Ground‐ and excited‐state proton transfer and antioxidant activity of 3‐hydroxyflavone in egg yolk phosphatidylcholine liposomes: absorption and fluorescence spectroscopic studies

Excited‐state intramolecular proton transfer (ESIPT) and dual luminescence behaviour of 3‐hydroxyflavone (3‐HF) have been utilized to monitor its binding to liposomal membranes prepared from egg yolk phosphatydilcholine (EYPC). Additionally, absorption spectrophotometric assay has been performed to evaluate the antioxidant activity of 3‐HF against lipid peroxidation in this membrane system. When 3‐HF molecules are partitioned into EYPC liposomes, a weak long‐wavelength absorption band with λ ～410 nm appears in addition to the principal absorption at ～λ = 345 nm. Selective excitation of the 410 nm band produces the characteristic emission (λ～460 nm) of the ground‐state anionic species, whereas excitation at the higher energy absorption band leads to dual emission with predominatly ESIPT tautomer fluorescence (λ = 528 nm). Both ESIPT tautomer and the anionic species exhibit fairly high fluorescence anisotropy (r) values (r = 0.122 and 0.180, respectively). Biexponential fluorescence decay kinetics are observed for the ESIPT tautomer as well as the ground‐state anionic forms, indicating heterogeneity in the microenvironments of the corresponding emitting species. Furthermore, we demonstrate that lipid peroxidation of EYPC liposomes is significantly inhibited upon 3‐HF binding, suggesting that 3‐HF can be potentially useful as an inhibitor of peroxidative damage of cell membranes. Copyright © 2008 John Wiley & Sons, Ltd.     

Changes in the fluorescence parameters of bound N-(1-pyrene) maleimide by lipid peroxidation of intestinal brush-border membranes

Using a fluorogenic thiol reagent, N-(1-pyrene)maleimide (NPM), we have examined of lipid peroxidation on the microenvironment around SH groups of the membrane proteins in porcine intestinal brush-border membrane vesicles. The lipid peroxidation of the membranes was performed with various concentrations of t-butylhydroperoxide (t-BuOOH) in the presence of 100 microM ascorbic acid and 10 microM Fe2+. Treatment of NPM-labeled membranes with these oxidizing agents resulted in a decrease of the fluorescence lifetime, suggesting modification of the environmental properties around the bound dye. Measurement of the steady-state fluorescence anisotropy of the labeled membranes indicated restriction of the motion of the bound dye by the lipid peroxidation membranes. This interpretation was further supported by an elevation of the transition temperature of the anisotropy, a decrease in the quenching rate constant of the fluorescence with acrylamide and a decrease in the SH reactivity of the membrane proteins for NPM by lipid peroxidation. Based on these results, the possibility of conformation changes in the vicinity of SH groups in the membrane proteins associated with lipid peroxidation has been discussed.     

Pyrene derivatives as markers of transbilayer effect of lipid peroxidation on neuronal membranes

Two different pyrene derivatives, namely 12-(1-pyrene)dodecanoic acid (P12-FA) and N-(12-(1-pyrene)dodecanoyl)-galactosylsphingosine I3-sulfate (P12-CS) have been used to follow lipid peroxidation both in model and natural membranes. The malondialdehyde (MDA) production in small unilamellar vesicles of dipalmitoylphosphatidylcholine/arachidonic acid (80:20, molar ratio), symmetrically labelled with both probes determined a progressive decrease of pyrene fluorescence due to an involvement of pyrene in the peroxidative reaction. Nervous membranes are particularly sensitive to lipid oxidation which differentially acts on the two layers of the membrane determining a greater rigidity of the exofacial one. Thus, we consider the possibility to asymmetrically introduce the pyrene ring, as P12-FA or P12-CS, in synaptosomes for monitoring lipid peroxidation in each layer of the membrane. The amount of the two probes incorporated in the membrane was 20 +/- 3 and 10 +/- 2 nmol/mg of protein for P12-FA and P12-CS, respectively. P12-FA was symmetrically distributed in the two layers, whereas 95% of P12-CS was incorporated in the exofacial layer of the membrane as determined by TNBS measurements. The decrease in fluorescence of synaptosome associated pyrene was, in the early stages of lipid peroxidation, greater for P12-CS than for P12-FA labelled membranes, indicating a greater susceptibility of the exofacial layer to iron-induced peroxidation.     

Lipid peroxidation in small and large phospholipid unilamellar vesicles induced by water-soluble free radical sources

The susceptibility of small and large egg yolk phosphatidylcholine unilamellar vesicles to Fe(2+)/histidine-Fe(3+)- and Fenton reagent (Fe(2+)-H(2)O(2))-induced lipid peroxidation was evaluated by measuring the formation of thiobarbituric acid reactive substances (TBARS). It has been found that surface curvature or phospholipid packing exerts significant effect on the oxidative susceptibility of the unsaturated lipid bilayers and the highly curved and loosely packed small unilamellar vesicles (SUVs) exhibit much less resistance to the oxidative stress induced by the water-soluble free radical sources. The presence of lipid hydroperoxides in sonicated vesicles was excluded as the cause for higher level of lipid peroxidation in the phospholipid SUVs. Instead, the experimental results can be explained by the difference in ability of the water-soluble oxidants to penetrate the two types of lipid membranes. This hypothesis is supported by data obtained from fluorescence lifetime and quenching studies.     

Interaction of flavonoids with red blood cell membrane lipids and proteins: antioxidant and antihemolytic effects

Plant flavonoids are emerging as potent therapeutic drugs effective against a wide range of free radical mediated diseases. Hence their interactions with cell membranes, which generally serve as targets for lipid peroxidation, are of enormous interest. Here we report in vitro studies, via absorption and fluorescence spectroscopy, on the effects of several flavonoids (namely fisetin, quercetin, chrysin, morin, and 3-hydroxyflavone, 3-HF) in goat RBC membranes. Owing to the presence of functionally relevant membrane protein components embedded in the lipid bilayer RBC ghosts provide a more realistic system for exploring drug actions in biomembranes than simpler membrane models like phosphatidylcholine liposomes used in our previous studies [e.g. B. Sengupta, A. Banerjee, P.K. Sengupta, FEBS Lett. 570 (2004) 77-81]. Here, we demonstrate that binding of the flavonoids to the RBC membranes significantly inhibits lipid peroxidation, and at the same time enhances their integrity against hypotonic lysis. Interestingly, the antioxidant and antihemolytic activities are found to be crucially dependent on the locations of the flavonoids in the membrane matrix as revealed by fluorescence studies. Furthermore, we observe that FRET (from membrane protein tryptophans to flavonoids) occurs with significant efficiency indicating that the flavonoid binding sites lie in close proximity to the tryptophan residues in the ghost membrane proteins.     

7-Hydroxycholestrol as a possible biomarker of cellular lipid peroxidation: Difference between cellular and plasma lipid peroxidation

Polyunsaturated fatty acids and their esters are known to be susceptible to free radical-mediated oxidation, whereas cholesterol is thought to be more resistant to oxidation. In fact, it has been observed that in the case of plasma lipid peroxidation, the amount of oxidation products of polyunsaturated fatty acids such as linoleic acid was higher than that of cholesterol. In contrast, during oxidative stress-induced cellular lipid peroxidation, oxidation products of cholesterol such as 7-hydroxycholesterol (7-OHCh) were detected in greater amounts than those of linoleates such as hydroxyoctadecadienoic acid (HODE). There are several forms of oxidation products of cholesterol and linoleates in vivo, namely, hydroperoxides, as well as the hydroxides of both the free and ester forms of cholesterol and linoleates. To evaluate these oxidation products, a method used to determine the lipid oxidation products after reduction and saponification was developed. With this method, several forms of oxidation products of cholesterol and linoleates are measured as total 7-OHCh (t7-OHCh) and total HODE (tHODE), respectively. During free radical-mediated lipid peroxidation in plasma, the amount of tHODE was 6.3-fold higher than that of t7-OHCh. In contrast, when Jurkat cells were exposed to free radicals, the increased amount of cellular t7-OHCh was 5.7-fold higher than that of tHODE. Higher levels of t7-OHCh than those of tHODE have also been observed in selenium-deficient Jurkat cells and glutamate-treated neuronal cells. These results suggest that, in contrast to plasma oxidation, cellular cholesterol is more susceptible to oxidation than cellular linoleates. Collectively, cholesterol oxidation products at the 7-position may be a biomarker of cellular lipid peroxidation.     

Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: Only the myristoylated form translocates into the lipid bilayer

Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.     

Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.     

The effect of ionic strength on the lipid peroxidation of porcine intestinal brush-border membrane vesicles

The effects of salt concentration gradient (inside to outside) on the lipid peroxidation of porcine intestinal brush-border membrane vesicles have been studied and several interesting features of the peroxidation have been elucidated. The addition of dithiothreitol and Fe2+ is far more effective in induction of the lipid peroxidation than any of the other metal ion species tested (Fe3+, Cu2+, Ni2+, Zn2+ and Cr3+). The peroxidation rate of the membrane vesicles induced by dithiothreitol plus Fe2+ was sensitive for the incubation temperature and was increased with increase of the temperature. Imposition of an inward salt concentration gradient on the membrane vesicles preloaded with 300 mM mannitol by addition of 100 mM chloride of K+, Na+, Li+, Rb+, NH4+ or choline to medium produces a very large reduction of the lipid peroxidation induced by dithiothreitol plus Fe2+. The membrane peroxidation is depressed more with the mannitol (300 mM)-preloaded vesicles than with the K2SO4 (100 mM)-preloaded vesicles when they are incubated in medium containing 20-100 mM of K2SO4. Addition of membrane-permeant anions such as SCN- and I-, but not addition of NO3-, to incubation medium has been found to decrease markedly the lipid peroxidation of the mannitol-preloaded vesicles. From these results it is suggested that the lipid peroxidation of the brush-border membranes by addition of dithiothreitol plus Fe2+ is sensitively changed with change in ionic strength.     

Changes in photosynthetic electron transfer and state transitions in an herbicide-resistant D1 mutant from soybean cell cultures

Anomalies in photosynthetic activity of the soybean cell line STR7, carrying a single mutation (S268P) in the chloroplastic gene psbA that codes for the D1 protein of the photosystem II, have been examined using different spectroscopic techniques. Thermoluminescence emission experiments have shown important differences between STR7 mutant and wild type cells. The afterglow band induced by both white light flashes and far-red continuous illumination was downshifted by about 4 °C and the Q band was upshifted by 5 °C. High temperature thermoluminescence measurements suggested a higher level of lipid peroxidation in mutant thylakoid membranes. In addition, the reduction rate of P700⁺ was significantly accelerated in STR7 suggesting that the mutation led to an activation of the photosystem I cyclic electron flow. Modulated fluorescence measurements performed at room temperature as well as fluorescence emission spectra at 77 K revealed that the STR7 mutant is defective in state transitions. Here, we discuss the hypothesis that activation of the cyclic electron flow in STR7 cells may be a mechanism to compensate the reduced activity of photosystem II caused by the mutation. We also propose that the impaired state transitions in the STR7 cells may be due to alterations in thylakoid membrane properties induced by a low content of unsaturated lipids.     

Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes

The liver is responsible for the clearance and metabolism of unconjugated bilirubin, the hydrophobic end-product of heme catabolism. Although several putative bilirubin transporters have been described, it has been alternatively proposed that bilirubin enters the hepatocyte by passive diffusion through the plasma membrane. In order to elucidate the mechanism of bilirubin uptake, we measured the rate of bilirubin transmembrane diffusion (flip-flop) using stopped-flow fluorescence techniques. Unconjugated bilirubin rapidly diffuses through model phosphatidylcholine vesicles, with a first-order rate constant of 5.3 s-1 (t(1)/(2) = 130 ms). The flip-flop rate is independent of membrane cholesterol content, phospholipid acyl saturation, and lipid packing, consistent with thermodynamic analyses demonstrating minimal steric constraint to bilirubin transmembrane diffusion. The coincident decrease in pH of the entrapped vesicle volume supports a mechanism whereby the bilirubin molecule crosses the lipid bilayer as the uncharged diacid. Transport of bilirubin by native rat hepatocyte membranes exhibits kinetics comparable with that in model vesicles, suggesting that unconjugated bilirubin crosses cellular membranes by passive diffusion through the hydrophobic lipid core. In contrast, there is no demonstrable flip-flop of bilirubin diglucuronide or bilirubin ditaurate in phospholipid vesicles, yet these compounds rapidly traverse isolated rat hepatocyte membranes, confirming the presence of a facilitated uptake system(s) for hydrophilic bilirubin conjugates.     

Protein-lipid interactions in antipodal plasma membranes of rat colonocytes

The isolation of apical membranes from rat proximal colonic epithelial cells is described. Differential centrifugation yielded a ‘crude’ membrane fraction which was further purified using sucrose density centrifugation. The final membrane fraction was enriched 20–28-fold over homogenate in alkaline phosphatase and cysteine-sensitive alkaline phosphatase specific activities. Lipid-protein interactions and lipid dynamics examined in apical and basolateral membranes prepared from colonocytes demonstrated: (1) apical membrane, as assessed by steady-state fluorescence polarization studies have a low lipid fluidity; (2) colonic basolateral membranes possess a greater lipid fluidity than apical membranes; (3) compositional differences in these antipodal membranes appear to explain these differences in lipid fluidity; (4) fluorescence polarization studies using diphenylhexatriene detect a thermotropic transition at 21–23°C in apical membranes and liposomes prepared from lipid extracts of these membranes; (5) alkaline phosphatase and l-cysteine-sensitive alkaline phosphatase activities appear to be functionally dependent on the physical state of the apical membrane's lipid.     

HDLs induce raft domain vanishing in heterogeneous giant vesicles

Cholesterol efflux from the plasma membrane to HDLs is essential for cell cholesterol homeostasis. Recently, cholesterol-enriched ordered membrane domains, i.e. lipid rafts have been proposed to play an important role in this process. Here we introduce a new method to investigate the role of HDL interactions with the raft lipid phase and to directly visualize the effects of HDL-induced cholesterol efflux on rafts in model membranes. Addition of HDLs to giant lipid vesicles containing raft-type domains promoted decrease in size and disappearance of such domains as visualized by fluorescence microscopy. This was interpreted as resulting from cholesterol efflux from the vesicles to the HDLs. The raft vanishing rate was directly related to the HDL concentration. Evidence for a direct interaction of HDLs with the membrane was obtained by observing mutual adhesion of vesicles. It is suggested that the present method can be used to study the selective role of the bilayer lipid phase (raft and non-raft) in cholesterol efflux and membrane-HDL interaction and their underlying mechanisms. Such mechanisms may contribute to cholesterol efflux in vivo.     

Aluminum-induced lipid phase separation and membrane fusion does not require the presence of negatively charged phospholipids

The interaction of Aluminum with phosphatidyl serine lipid vesicles containing variable amounts of phosphatidyl ethanolamine, phosphatidyl choline and cholesterol has been studied by lipid phase separation monitored by fluorescence quenching. The interaction of Al3+ with neutral phospholipid membranes has also been investigated. Maximal lipid phase separation can be demonstrated in mixed phosphatidyl ethanolamine-cholesterol vesicles when using concentrations of aluminum between 87.5 and 125 microM. Millimolar concentrations of Ca2+, Mn2+, Cd2+ and Zn2+ were without any effect. Aluminum also induced fusion of phospholipid membranes monitored by resonance energy transfer between N-(7-nitro-2,1,3, benzoxadiazol-4 yl) phosphatidyl ethanolamine and N-(lissamine Rhodamine B-sulfonyl) phosphatidyl ethanolamine, either when containing low amounts of phosphatidyl serine (12.5%) or without any negatively charged phospholipid. Aluminum-induced fusion of liposomes was also monitored by the fluorescence of the terbium-dipicolinic acid complex (Tb-DPA3-) formed during fusion of vesicles containing either Tb-(citrate)6- complex or sodium salt of dipicolinic acid.     

Interactions Of Vitamin E With Free Radicals And Membranes

-Tocopherol performs an antioxidant role in biological membranes by acting as a one-electron reductant. In micellar solutions it has been observed by pulse radiolysis that the micellar charge has a pronounced effect on the rate constant for repair of organic free radicals by -tocopherol. The interactions between -tocopherol and model bilayer lipid membranes have been studied by fluorescence spectroscopy. Quencing of -tocopherol fluorescence by acrylamide and some n-doxyl stearates shows the transverse distribution of -tocopherol in membranes to be affected by the physical state of the membrane lipids and by the salt concentration in the aqueous phase. Time-resolved fluorescence depolarization measurements, with a diphenylhexatriene-phospholipid conjugate as probe. demonstrate an increase in the bilayer order parameter on incorporation of -tocopherol into a membrane     

Fluorescence anisotropy measurements of lipid order in plasma membranes and lipid rafts from RBL-2H3 mast cells

Specialized plasma membrane domains known as lipid rafts participate in signal transduction and other cellular processes, and their liquid ordered (L(o)) phase appears to be important for their function. To quantify ordered lipids in biological membranes, we investigated steady-state fluorescence anisotropy of two lipid probes, 2-[3-(diphenylhexatrienyl)propanoyl]-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). We show using model membranes with varying amounts of cholesterol that steady-state fluorescence anisotropy is a sensitive measure of cholesterol-dependent ordering. The results suggest that DPH-PC is a more sensitive probe than NBD-PE. In the presence of cholesterol, ordering also depends on the degree of saturation of the phospholipid acyl chains. Using DPH-PC, we find that the plasma membrane of RBL-2H3 mast cells is substantially ordered, roughly 40%, as determined by comparison with anisotropy values for model membranes entirely in a liquid ordered (L(o)) phase and in a liquid disordered (L(alpha)) phase. This result is consistent with the finding that approximately 30% of plasma membrane phospholipids are insoluble in 0.5% Triton X-100. Furthermore, detergent-resistant membranes isolated by sucrose gradient fractionation of Triton X-100 cell lysates are more ordered than plasma membrane vesicles, suggesting that they represent a more ordered subset of the plasma membrane. Treatment of plasma membrane vesicles with methyl-beta-cyclodextrin resulting in 75% cholesterol depletion leads to commensurate decreases in lipid order as measured by anisotropy of DPH-PC and NBD-PE. These results demonstrate that steady-state fluorescence anisotropy of DPH-PC is a useful way to measure the amount of lipid order in biological membranes.     

Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone

We characterized the recently introduced environment-sensitive fluorescent membrane probe based on 3-hydroxyflavone, F2N12S, in model lipid membranes displaying liquid disordered (Ld) phase, liquid ordered (Lo) phase, or their coexistence. Steady-state fluorescence studies in large unilamellar vesicles show that the probe dual emission drastically changes with the lipid bilayer phase, which can be correlated with the difference in their hydration. Using two-photon excitation microscopy on giant unilamellar vesicles, the F2N12S probe was found to bind both Ld and Lo phases, allowing visualization of the individual phases from the fluorescence intensity ratio of its two emission bands. By using a linearly polarized excitation light, a strong photoselection was observed for F2N12S in the Lo phase, indicating that its fluorophore is nearly parallel to the lipid chains of the bilayer. In contrast, the absence of the photoselection with the Ld phase indicated no predominant orientation of the probe in the Ld phase. Comparison of the present results with those reported previously for F2N12S in living cells suggests a high content of the Lo phase in the outer leaflet of the cell plasma membranes. Taking into account the high selectivity of F2N12S for the cell plasma membranes and its suitability for both single- and two-photon excitation, applications of this probe to study membrane lateral heterogeneity in biological membranes are foreseen.     

Organization and dynamics of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: a fluorescence approach

Lipids that are labeled with the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group are widely used as fluorescent analogues of native lipids in biological and model membranes to monitor a variety of processes. NBD-labeled lipids have previously been used to monitor the organization and dynamics of molecular assemblies such as membranes, micelles and reverse micelles utilizing the wavelength-selective fluorescence approach. In this paper, we have characterized the organization and dynamics of various NBD-labeled lipids using red edge excitation shift (REES) and other fluorescence approaches which include analysis of membrane penetration depths of the NBD group using the parallax method. We show here that the environment and location experienced by the NBD group of the NBD-labeled lipids could depend on the ionization state of the lipid. This could have potentially important implications in future studies involving NBD-labeled lipids as tracers in a cellular context.     

The Interaction of Equine Lysozyme:Oleic Acid Complexes with Lipid Membranes Suggests a Cargo Off-Loading Mechanism

The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to α-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of α-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOAs) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appear to be central to its biological action, similar to human α-lactalbumin made lethal to tumor cells. Here, we describe the interaction of ELOA with phospholipid membranes. Confocal scanning laser microscopy shows that ELOA, but not native EL, accumulates on the surface of giant unilamellar vesicles, without inducing significant membrane permeability. Quartz crystal microbalance with dissipation data indicated an essentially non-disruptive binding of ELOA to supported lipid bilayers, leading to formation of highly dissipative and “soft” lipid membrane; at higher concentrations of ELOA, the lipid membrane desorbs from the surface probably as bilayer sheets of vesicles. This membrane rearrangement occurred to a similar extent when free oleic acid (OA) was added, but not when free OA was removed from ELOA by prior incubation with bovine serum albumin, emphasizing the role of OA in this process. NMR data indicated an equilibrium between free and bound OA, which shifts towards free OA as ELOA is progressively diluted, indicating that OA is relatively loosely bound. Activity measurements together with fluorescence spectroscopy and circular dichroism suggested a conversion of ELOA towards a more native-like state on interaction with lipid membranes, although complete refolding was not observed. Altogether, these results suggest that ELOA may act as an OA carrier and facilitate OA transfer to the membrane. ELOA's properties illustrate that protein folding variants may possess specific functional properties distinct from the native protein.     

Photoaffinity labelling of submitochondrial membranes with the 3-azido analogue of 9-amino-3-chloro-7-methoxyacridine

9-Amino-3-azido-7-methoxyacridine has been synthesized and shown to be a suitable photoaffinity probe for the site(s) of interaction of 9-amino-3-chloro-7-methoxyacridine with submitochondrial membranes. Both the excitation and emission spectra of the azido analogue covalently bound to membranes in the energized state display distinctive differences from the spectra of labelled, non-energized membranes (i.e., in the absence of oxidizable substrate, or its presence when uncoupler (FCCP) is also present during photolysis). Enzymatic analyses indicate that the probe interacts with the ATPase and the respiratory chain enzymes; energization appears to afford some protection against inactivation. Electrophoresis of the labelled membranes and isolation of their lipid and protein components indicate that the spectral differences are attributable to differing interactions with the lipid components of energized, relative to non-energized, membranes. Similar results have been obtained with the 3-azido analogue of quinacrine (Mueller, D.M., Hudson, R.A. and Lee, C.P. (1982) Biochemistry 21, 1445-1453), which differs significantly, however, in the extent of its interactions with the enzymes of the respiratory chain and the ATPase. These results indicate that the energy-linked fluorescence responses of 9-aminoacridines with submitochondrial membranes arise from direct interactions with membrane components and may involve redistribution of the probe molecules and/or alteration of their microenvironments upon energization.