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BBA - Biomembranes (v.1758, #1)

Editorial Board (pp. ii).

Acknowledgment (pp. 1-3).

Cholesterol affects spectrin–phospholipid interactions in a manner different from changes resulting from alterations in membrane fluidity due to fatty acyl chain composition by Witold Diakowski; �?ukasz Ozimek; Ewa Bielska; Sylwia Bem; Marek Langner; Aleksander F. Sikorski (pp. 4-12).

We previously showed that erythrocyte and brain spectrins bind phospholipid vesicles and monolayers prepared from phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (Review: A.F. Sikorski, B. Hanus-Lorenz, A. Jezierski, A. R. Dluzewski, Interaction of membrane skeletal proteins with membrane lipid domain, Acta Biochim. Polon. 47 (2000) 565). Here, we show how changes in the fluidity of the phospholipid monolayer affect spectrin–phospholipid interaction. The presence of up to 10%–20% cholesterol in the PE/PC monolayer facilitates the penetration of the monolayer by both types of spectrin. For monolayers constructed from mixtures of PI/PC and cholesterol, the effect of spectrins was characterised by the presence of two maxima (at 5 and 30% cholesterol) of surface pressure for erythroid spectrin, and a single maximum (at 20% cholesterol) for brain spectrin. The binding assay results indicated a small but easily detectable decrease in the affinity of erythrocyte spectrin for FAT-liposomes prepared from a PE/PC mixture containing cholesterol, and a 2- to 5-fold increase in maximal binding capacity ( B_max) depending on the cholesterol content. On the other hand, the results from experiments with a monolayer constructed from homogenous synthetic phospholipids indicated an increase in Δ π change with the increase in the fatty acyl chain length of the phospholipids used to prepare the monolayer. This was confirmed by the results of a pelleting experiment. Adding spectrins into the subphase of raft-like monolayers constructed from DOPC, SM and cholesterol (1/1/1) induced an increase in surface pressure. The Δ π change values were, however, much smaller than those observed in the case of a natural PE/PC (6/4) monolayer. An increased binding capacity for spectrins of liposomes prepared from a “raft-like�? mixture of lipids could also be concluded from the pelleting assay. In conclusion, we suggest that the effect of membrane lipid fluidity on spectrin–phospholipid interactions is not simple but depends on how it is regulated, i.e., by cholesterol content or by the chemical structure of the membrane lipids.

Keywords: Abbreviations; DTT; dithiothreitol; EGTA; ethylene glycol bis-(β-aminoethyl ether); N; ,; N; ,; N; ′,; N; ′-tetraacetic acid; DMSO; dimethyl sulfoxide; DMPE; 1,2-dimyristoyl-phosphatidylethanolamine; DPPE; 1,2-dipalmitoyl-phosphatidylethanolamine; DSPE; 1,2-distearoyl-phosphatidylethanolamine; DMPC; 1,2-dimyristoyl-phosphatidylcholine; DPPC; 1,2-dipalmitoyl-phosphatidylcholine; DSPC; 1,2-distearoyl-phosphatidylcholine; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PI; phosphatidylinositol; SM; sphingomyelin; DOPC; 1,2-dioleoyl-phosphatidylcholine; DOPE; 1,2-dioleoyl-phosphatidylethanolamineSpectrin; Erythroid and non-erythroid spectrin; Membrane skeleton; Membrane fluidity; Lipid raft; Cholesterol

Functional and molecular characterization of adenosine transport at the rat inner blood–retinal barrier by Katsuhiko Nagase; Masatoshi Tomi; Masanori Tachikawa; Ken-ichi Hosoya (pp. 13-19).

The purpose of the present study was to characterize the adenosine transport system(s) at the inner blood–retinal barrier (inner BRB). A conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2), used as an in vitro model of the inner BRB, expresses equilibrative nucleoside transporter 1 (ENT1), ENT2, concentrative nucleoside transporter 2 (CNT2), and CNT3 mRNAs. TR-iBRB2 cells exhibited an Na⁺-independent and concentration-dependent [³H]adenosine uptake with a Michaelis–Menten constant of 28.5 μM and a maximum uptake rate of 814 pmol/(min mg protein). [³H]Adenosine uptake by TR-iBRB2 cells was strongly inhibited by 2 mM adenosine, inosine, uridine, and thymidine. On the other hand, this process was not inhibited by 100 nM nitrobenzylmercaptopurine riboside and dipyridamole. These uptake studies suggest that ENT2 is involved in [³H]adenosine uptake by TR-iBRB2 cells. Quantitative real-time PCR revealed that the expression of ENT2 mRNA is 5.5-fold greater than that of ENT1 mRNA. An in vivo study suggested that [³H]adenosine is transported from the blood to the retina and significantly inhibited by adenosine and thymidine. The results of this study show that ENT2 most likely mediates adenosine transport at the inner BRB and is expected to play an important role in regulating the adenosine concentration in the retina.

Keywords: Abbreviations; BRB; blood–retinal barrier; TR-iBRB2; conditionally immortalized rat retinal capillary endothelial cell line; NBMPR; nitrobenzylmercaptopurine riboside; ENT; equilibrative nucleoside transporter; CNT; concentrative nucleoside transporter; ECF; extracellular fluid; V; _d; apparent retina-to-plasma concentration ratio; R; _B; apparent blood-to-plasma concentration ratio; K; _{in, retina}; apparent retinal uptake clearance of [; ³; H]adenosine; RUI; retinal uptake index; BBB; blood–brain barrierBlood–retinal barrier; Adenosine; Nucleoside transporter

Tresylated PEG-sterols for coupling of proteins to preformed plain or PEGylated liposomes by Thomas Steenpaß; Andreas Lung; Rolf Schubert (pp. 20-28).

A simple and inexpensive method for functionalization of preformed liposomes is presented. Soy sterol–PEG₁₃₀₀ ethers are activated by tresylation at the end of the PEG chain. Coupling of bovine serum albumin as an amino group containing model ligand to the activated lipids can be performed at pH 8.4 with high efficiency. At room temperature, the mixture of sterol–PEG and sterol–PEG–protein inserts rapidly into the outer liposome monolayer with high efficiency (>100 μg protein/μmol total lipid). This method of post-functionalization is shown to be effective with fluid or rigid and plain or pre-PEGylated liposomes (EPC/Chol, 7:3; HSPC/Chol 2:1, and EPC/Chol/MPEG₂₀₀₀–DSPE 2:1:0.16 molar ratios). The release of entrapped calcein upon the insertion of 7.5 mol% of the functionalized sterols is lower than 4%. Incubation of post-functionalized liposomes with serum for 20 h at 37 °C shows stable protein attachment at the liposome surface.

Keywords: Abbreviations; BSA; bovine serum albumin; Chol; cholesterol; DSPE; 1,2-distearoyl-; sn; -glycero-3-phosphoethanolamine; EPC; egg phosphatidylcholine; HBS; HEPES-buffered saline; HSPC; hydrogenated soy phosphatidylcholine; MAL-PEG–DSPE; 1,2-distearoyl-; sn; -glycero-3-phosphoethanolamine-N-[maleimido-poly(ethyleneglycol)]; MPEG; ₂₀₀₀; –DSPE; 1,2-distearoyl-; sn; -glycero-3-phosphoethanolamine-N-[methoxy-poly(ethyleneglycol)-2000]; PCS; photon correlation spectroscopy; PEG; poly(ethylene glycol); sterol–PEG1300; soy sterol-poly(ethyleneglycol)-1300-ether; TEA; triethylamine; THF; tetrahydrofuran; tresylchloride; 2,2,2-trifluoroethanesulfonylchloride; TL; total lipid; TLC; thin layer chromatographyLiposome; Sterical stabilization; Soy sterol-poly(ethyleneglycol); Tresylation; Protein coupling; Specific targeting

Transient permeability induced by alkyl derivatives of amphotericin B in lipid membranes by Vafa Ibragimova; Irada Alieva; Khalil Kasumov; Vitaly Khutorsky (pp. 29-37).

Individual ionic channels were shown to be formed in the brain cholesterol containing phospholipid membranes by two-sided addition of the amphotericin B alkyl derivatives. At concentrations between 10⁻⁸ and 10⁻⁷ M, the resulting conductance appeared to be transient. Existence of different antibiotic assemblies was justified by the kinetic analysis of the membrane conductance decline following the antibiotic washing out. In order to account for the transient characteristics of the induced conductance, it was proposed that the antibiotic oligomers incorporate into the membrane from the aqueous phase, form channels aggregating with cholesterol, and then dissociate in the bilayer into non-active degraded oligomeric or monomeric forms.

Keywords: Amphotericin B; Alkyl derivatives of amphotericin B; Transient permeability in membrane; The kinetic of the membrane conductance; Washing out the antibiotic; Dissociate; Non active monomeric form

Spectroscopic studies of amphotericin B solubilized in nanoscale bilayer membranes by Peter L. Hargreaves; Thanh-Son Nguyen; Robert O. Ryan (pp. 38-44).

Nanodisks (ND) are discrete nanometer scale phospholipid bilayers whose perimeter is circumscribed by amphipathic apolipoproteins. The membranous environment of ND serves as a matrix for solubilizing the polyene antibiotic amphotericin B (AMB). The spectral properties of AMB in ND are dependent upon AMB concentration. Whereas AMB-ND prepared at a concentration of 2.5 mg AMB per 10 mg phospholipid are consistent with AMB self association in the ND membrane environment, AMB-ND prepared at 0.25 or 0.025 mg AMB per 10 mg phospholipid give rise to spectra reminiscent of AMB in organic solvent. Incubation of ND prepared at a phospholipid/AMB ratio of 400:1 (w/w) at 37 °C for 1 h induced a shift in absorbance and near UV circular dichroism spectra consistent with antibiotic self-association. The kinetics of this spectral transition were investigated as a function of incubation temperature. While no change in A₃₈₈ nm occurred in incubations at 20 °C, a time-dependent decrease in A₃₈₈ nm was observed at 25, 30 and 37 °C. Inclusion of ergosterol in the ND membrane attenuated temperature-induced AMB spectral changes. In Saccharomyces cerevisiae growth inhibition assays, ND containing self associated AMB were somewhat less effective than ND possessing a greater proportion of monomeric AMB. On the other hand, inclusion of ergosterol or cholesterol in the ND particle did not alter the growth inhibition properties of AMB-ND. The miniature membrane environment of ND provides a novel milieu for solubilization and characterization of lipophilic biomolecules.

Keywords: Abbreviations; AMB; amphotericin B; ND; nanodisk; DMPC; dimyristoylphosphatidylcholine; DMPG; dimyristoylphosphatidylglycerol; PBS; phosphate buffered saline; DMSO; dimethylsulfoxide; PAGE; polyacrylamide gel electrophoresis; CD; circular dichroism; apo; apolipoproteinAmphotericin B; Phospholipid; Apolipoprotein; Spectroscopy; Nanodisk

Rabbit small intestine does not contain an annexin II/caveolin 1 complex as a target for 2-azetidinone cholesterol absorption inhibitors by Werner Kramer; Daniel Corsiero; Frank Girbig; Gerhard Jähne (pp. 45-54).

Intestinal cholesterol absorption is specifically inhibited by the 2-azetidinone cholesterol absorption inhibitor ezetimibe. Photoreactive ezetimibe analogues specifically label a 145-kDa protein in the brush border membrane of enterocytes from rabbit small intestine identified as aminopeptidase N (CD13). In zebrafish and mouse small intestinal cytosol, a heterocomplex of M_r 52 kDa between annexin II and caveolin 1 was suggested as a target of ezetimibe. In contrast, in the cytosol and brush border membrane vesicles (BBMV) from rabbit small intestine of control animals or rabbits treated with the nonabsorbable cholesterol absorption inhibitor AVE 5530, both annexin II and caveolin 1 were exclusively present as monomers without any heterocomplex formation. Upon immunoprecipitation with annexin II a 52-kDa band was observed after immunostaining with annexin II antibodies, whereas no staining of a 52-kDa band occurred with anti-caveolin 1 antibodies. Vice versa, a 52-kDa band obtained by immunoprecipitation with caveolin 1 antibodies did not stain with annexin II-antibodies. The intensity of the 52-kDa band was dependent on the amount of antibody and was also observed with anti-actin or anti-APN antibodies suggesting that the 52-kDa band is a biochemical artefact. After incubation of cytosol or BBMV with radioactively labelled ezetimibe analogues, no significant amounts of the ezetimibe analogues could be detected in the immunoprecipitate with caveolin-1 or annexin II antibodies. Photoaffinity labelling of rabbit small intestinal BBMV with ezetimibe analogues did not result in labelling of proteins being immunoreactive with annexin II, caveolin 1 or a 52-kDa heterocomplex. These findings indicate that the rabbit small intestine does not contain an annexin II/caveolin 1 heterocomplex as a target for ezetimibe.

Keywords: Abbreviations; APN; Aminopeptidase N; BBMV; Brush border membrane vesicles; DTT; Dithiothreitol; PBS; Phosphate buffered salineCholesterol absorption; Ezetimibe; Annexin II/caveolin1; Photoaffinity labelling; Brush border membrane (rabbit)

Therapeutically optimized rates of drug release can be achieved by varying the drug-to-lipid ratio in liposomal vincristine formulations by Michael J.W. Johnston; Sean C. Semple; Sandra K. Klimuk; Katarina Edwards; Merete L. Eisenhardt; Esther C. Leng; Göran Karlsson; Daniel Yanko; Pieter R. Cullis (pp. 55-64).

The anti-tumor efficacy of liposomal formulations of cell cycle dependent anticancer drugs is critically dependent on the rates at which the drugs are released from the liposomes. Previous work on liposomal formulations of vincristine have shown increasing efficacy for formulations with progressively slower release rates. Recent work has also shown that liposomal formulations of vincristine with higher drug-to-lipid ( D/ L) ratios exhibit reduced release rates. In this work, the effects of very high D/ L ratios on vincristine release rates are investigated, and the antitumor efficacy of these formulations characterized in human xenograft tumor models. It is shown that the half-times ( T_1/2) for vincristine release from egg sphingomyelin/cholesterol liposomes in vivo can be adjusted from T_1/2 = 6.1 h for a formulation with a D/ L of 0.025 (wt/wt) to T_1/2 = 117 h (extrapolated) for a formulation with a D/ L ratio of 0.6 (wt/wt). The increase in drug retention at the higher D/ L ratios appears to be related to the presence of drug precipitates in the liposomes. Variations in the D/ L ratio did not affect the circulation lifetimes of the liposomal vincristine formulations. The relationship between drug release rates and anti-tumor efficacy was evaluated using a MX-1 human mammary tumor model. It was found that the antitumor activity of the liposomal vincristine formulations increased as D/ L ratio increased from 0.025 to 0.1 (wt/wt) ( T_1/2 = 6.1–15.6 h respectively) but decreased at higher D/ L ratios ( D/ L = 0.6, wt/wt) ( T_1/2 = 117 h). Free vincristine exhibited the lowest activity of all formulations examined. These results demonstrate that varying the D/ L ratio provides a powerful method for regulating drug release and allows the generation of liposomal formulations of vincristine with therapeutically optimized drug release rates.

Keywords: Abbreviations; SM; Egg sphingomyelin; [; ³; H]-CHE; [; ³; H]-cholesterylhexadecyl ether; MLV; multilamellar vesicles; LUV; large unilamellar vesicles; FBS; fetal bovine serum; D; /; L; ratio; drug-to-lipid ratioLiposome; Vincristine; Regulated drug release; Efficacy; Drug precipitation

Effects of trehalose on the phase behavior of DPPC–cholesterol unilamellar vesicles by Satoshi Ohtake; Carolina Schebor; Juan J. de Pablo (pp. 65-73).

A systematic study is presented of the effects of trehalose on the physical properties of extruded DPPC–cholesterol unilamellar vesicles. Particular emphasis is placed on examining how the interactions present in the hydrated state translate into those in the dehydrated state. Observations from HSDSC and DSC are used to examine the phase behavior of hydrated and dehydrated vesicles, respectively. The concentration of trehalose inside and outside the vesicles is manipulated, and is shown to affect the relative stability of the membranes. Our results show for the first time that a combination of high inner and low outer trehalose concentration is able to decrease the gel-to-liquid crystalline phase temperature ( T_m), while any other combination will not. Upon dehydration, the T_m of all lipid mixtures increases. The extent of the increase depends on the trehalose distribution across the bilayer. The T_m changes in the same direction with trehalose concentration for both freeze-dried and fully hydrated samples, suggesting that the trehalose distribution across the vesicle membrane, as well as the trehalose–phospholipid interaction, is maintained upon lyophilization. The results presented in this work may aid in the formulation of systems to be used in the lyophilization of liposomes for drug delivery applications.

Keywords: Trehalose; DPPC; Cholesterol; Liposome

Characterization of Ni transport into brush border membrane vesicles (BBMVs) isolated from the kidney of the freshwater rainbow trout ( Oncorhynchus mykiss) by Eric F. Pane; Chris N. Glover; Monika Patel; Chris M. Wood (pp. 74-84).

The transport of nickel (Ni) across the renal brush border membrane of the rainbow trout was examined in vitro using brush border membrane vesicles (BBMVs). Both transmembrane transport of Ni into an osmotically active intravesicular space, and binding of Ni to the brush border membrane itself, were confirmed. Nickel (Ni) uptake fitted a two component kinetic model. Saturable, temperature-dependent transport dominated at lower Ni concentrations, with a moderate linear diffusive component of Ni transport apparent at higher Ni concentrations. An affinity constant ( K_m) for Ni transport within the specifically described vesicular media was calculated as 17.9±1.9 μM, the maximal rate of transport ( J_max) was calculated as 108.3±3.7 nmol mg protein⁻¹ min⁻¹, and the slope of the linear diffusive component was calculated as 0.049±0.005 nmol mg protein⁻¹ min⁻¹ per μM of Ni. Efflux of Ni from BBMVs was fitted to an exponential decay curve with a half-time ( T_1/2) of 125.2±7.3 s. Ni uptake into renal BBMVs was inhibited by magnesium at a 100:1 Mg to Ni molar ratio, and by magnesium and calcium at a 1000:1 molar ratio. In the presence of histidine at a 100:1 histidine to Ni ratio, Ni uptake was almost completely abolished. At a 1:1 molar ratio, histidine inhibited Ni uptake by approximately 50%. Ni–histidine complexation was rapid, with a T_1/2 of 12.2 s describing the Ni–histidine equilibration time needed to inhibit Ni uptake into renal BBMVs by 50%. Characterization of Ni transport across cellular membranes is an important step in understanding both the processes underlying homeostatic regulation of Ni, and the toxicological implications of excessive Ni exposure in aquatic ecosystems.

Keywords: Ni; Transport; BBMVs; Rainbow trout; Kidney

Lysophosphatidic acid and lipopolysaccharide bind to the PIP₂-binding domain of gelsolin by Evan Mintzer; Hasmik Sargsyan; Robert Bittman (pp. 85-89).

The binding of the gelsolin P2 peptide (residues 150–169) with lysophosphatidic acid (LPA) and lipopolysaccharide (LPS) was investigated by isothermal titration calorimetry. P2 binds to LPS with higher affinity than to LPA. For the interaction of 1-oleoyl-LPA with P2 in the absence of salt, K_d and ΔH° were 920 nM and −2.07 kcal/mol, respectively, at pH 7.4 and 25 °C. For the interaction of lipopolysaccharide (LPS) from P. aeruginosa with P2 under the same conditions, K_d was 177 nM and ΔH° was −7.6 kcal/mol.

Keywords: Binding; Gelsolin; Lipopolysaccharide; Lysophospholipid; Thermodynamics

Development of wrapped liposomes: Novel liposomes comprised of polyanion drug and cationic lipid complexes wrapped with neutral lipids by Masahiro Yamauchi; Hiroko Kusano; Etsuko Saito; Takeshi Iwata; Masashi Nakakura; Yasuki Kato; Noboru Aoki (pp. 90-97).

Novel wrapped liposomes comprised of polyanion drug and cationic lipid complexes wrapped with neutral lipids were prepared using an efficient, innovative procedure. In this study, dextran fluorescein anionic (DFA) was used as an example of a polyanionic compound. During the process, neutral lipids accumulated around the complexes and eventually covered the complexes. The resulting liposomes were 120–140 nm in diameter and the encapsulation efficiency was up to 90%. In fetal bovine serum, DFA/cationic lipid complexes degraded rapidly but the wrapped liposomes were considerably more stable. Following intravenous administration to rats, DFA/cationic lipid complexes were rapidly eliminated whereas the wrapped liposomes exhibited a much longer blood half-life. These data suggest that DFA is located on the surface of the complexes, but DFA is present inside the wrapped liposomes. The drug-delivery properties of the wrapped liposomes established in the present study suggests that formulations based on this technology could offer important advantages for the administration of many types of drug including antisense oligonucleotides, plasmids and siRNAs which may therefore lead to improved therapeutic effectiveness of this range of drugs. The method of preparation of the wrapped liposomes is so simple that it should be straightforward to adapt to a manufacturing scale.

Keywords: Liposome; Cationic lipid; Drug delivery; Formulation; DOTAP; PEG

Insights into the molecular mechanism of action of Celastraceae sesquiterpenes as specific, non-transported inhibitors of human P-glycoprotein by Francisco Muñoz-Martínez; Carolina P. Reyes; Antonio L. Pérez-Lomas; Ignacio A. Jiménez; Francisco Gamarro; Santiago Castanys (pp. 98-110).

Dihydro-β-agarofuran sesquiterpenes from Celastraceae have been recently shown to bind to human P-glycoprotein (Pgp), functioning as specific, mixed-type inhibitors of its drug transport activity, as well as multidrug resistance (MDR) modulators in vitro. However, nothing is known about whether such compounds are themselves transported by Pgp, or whether they affect Pgp expression as well as its activity, or about the location of their binding site within the protein. We performed transport experiments with a newly synthesized fluorescent sesquiterpene derivative, which retains the anti-Pgp activity of its natural precursor. This probe was poorly transported by Pgp, MRP1, MRP2 and BCRP transporters, compared with classical MDR substrates. Moreover, Pgp did not confer cross-resistance to the most potent dihydro-β-agarofurans, which did not affect Pgp expression levels in several MDR cell lines. Finally, we observed competitive and non-competitive interactions between one of such dihydro-β-agarofurans (Mama12) and classical Pgp modulators such as cyclosporin A, verapamil, progesterone, vinblastine and GF120918. These findings suggest that multidrug ABC transporters do not confer resistance to dihydro-β-agarofurans and could not affect their absorption and biodistribution in the body. Moreover, we mapped their binding site(s) within Pgp, which may prove useful for the rational design of improved modulators based on the structure of dihydro-β-agarofurans.

Keywords: P-glycoprotein; Dihydro-β-agarofurans; Fluorescent derivative; Fluorescent dye extrusion; Binding site mapping

The membranotropic regions of the endo and ecto domains of HIV gp41 envelope glycoprotein by Miguel R. Moreno; Marcela Giudici; José Villalaín (pp. 111-123).

We have identified the membranotropic regions of the full sequence of the HIV gp41 envelope glycoprotein by performing an exhaustive study of membrane rupture, phospholipid-mixing and fusion induced by two 15-mer gp41-derived peptide libraries from HIV strains HIV_MN and HIV_consensus_B on model membranes having different phospholipid compositions. The data obtained for the two strains and its comparison have led us to identify different gp41 membranotropic segments in both ecto- and endodomains which might be implicated in viral membrane fusion and/or membrane interaction. The membranotropic segments corresponding to the gp41 ectodomain were the fusion domain, a stretch located on the N-heptad repeat region adjacent to the fusion domain, part of the immunodominant loop, the pre-transmembrane domain and the transmembrane domain. The membranotropic segments corresponding to the gp41 endodomain were mainly located at some specific parts of the previously described lentivirus lytic sequences. Significantly, the C-heptad repeat region and the Kennedy sequence located in the ectodomain and in the endodomain, respectively, presented no membranotropic activity in any model membrane assayed. The identification of these gp41 segments as well as their membranotropic propensity sustain the notion that different segments of gp41 provide the driving force for the merging of the viral and target cell membranes as well as they help us to define those segments as attractive targets for further development of new anti-viral compounds.

Keywords: Abbreviations; CF; 5-Carboxyfluorescein; Chol; Cholesterol; CHR; C-Terminal heptad repeat region; FP; Fusion peptide; HIV; Human immunodeficiency virus; LLP; Lentivirus lytic peptide; LUV; Large unilamellar vesicles; NBD-PE; N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-; sn; -glycero-3-phosphoethanolamine; NHR; N-Terminal heptad repeat region; N-RhB-PE; Lissamine™ rhodamine B 1,2-dihexadecanoyl-; sn; -glycero-3-phosphoethanolamine; PA; Egg; l; -α-phosphatidic acid; PC; Egg; l; -α-phosphatidylcholine; PE; Egg; trans; -sterified; l; -α-phosphatidylethanolamine; PI; Bovine brain; l; -α-phosphadidylinositol; PS; Bovine brain; l; -α-phosphatidylserine; PTM; Pre-transmembrane; SM; Egg sphingomyelin; TM; TransmembraneMembrane fusion; gp41; HIV

Functional reconstitution into liposomes and characterization of the carnitine transporter from rat liver microsomes by Annamaria Tonazzi; Michele Galluccio; Francesca Oppedisano; Cesare Indiveri (pp. 124-131).

The carnitine transporter was solubilized from rat liver microsomes with Triton X-100 and reconstituted into liposomes, after addition of Triton X-114, by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite (Bio-Beads SM 2). The reconstitution was optimized with respect to the detergent/phospholipid ratio, the protein concentration, and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalyzed a first-order uniport reaction inhibited by HgCl₂ and DIDS. The IC₅₀ for HgCl₂ was 0.16±0.03 mM. The reconstituted transporter also catalyzed carnitine efflux from the proteoliposomes; the efflux was stimulated by externally added long-chain acylcarnitines. Besides carnitine, ornithine, arginine, glutamine and lysine were taken up by the reconstituted liposomes with lower efficiency respect to carnitine. Optimal activity was found at pH 8.0. The Km for carnitine on the external side of the transporter was 10.9±0.16 mM. The activation energy of the carnitine transport derived by Arrhenius plot was 16.1 kJ/mol.

Keywords: Abbreviations; NEM; N-ethylmaleimide; NPheM; N-phenylmaleimide; PheAsO; phenylarsine oxide; DTE; 1.4-dithioerythritol; PLP; pyridoxal 5-phosphate; SITS; 4-acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid; DIDS; 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid; WRK; Woodward's reagent K; EDC; 1-ethyl-3(3-dimethylaminopropyl)carbodiimmide; C; ₁₂; E; ₈; octaethylene glycol monododecyl ether; NDP40; Nonidet P40Endoplasmic reticulum; Microsome; Membrane; Transport; Liposome; Carnitine; Reconstitution

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BBA - Biomembranes (v.1758, #1)