid using chloroform/methanol/water/ammonia (65:20:2:2, v/v) as the solvent system and examination of the plates for pyrene fluorescence or after iodine staining. All other reagents were of reagent grade from Sigma or Merck. Oleic acid and POPG were reacted with Br2 to produce 9,10-dibromostearic acid and the corresponding phospholipid derivate (14Dawidowicz E.A. Rothman J.E. Biochim. Biophys. Acta. 1976; 455: 621-630Crossref PubMed Scopus (34) Google Scholar). Briefly, Br2reacts with the double bond of oleic acid moiety by addition. Lipids (approximately 1 μmol) were dissolved into 1 ml of octane, and 5% Br2 (w/v) in this solvent was added in 2-μl aliquots until the solution began accumulating the color of molecular bromine. Solutions were immediately stored in −20 °C for at least 24 h. Unreacted bromine was removed by evaporation under reduced pressure, and by chromatography on a silicic acid column eluted with CHCl3:MeOH (7:3, v/v). The purity of the products was verified by thin layer chromatography in chloroform/methanol/water/ammonia (65:20:2:2, v/v) where a single band was evident upon iodine staining. Required amounts of cyt cwere weighed and dissolved in 20 mm Hepes, 0.1 mm EDTA, pH 7.0. To obtain reduced cyt c, excess solid sodium dithionite was added to the solution. Subsequently, the protein solutions were eluted through a disposable gel column (Sephadex PD-10, Amersham Biosciences, Inc., Uppsala, Sweden) to remove the inorganic reaction products and excess of the reducing agent. The redox state was verified by recording the absorption spectrum, and concentrations were determined from reduced solutions using an extinction coefficient of 106.100 m−1 cm at 410 nm (15Margoliash E. Frohwirt N. Biochem. J. 1959; 71: 570-572Crossref PubMed Scopus (681) Google Scholar). Substitution of Zn2+ for Fe2+ in the porphyrin of cyt c yields an intensely fluorescent derivative (16Vanderkooi J.M. Adar F. Erecinska M. Eur. J. Biochem. 1976; 64: 381-387Crossref PubMed Scopus (167) Google Scholar). This analog has been characterized in considerable detail and has been shown to closely resemble the parent protein in most qualities, thus representing a good model to study the conformation of cyt c (11Tuominen E.K.J. Zhu K. Wallace C.J.A. Clark-Lewis I. Craig D.B. Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 2001; 276: 19356-19362Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 17Anni H. Vanderkooi J.M. Mayne L. Biochemistry. 1995; 34: 5744-5753Crossref PubMed Scopus (73) Google Scholar). The [Zn2+-heme] cyt c derivative was prepared from horse cyt c according to Vanderkooi and Erecinska (18Vanderkooi J.M. Erecinska M. Eur. J. Biochem. 1975; 60: 199-207Crossref PubMed Scopus (106) Google Scholar). To obtain iron-free cyt c, porphyrin cyt c was first made and then subsequently treated with ZnCl2 to yield [Zn2+-heme] cyt c (16Vanderkooi J.M. Adar F. Erecinska M. Eur. J. Biochem. 1976; 64: 381-387Crossref PubMed Scopus (167) Google Scholar). Lipids were dissolved in chloroform. After mixing to obtain the desired compositions, the solvent was removed under a stream of nitrogen and the lipid residue subsequently maintained under reduced pressure for at least 2 h. The dry lipids were then hydrated in 20 mm Hepes, 0.1 mm EDTA, pH 7.0 or 4.0, at room temperature to yield a lipid concentration of 1 mm. To obtain unilamellar vesicles, the hydrated lipid dispersions were extruded with a LiposoFast pneumatic small volume homogenizer (Avestin, Ottawa, Canada). Samples were subjected to 19 passes through two polycarbonate filters (100-nm pore size, Nucleopore, Pleasanton, CA) installed in tandem (19MacDonald R.C. MacDonald R.I. Menco B.Ph. M. Takeshita K. Subbarao N.K. Hu L. Biochim. Biophys. Acta. 1991; 1061: 297-303Crossref PubMed Scopus (1366) Google Scholar). Subsequently, the liposome solution was divided into proper aliquots and diluted with the buffer to desired final lipid concentration. Steady-state absorbance spectra were recorded on a Cary 100 Bio UV-visible spectrophotometer (Varian, Walnut Creek, CA). Measurements were conducted in dual beam configuration at ambient temperature. Baseline was recorded for each experiment prior to the addition of the indicated amounts of cyt c into the measurement cuvette. Interactions of fatty acids and phospholipids with [Zn2+-heme] cyt c were assessed by fluorescence spectroscopy. These measurements were conducted with a PerkinElmer Life Sciences LS50B spectrofluorometer using 5- and 10-nm band passes for excitation at 412 nm and emission at 588 nm, respectively. Two milliliters of 0.7 μm[Zn2+-heme] cyt c in 20 mm Hepes, 0.1 mm EDTA, pH 7.0, were placed into a magnetically stirred four-window quartz cuvette in a holder thermostatted with a circulating water bath at 25 °C. Subsequently, the indicated lipids (fatty acid or liposomes) were added to yield the desired final concentrations and the fluorescence of the Zn2+-heme moiety was observed. Fatty acids were added in 1- to 5-μl aliquots in ethanol solution. Control experiments indicated no detectable changes in fluorescence intensities by this solvent (data not shown). Because of the low concentrations of both lipids and cyt c used minimal interference by the inner filter effect is expected. In keeping with a previous study (20Stewart J.M. Blakely J.A. Johnson M.D. Biochem. Cell Biol. 2000; 78: 675-681Crossref PubMed Google Scholar), a change in the absorption spectrum of reduced cyt c was detected upon interaction with oleic acid (Fig. 1). In brief, the intensity of the Soret band at 415 nm decreased by approximately 30%, concomitant with a shift to a lower wavelength, from 415 to 407 nm. The double peak characteristic for reduced cyt c disappeared, and the resulting spectrum with the bound fatty acid thus resembles that for oxidized cyt c. The above change due to fatty acid binding to cyt c was interpreted to indicate a shift of the heme iron to a high spin state (20Stewart J.M. Blakely J.A. Johnson M.D. Biochem. Cell Biol. 2000; 78: 675-681Crossref PubMed Google Scholar). Spectral changes have recently been reported for cyt c interacting with cardiolipin-containing liposomes (21Nantes I.L. Zucchi M.R. Nascimento O.R. Faljoni-Alario A. J. Biol. Chem. 2001; 276: 153-158Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), and based on EPR and magnetic CD measurements these authors suggested also that these spectral alterations result from spin-state changes. To investigate if the changes in the spectra for cyt cresult from an interaction of the protein with phospholipid acyl chains we studied the binding of cyt c to large unilamellar phospholipid vesicles, LUVs. cyt c was equilibrated with LUVs consisting of different lipid compositions and at excess phospholipid concentration (1:100 cyt c:phospholipid stoichiometry). These conditions were selected on the basis of our earlier fluorescence studies (9Rytömaa M. Mustonen P. Kinnunen P.K.J. J. Biol. Chem. 1992; 267: 22243-22248Abstract Full Text PDF PubMed Google Scholar, 11Tuominen E.K.J. Zhu K. Wallace C.J.A. Clark-Lewis I. Craig D.B. Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 2001; 276: 19356-19362Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The absorption spectra of cytc were then recorded. To avoid difficulties in the interpretation of the data, PG was used instead of cardiolipin in these experiments, similarly to our earlier studies (9Rytömaa M. Mustonen P. Kinnunen P.K.J. J. Biol. Chem. 1992; 267: 22243-22248Abstract Full Text PDF PubMed Google Scholar, 10Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1995; 270: 3197-3202Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 11Tuominen E.K.J. Zhu K. Wallace C.J.A. Clark-Lewis I. Craig D.B. Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 2001; 276: 19356-19362Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 22Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1994; 269: 1770-1774Abstract Full Text PDF PubMed Google Scholar). At low molar fractions (X PG < 0.5) of acidic phospholipid and neutral pH, cyt c interacts with membranes electrostatically via its so-called A-site (22Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1994; 269: 1770-1774Abstract Full Text PDF PubMed Google Scholar). Interestingly, the A-site binding does not cause detectable changes in the absorbance spectrum (Fig. 2). In contrast, with neat PG liposomes (X PG = 1.0), spectral changes similar to those induced by oleic acid were evident (Fig. 2). The extent of the spectral changes was dependent on the lipid composition. More specifically, with X PGincreasing from 0 to 0.5, no indications for an interaction between cytc and phospholipid acyl chains were seen. BetweenX PG = 0.5 and 0.6 an effect on the spectra became evident, with maximal change at X PGbetween 0.8 and 0.9. The dependence on X PG of the spectral change (Fig. 3) in reduced cyt c on X PG is identical to the switching from the A-site to C-site binding with increasing acidic phospholipid content (10Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1995; 270: 3197-3202Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). This dependence onX PG is also reflected in the kinetics of association of cyt c with liposomes (23Subramanian M. Jutila A. Kinnunen P.K.J. Biochemistry. 1998; 37: 394-402Crossref Scopus (43) Google Scholar). Binding to the C-site is attributed to the protonation of the acidic phospholipid headgroups in the membrane. More specifically, upon increasing headgroup protonation (i.e. decreasing degree of dissociation), cyt c's binding to liposomes shifts from an electrostatic interaction to a hydrophobicity-driven membrane association and has also been suggested to involve hydrogen bonding (10Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1995; 270: 3197-3202Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The C-site binding of cyt c cannot be reversed by anions such as ATP while this nucleotide readily dissociates A-site bound cyt c (10Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1995; 270: 3197-3202Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 22Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1994; 269: 1770-1774Abstract Full Text PDF PubMed Google Scholar). The above data strongly suggest that the C-site interaction with membrane phospholipids shares a common mechanism with the binding of fatty acids to cyt c. To investigate the fatty acid binding of cyt cin more detail, we employed a cyt c analog in which the heme iron is changed to Zn2+. The [Zn2+-heme] moiety is intrinsically fluorescent (18Vanderkooi J.M. Erecinska M. Eur. J. Biochem. 1975; 60: 199-207Crossref PubMed Scopus (106) Google Scholar) and thus provides an excellent tool for monitoring changes occurring in the heme environment (11Tuominen E.K.J. Zhu K. Wallace C.J.A. Clark-Lewis I. Craig D.B. Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 2001; 276: 19356-19362Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Bromine is a well known collisional quencher for fluorophores, its action necessitating a contact with the fluorophore. Accordingly, brominated lipids such as 9,10-dibromostearic acid and P(Br2)SPG can be used to investigate the binding of cytc to lipids, because possible direct interactions between the brominated acyl chain and the fluorescent [Zn2+-heme] moiety can be detected. To assess the extent of fluorescence quenching, we first measured changes caused by the corresponding non-brominated lipids. The addition of oleic acid induced pronounced and biphasic changes in the fluorescence of [Zn2+-heme] cyt c. More specifically, with increasing oleic acid concentrations up to 15 μm (corresponding to approximately 1:20 of cytc:fatty acid molar ratio) relative fluorescence intensity first decreased to approx. 80% of the initial value (Fig.4). However, increasing the oleic acid concentration further led to an increased RFI, to approximately 90% of its initial value. This effect saturated at 30 μm fatty acid. When the brominated derivative of oleic acid, 9,10-dibromostearic, acid was used, a significant quenching of the [Zn2+-heme] cyt c fluorescence was evident (Fig. 4) with RFI decreasing maximally by approximately 45% at 15–20 μm 9,10-dibromostearic acid (Fig. 4). Yet, also for 9,10-dibromostearic acid, the changes in fluorescence were biphasic, with a minor increase being evident upon increasing the concentration of the brominated fatty acid from approximately 25–30 μm. With neat POPG liposomes, the alterations in the [Zn2+-heme] cyt c fluorescence were similar to those induced by oleic acid. At low (5 μm) phospholipid concentrations an initial decrement in RFI of 20% was seen. Increasing phospholipid concentration further caused RFI to exceed the initial value, with the signal approaching its maximum at 30–40 μm phospholipid. In contrast, the brominated phospholipid P(Br2)SPG efficiently quenched the Zn2+-heme fluorescence, similarly to 9,10-dibromostearic acid. Emission intensity decreased with increasing phospholipid concentration up to 20 μm whereafter a slight increase in fluorescence was seen with further increase in [P(Br2)SPG] up to 40 μm (Fig. 5). Normalizing the fluorescence intensity relative to the emission intensity measured in the presence of POPG assesses the effect of collisional quenching by bromine. These data reveal pronounced quenching by P(Br2)SPG, which saturates at 20–30 μm phospholipid and correspondes to cyt c:PG molar ratio of 1:30–1:40 (Fig. 6). The efficient quenching by the brominated fatty acid moiety of the PG derivative provides direct evidence for its interaction with the [Zn2+-heme] moiety residing in the interior of cytc. Cyt c has been shown to bind free fatty acids (20Stewart J.M. Blakely J.A. Johnson M.D. Biochem. Cell Biol. 2000; 78: 675-681Crossref PubMed Google Scholar, 21Nantes I.L. Zucchi M.R. Nascimento O.R. Faljoni-Alario A. J. Biol. Chem. 2001; 276: 153-158Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar,24Hardesty B.A. Mitchell H.K. Arch. Biochem. Biophys. 1963; 100: 1-8Crossref PubMed Scopus (5) Google Scholar). This interaction causes changes in the visual, NMR, CD, and MCD spectra, which indicate conformational changes in the protein, including disruption of the S-Fe coordination between Met-80 and the heme iron. The EPR spectra further indicated that the interaction of cyt c with lipids promoted a change of the heme iron spin state (21Nantes I.L. Zucchi M.R. Nascimento O.R. Faljoni-Alario A. J. Biol. Chem. 2001; 276: 153-158Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Together, these findings reveal the binding of free fatty acids as well as acidic phospholipids to cyt c to directly affect the heme environment. Binding to acidic phospholipid-containing membranes results in significant changes in the Soret band CD spectrum of cyt c, along with alterations in the [Zn2+-heme] fluorescence lifetimes for the derivatized cyt c (11Tuominen E.K.J. Zhu K. Wallace C.J.A. Clark-Lewis I. Craig D.B. Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 2001; 276: 19356-19362Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). These observations demonstrating alterations in the heme environment only occur upon binding of cyt c to bilayers with high lipid contents of acidic phospholipid, with the mole fraction X for PG exceeding 0.5 (11Tuominen E.K.J. Zhu K. Wallace C.J.A. Clark-Lewis I. Craig D.B. Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 2001; 276: 19356-19362Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Importantly, although the above studies demonstrate an effect by fatty acids and acidic phospholipids on the environment of the heme moiety of cytc, they do not distinguish between two possibilities: either direct contact between the heme and lipids or these alterations could originate from changes produced in the conformation of cytc. In this study we compared the effects in cyt c induced by the free fatty acids and acidic phospholipids. Our present data provide further evidence that these changes in the spectra of cyt cdepend on the content of the acidic phospholipids in the liposomes. This dependence is identical to that determining the binding of cytc to phospholipids via either its A- or C-site (10Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1995; 270: 3197-3202Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). We have previously suggested that C-site binding involves the so-called extended lipid conformation (12Kinnunen P.K.J. Kõiv A. Lehtonen J.Y.A. Rytömaa M. Mustonen P. Chem. Phys. Lipids. 1994; 73: 181-207Crossref PubMed Scopus (137) Google Scholar), with one of the acyl chains of a phospholipid molecule protruding out of the membrane, and accommodated into a hydrophobic channel in the protein (10Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1995; 270: 3197-3202Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 22Rytömaa M. Kinnunen P.K.J. J. Biol. Chem. 1994; 269: 1770-1774Abstract Full Text PDF PubMed Google Scholar). In this mechanism, the other acyl chain of the phospholipid resides in the membrane. This mechanism thus results in hydrophobic anchoring of cyt c on the membrane surface, without penetration into the bilayer. The first report on the structure of cyt c resolved by x-ray crystallography revealed a hydrophobic channel from the surface to the heme crevice (25Dickerson R.E. Takano T. Eisenberg D. Kallai O.B. Samson L. Cooper A. Margoliash E. J. Biol. Chem. 1971; 246: 1511-1535Abstract Full Text PDF PubMed Google Scholar). The latter authors also suggested this channel to provide a putative binding site for a non-defined hydrophobic moiety. The opening of this channel is located at the surface of cytc between the side chains of Tyr-97 and Phe-10 (25Dickerson R.E. Takano T. Eisenberg D. Kallai O.B. Samson L. Cooper A. Margoliash E. J. Biol. Chem. 1971; 246: 1511-1535Abstract Full Text PDF PubMed Google Scholar). This opening lined by hydrophobic amino acids is evident also in the crystal structures published later (26Bushnell G.W. Louie G.V. Brayer G.D. J. Mol. Biol. 1990; 214: 585-595Crossref PubMed Scopus (957) Google Scholar). The experiments described here also demonstrate direct contact between brominated oleic acid as well as the brominated sn-2 acyl chain of P(Br2)SPG and the [Zn2+-heme] moiety of cyt c. The biphasic changes in the [Zn2+-heme] fluorescence, i.e. an initial decrement followed by a smooth yet significant increase, are of interest. It is possible that this behavior could reflect occupancy of the hydrophobic channel by acyl chains inserted into it from two different directions, the observed concentration differences being due to distinct and different binding affinities of the lipid headgroups and the binding sites on protein surface. To elucidate the underlying mechanisms(s) requires further studies. Importantly, in keeping with the earlier results by Stewart et al. (20Stewart J.M. Blakely J.A. Johnson M.D. Biochem. Cell Biol. 2000; 78: 675-681Crossref PubMed Google Scholar) these changes in the [Zn2+-heme] fluorescence are specific for lipids having a charged headgroup and zwitterionic detergents such as dodecylmaltoside and CHAPS were without effect. However, SDS produced changes similar to those due to oleic acid (data not shown). In retrospect the extended phospholipid anchorage also readily explains also the early observations on the interaction of cyt c with phospholipid monolayers (27Quinn P.J. Dawson R.M.C. Biochem. J. 1969; 113: 791-803Crossref PubMed Scopus (59) Google Scholar, 28Quinn P.J. Dawson R.M.C. Biochem. J. 1969; 115: 65-75Crossref PubMed Scopus (101) Google Scholar). More specifically, the latter authors concluded that two types of interaction could occur with cytc and phosphatidylethanolamine (27Quinn P.J. Dawson R.M.C. Biochem. J. 1969; 113: 791-803Crossref PubMed Scopus (59) Google Scholar). At low surface pressures, cyt c penetrated into the monolayer, whereas at higher surface pressures no penetration was observed. Yet, a considerable amount of cyt c was adsorbed at the interface. Interestingly, the adsorption of cyt c to membranes at high surface pressures was found not to be a true equilibrium process and could be reversed by increasing salt concentrations or by perfusing the subphase with fresh buffer. In the experiments on cyt cinteracting with cardiolipin an unexpected behavior of the surface pressure and potential was observed, again leading to binding, which was irreversible by increasing salt concentrations (28Quinn P.J. Dawson R.M.C. Biochem. J. 1969; 115: 65-75Crossref PubMed Scopus (101) Google Scholar). These authors concluded that the cyt c-cardiolipin complex undergoes a time-dependent conformational change caused by interaction with the fatty acid residues of the phospholipid. These effects comply with the extended anchorage involving phospholipid acyl chains. Pachence and Blasie (29Pachence J.M. Blasie J.K. Biophys. J. 1991; 59: 894-900Abstract Full Text PDF PubMed Scopus (15) Google Scholar) observed the average electron density of the hydrocarbon chain methylene group region of the surface phospholipid layer to decrease upon the binding of cyt c to Langmuir-Blodgett type multilayers containing phosphatidylserine. At the same time the average distance of the terminal methyl group and the polar headgroup was increased, suggesting a conformational change in the monolayer phospholipids. Interestingly, the fatty acid moieties of cardiolipin in isolated mitochondrial membranes were found to be protected from double-bond hydrogenation catalyzed by a palladium complex (30Schlame M. Horvath L. Vigh L. Biochem. J. 1990; 265: 79-85Crossref PubMed Scopus (58) Google Scholar). The presence of peripheral membrane proteins was found to be required for this selective protection of cardiolipins fatty acids, as compared with phosphatidylcholine fatty acids. The authors concluded that the hydrophobic moieties of the cardiolipin are critical requirements for the high protein affinity of this phospholipid (30Schlame M. Horvath L. Vigh L. Biochem. J. 1990; 265: 79-85Crossref PubMed Scopus (58) Google Scholar). Extended lipid anchorage would readily explain these results, because a fatty acid moiety residing in a hydrophobic cavity in a protein would be sterically protected from the catalyst. As has already been pointed out (13Kinnunen P.K.J. Chem. Phys. Lipids. 1996; 81: 151-166Crossref Scopus (91) Google Scholar) the extended lipid anchorage could represent a generic mechanism. A common requirement for proteins in this category should be the presence of a free fatty acid binding site. To this end, Kahana et al. have suggested (31Kahana E. Pinder J.C. Smith K.S. Gratzer W.B. Biochem. J. 1992; 282: 75-80Crossref PubMed Scopus (49) Google Scholar) that fatty acid binding is a general characteristic for peripheral membrane proteins. Regarding the structure of membrane phospholipids, the presence of a saturated acyl chain in the sn-1 and unsaturated acyl chain in the sn-2 positions is conspicuous (32Gennis R.B. Biomembranes: Molecular Structure and Function. Springer-Verlag, New York1989: 23-27Google Scholar). The adoption of the extended conformation only requires the rotation of the C2–C3 carbon bond of the phospholipid glycerol backbone (33Hauser H. Guyer W.G. Skrabal P.P. Sundell S. Biochemistry. 1980; 19: 366-373Crossref PubMed Scopus (120) Google Scholar, 34Hauser H. Pascher I. Sundell S. Biochemistry. 1988; 27: 9166-9174Crossref PubMed Scopus (96) Google Scholar). Accordingly, it would be thesn-2 chain, which would protrude out of the lipid bilayer. Obviously, the cis double bond readily allows for greater conformational flexibility. The above considerations are in keeping with the quenching of Trp-187 of annexin V by phospholipids bearing a fluorescence quenching nitroxyl moiety linked to acyl chain in thesn-2 position, although this was not observed for thesn-1 nitroxyl acyl chain (35Meers P. Mealy T. Biochemistry. 1994; 33: 5829-5837Crossref PubMed Scopus (54) Google Scholar). Likewise, long chain fatty acids in the sn-2 position had a greater effect on Trp fluorescence, when compared with shorter acyl chains. These authors concluded that specific hydrophobic interactions with thesn-2 acyl chain are a major determinant for the binding of annexin V to membranes (35Meers P. Mealy T. Biochemistry. 1994; 33: 5829-5837Crossref PubMed Scopus (54) Google Scholar). Finally, the lipid binding properties of milk protein α-lactalbumin closely resemble those of cyt c(36Carwthern K.M. Permyakov E. Berliner L.J. Protein Sci. 1996; 4: 1394-1405Crossref Scopus (53) Google Scholar). Moreover, lipid binding to α-lactalbumin has been suggested to induce the molten globule state, similarly to cyt c (37Dolgikh D.A. Gilmanshin R.I. Brazhnikov E.V. Bychkova V.E. Semisotnov G.V. Venjaminov S.Y. Ptitsyn O.B. FEBS Lett. 1981; 136: 311-315Crossref PubMed Scopus (582) Google Scholar,38Hamada D. Kidokoro A.-I. Fukada H. Takahashi K. Goto Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10325-10329Crossref PubMed Scopus (101) Google Scholar). This is particularly intriguing when taking into account the role of cyt c in apoptosis (2Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4405) Google Scholar, 3Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4362) Google Scholar, 4Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4241) Google Scholar) and the finding that it is the lipid-bound cyt c that is active in triggering the activation of caspases (6Jemmerson R. Liu J. Hausauer D. Lam K.P. Mondino A. Nelson R.D. Biochemistry. 1999; 38: 3599-3609Crossref PubMed Scopus (113) Google Scholar). Analogously, α-lactalbumin with bound oleic acid has been recently reported to induce apoptosis in cancer cells (39Svensson M. Håkansson A. Mossberg A.-K. Svanborg C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4221-4226Crossref PubMed Scopus (307) Google Scholar). In the light of the above findings, a thorough understanding of lipid-protein interactions could shed light on a number of central cellular processes, including apoptosis." @default.
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