[page 24↓]

Material and Methods

3.1 Chemicals

M-C6-NBD-PE, M-C6-NBD-PG, M-C6-NBD-PC, P-C6-NBD-PE and P-C12-NBD-PE were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Egg PC, BSA, EDTA, NEM, ascorbic acid, PMSF, DTT, sodium dithionite were from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany). TRIS, Na2HPO4, NaH2PO4, ammonium molybdate, sodium chloride, HEPES, Triton X-100 analytical grade and sucrose were purchased from Fluka Chemie AG (Buchs, Switzerland). Triton X-100 ultra clean was from Boehringer Mannheim GmbH (Mannheim, Germany). SM-Bio-Beads was obtained from Bio-Rad Laboratories (Hercules, USA). Fluka Chemie AG (Buchs, Switzerland) provided Polyacryl amide, TEMED, ammonium persulfate and SDS.

3.2  Preparation of inverted inner membrane vesicles from E.coli

Inner membrane vesicles were isolated from the E.coli wild type strain MG 1655. Cell growth and membrane isolation were essentially performed as described by Huijbregts et al. (Huijbregts, et al., 1996) with minor modifications.

A small volume of Luria Broth (LB) was inoculated with a pipette tip that was dipped into a deep freeze culture of MG 1655. This culture was grown at 37°C on a shaker at 140 U/min overnight (12 h-16 h). Nine milliliters of the overnight culture were diluted into three liters LB and grown to an optical density (OD660) of 0.7-0.8 (late log phase). The cells were harvested by centrifugation in a type JLA 10.500 Beckman rotor (10 min, 2,000 g, 3°C) and washed with ice cold medium A (33 mM KH2 PO 4 , 60 mM K 2 HPO 4 , 1.7 mM sodium citrate (hydrated) and 7.6 mM (NH 2 ) 4 SO 4 pH 7.5). The sedimented cells were resuspended in 30 ml of buffer S (50 mM triethanolamine pH 7.5, 250 mM sucrose, 1 mM EDTA). DTT and PMSF were added to a final concentration of 1 mM and 0.375 mM, respectively. Cells were broken by 2-3 passages in a French press at a cell pressure of 1,100 psi. Subsequently additional DTT and PMSF were added to a final concentration 1 mM and 0.375 mM, respectively. Cell debris was removed [page 25↓] by centrifugation (2x for 10 min at 6000 g in a 45Ti Beckman rotor at 3°C). The outer membrane fraction was pelleted by centrifugation of the suspension in a SW 28 Beckman rotor for one minute on the maximum speed (165,000 g). To sediment the crude inverted inner membrane vesicles (IIMV), the supernatant was centrifuged at 165,000 g, for 90 min in a SW 28 Beckman rotor at 3°C. The resulting pellet was resuspended and homogenized with 15 strokes (one stroke corresponds to one down and one up move) in two milliliters ice cold buffer S and layered on top of a discontinuous sucrose gradient in buffer S0 (buffer S without sucrose) according to Osborn et al. 1972 (Osborn, et al., 1972). Subsequently, the gradients were centrifuged for 16 h to 18 h in a SW 40 Beckman rotor at 112,000 g at 3°C. Three bands were visible. The light band (corresponding to the purified IIMV, (Osborn, et al., 1972)) were collected and washed in 10 mM HPS (10 mM HEPES pH 7.5, 100 mM NaCl) at 165,000 g for 90 min and at 3°C (SW 28 rotor Beckman). Vesicles were resuspended in 10 mM HPS using a dounce homogenizer, quickly frozen in liquid nitrogen and stored at -80°C. IIMV suspensions were thawed immediately before use.

IIMV isolated as described above typically contained about 3 mg protein/µmol phospholipid. According to van Klompenburg et al. (van Klompenburg, et al., 1995), the vesicles are sealed and 100% inside-out.

3.3  Reconstitution of IIMV derived from E.coli

IIMV were solubilized and reconstituted according to the method described by Menon et al. (Menon, et al., 2000) and Hrafnsdóttir and Menon (Hrafnsdottir and Menon, 2000). In brief, the vesicle suspension was mixed with an equal amount of buffer DTEB (20 mM HEPES pH 7.5, 200 mM NaCl, 2% (w/v) Triton X-100). The mixture was incubated on ice for 30-60 min and subsequently centrifuged in a 70.1 Ti Beckman rotor at 175,000 g for 30 min to pellet unsolved proteins. The resulting triton extract (TE) was carefully collected and stored on ice until further reconstitution steps. The TE or chromatographic fraction was added to a solution of egg PC in buffer OTEB (10 mM HEPES pH 7.5, 100 mM NaCl, 1% (w/v) Triton X-100) with a final phospholipid concentration (after addition of the TE) of 4.5 µmol/ml. To incorporate fluorescent phospholipid analogues [page 26↓]symmetrically between the two leaflets of the reconstituted proteoliposomes the respective NBD-labeled phospholipids were added with a final concentration of max. two molpercent of total phospholipid content. For detergent removal 100 mg/ml (wet weight) SM-2 Bio-Beads® were added and gently rocked for three hours at room temperature. Subsequently, additional Bio-Beads were added (200 mg/ml wet weight), rocking was continued for additional two hours at room temperature. The mixture was transferred to 4°C and gently rocked for further 12 h-18 h. The resulting turbid suspension was separated from the beads using a glass Pasteur pipette and centrifuged for 45 min at 175,000 g, at 4°C (70.1 Ti Beckman rotor). The resulting pellet was resuspended in two milliliters 10 mM HPS, centrifuged again at 175,000 g for 45 min, 4°C, resuspended in the same buffer and homogenized with a dounce homogenizer (15 strokes) on ice.

3.4  Incorporation of NBD-labeled phospholipids into IIMV

The fluorescent phospholipid analogues (two molpercent of the total phospholipid content) in organic solvent were dried under a gentle stream of nitrogen. The resulting lipid film was dissolved in a small volume of ethanol (1% (w/v) of the final volume) and subsequently, suspended in 10 mM HPS. Two milliliters of the suspension were mixed with an aliquot of IIMV (12.5 µM final phospholipid concentration) and the fluorescence increase (excitation: 467 nm, emission: 540 nm, slid with: 4 nm) was monitored using an Aminco Bowman Series 2 spectrofluorometer (SLM Instruments Inc., Rochester, USA) for 1,600 s. All experiments were performed at room temperature.

3.5  The BSA back-exchange assay

NBD-labeled, short-chain lipid analogues were used to investigate the transmembrane movement and distribution of phospholipids across the inner membrane of E.coli. BSA is able to extract short-chain phospholipid analogues from given membranes. Back extraction of the phospholipid analogues by BSA from these membranes results in a fluorescence decrease because BSA-bound NBD-labeled phospholipid explores a different polarity with respect to [page 27↓]membranes. Extraction of fluorescent labeled, short-chain phospholipids can therefore be directly monitored by the fluorescence decrease.

3.5.1 Extraction of fluorescent labeled phospholipid analogues by BSA – cuvette experiments

To measure the rapid transbilayer movement of phospholipid analogues the BSA back-exchange assay was used. An aliquot of IIMV, proteoliposomes or liposomes labeled with max. two molpercent of total phospholipid content either M-C6-NBD-PC, -PE, -PG or P-C6-NBD-PS was diluted into two milliliters of 10 mM HPS. The suspension was placed in a quartz cuvette and the fluorescence was monitored (excitation: 467 nm, emission: 540 nm, slid with: 4 nm) using an Aminco Bowman Series 2 spectrofluorometer. After a constant fluorescence baseline was obtained (usually after 30 s), 20 µl of BSA in 10 mM HPS (20% w/v)) were added and the fluorescence decrease was measured for at least ten minutes at room temperature until a plateau of fluorescence intensity was reached.

3.5.2  The stopped-flow assay

All stopped-flow measurements were performed at room temperature. The time-dependent BSA back-exchange of NBD-labeled phospholipids was monitored by mixing the labeled IIMV, proteoliposomes or liposomes with 2% (w/v) BSA (final concentration) in 10 mM HPS using a stopped-flow accessory (RX 1000, Applied Photophysics, Leatherhead, UK) linked to an Aminco Bowman Series 2 spectrofluorometer. The dead time of mixing the two reactants amounts to about ten milliseconds. Extraction of analogues from membranes by BSA was followed by the decrease of fluorescence intensity. Fluorescence was recorded for 300 s at a time resolution of 0.2 s or 0.5 s, excitation wavelength λex=467 nm, emission wavelength λem=540 nm, slit widths 4 nm. For each preparation, five or more kinetic traces were recorded and averaged for kinetic analysis (see 3.14). However, scattering of (non-labeled) liposomes, IIMV and IIMV-derived proteoliposomes was significant. Therefore, traces were corrected to compensate for the light scattering contribution to the amplitude of the signal.


[page 28↓]

3.6  The dithionite assay

Alternatively to the BSA assay, where the fluorescence decrease due to extraction of phospholipid analogues by BSA was monitored, a chemical fluorescence quenching assay was used to characterize the flip-flop of fluorescent phospholipid analogues. In this assay, the fluorescence is quenched by the chemical reaction of non-permeable dithionite (S2O4 2-) with the NBD-group of the phospholipid analogue (Figure 3), resulting in a non-fluorescent NBD-lipid derivative (McIntyre and Sleight, 1991). When unilamellar vesicles containing NBD-labeled phospholipids are mixed with dithionite, only the fluorescent lipids located on the outer leaflet of the vesicle bilayer are reduced.

Figure 3: Chemical reaction of dithionite with the fluorescent NBD-group. The fluorescence of NBD is quenched by generation of a non-fluorescent derivative due to chemical interaction of the dithionite radical with the nitroxide group of the NBD-molecule.

To measure the transmembrane movement of fluorescent phospholipid analogues essentially the same experimental set-up as outlined in chapter 3.5 for the BSA back-exchange assay was used. Briefly, when performing cuvette experiments an aliquot of proteoliposomes derived from IIMV or chromatographic fractions from IIMV (see chapter 3.7) containing 0.5 mol% of the appropriate fluorescent analogue with respect to the total phospholipid content was suspended into two milliliters 10 mM HPS. Subsequently, the mixture was placed in a quartz cuvette and the fluorescence was monitored until a stable baseline was obtained. Then, freshly prepared dithionite in 40 mM Tris pH 8.0 was added to a final concentration of 10 mM and the fluorescence decay was measured for 600 s. Subsequently, the vesicles were disrupted by adding 1% (w/v) [page 29↓]Triton X-100 (final concentration) to test whether the concentration of dithionite was sufficient to quench the fluorescence completely.

When carrying out stopped-flow measurements, aliquots of the appropriate fluorescent labeled vesicles and 20 mM dithionite in 40 mM Tris pH 8.0 were mixed using the stopped-flow device and the resulting fluorescence decrease was measured.

All experiments were performed at 22°C to minimize the penetration of dithionite. The instrumental parameters used for both cuvette and stopped-flow measurements are described in chapter 3.5.

3.7  Ion exchange chromatography

In this study, ion exchange chromatography (IEC) was used to enrich flippase activity in a distinct fraction. All chromatographic steps were carried out at room temperature. An aliquot TE (see 3.3) was diluted 1:5 in buffer Z (25 mM TEA pH 8.0, 10 mM NaCl). This suspension was placed on a 1 ml Hi Trap Q HP column (Amersham-Pharmacia Biotech) equilibrated with buffer C (25 mM TEA acetate pH 8.0, 10 mM NaCl, 0.2% (w/v) Triton X-100). The Hi Trap column was operated using a peristaltic pump (BioRad) with a flow rate of 0.5 ml/min. The column was washed with three milliliters buffer A (10 mM HEPES pH 7.5, 100 mM NaCl, 0.2%(w/v) Triton X-100) and the wash was pooled with the flow-through. Bound proteins were eluted with buffer D (buffer A containing 1 M NaCl). Samples of each fraction were dialyzed against 1.5 l 10 mM HPS for one hour at room temperature and subsequently, reconstituted into proteoliposomes as described in chapter 3.3.

After dialysis, the resulting proteoliposomes were assayed by the dithionite approach as described in chapter 3.6.


[page 30↓]

The flippase activities of the reconstituted fraction were calculated as follows: The activity A is the percent of the fluorescent intensity above the pure liposome control, where Fred,lip is the normalized fluorescent intensity of the liposomes after dithionite treatment and Fred,prot is given by the final fluorescent intensity of the proteoliposome sample.

The specific activity (AS) describes the activity (A) of the probes relative to the protein/phospholipid ratio (P/PL) in %*µmol*µg-1.

3.8  SDS-PAGE analysis

Gels and buffers were made and the gel electrophoresis was run according to Laemmli (Laemmli, 1970). Aliquots of proteoliposomes were delipidated by the procedure of Bligh and Dyer (Bligh and Dyer, 1959) before subjecting to gel electrophoresis. The samples were mixed with sample buffer and heated to 95°C for several minutes. Subsequently, the samples were applied to sodiumdodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the gels were silver stained as follows: First, the gels were fixed for at least 30 min in 100 ml aqueous solution containing 30% (v/v) ethanol and 10% (v/v) acetic acid. Afterwards, the gels were incubated for 30 min in 100 ml of a solution of 30% (v/v) ethanol, 0.5% (v/v) glutaraldehyde, 0.2% (w/v) sodium thiosulfate and 0.5 M sodium acetate. Subsequently, the gels were washed with 100 ml aqua dest. for ten minutes. The wash was repeated twice. Then, the proteins were stained by incubation of the gel in the color solution containing 0.1% (v/v) silver nitrate and 0.02% (v/v) formaldehyde. Thereafter, the gel was rinsed with aqua dest. . Subsequently, the color was developed by incubation in 100 ml of an aqueous solution of 2.5% (v/v) sodium carbonate and 0.01% (v/v) formaldehyde until the silver stained bands became visible. The reaction was stopped by incubation of [page 31↓]the stained gels in a 0.5 M EDTA solution for ten minutes. All reactions were performed at room temperature and with gently shaking on a table rocker.

3.9 Methods for the determination of protein concentration

3.9.1 The Lowry method modified by Peterson

Protein determination was performed according to the Lowry method modified by Peterson ((Lowry, et al., 1951;Peterson, 1977)) using the Sigma Protein Assay Kit No. P5656 (Bensadoun and Weinstein, 1976). The procedure was performed as described in the users manual with minor modifications. Samples were incubated with deoxycholate (0.125 mg/ml final concentration) for ten minutes at room temperature. Then, trichloracetic acid was added to a final concentration of 6% (w/v), vortexed and the mixture was incubated for 15 min at room temperature. To pellet the precipitated protein, the suspension was centrifuged for 15 min at 13,000 g (Heraeus Biofuge fresco, Heraeus Instruments GmbH, Berlin, Germany) at 15°C. The resulting supernatant was discarded. The sedimented protein precipitates were dissolved and rigorous vortexed in one milliliter SDS containing Lowry Reagent Solution and incubated for 20 min at room temperature. Subsequently, 0.5 ml of Folin & Ciocalteu's Phenol Reagent Working Solution were added, immediately and intensive vortexed and the color was allowed to develop for 45 min at room temperature. The absorbance was measured versus a blank sample at a wavelength of 750 nm (UV 2102 PC spectrometer, Shimadzu Europe GmbH, Duisburg, Germany). The measurements were completed within ten minutes. The protein content was determined by equal treated references of BSA standards with known concentrations.

3.9.2 The bicinchoninic acid (BCA) Method

An alternative method to rapidly determine the protein content of lipid and detergent containing samples, is the bicinchoninic acid (BCA) method in combination with SDS treatment. For the determination of protein concentration, the BCA Kit obtained from Pierce (Pierce, Rockford, IL, USA) was used, strictly [page 32↓]following the manual instructions. To minimize the influence of lipids on the protein determination reactions, aliquots of samples were mixed with SDS to a final concentration of 1% (w/v) SDS. In parallel, BSA standards with known concentrations were treated identically to generate a standard curve for protein quantification.

3.10  The lipid extraction procedure

Lipid extraction was performed according to Bligh and Dyer (Bligh and Dyer, 1959). An aliquot of membrane suspension was dissolved in aqua dest. to a final volume of 400 µl. Then, 500 µl chloroform and 1000 µl methanol were added. The suspension was rigorously vortexed for one minute. Subsequently, 500 µl chloroform, 500 µl methanol and a drop of 1 N HCl were added and the mixture was vortexed again for one minute. After centrifugation (10 min, 1,000 g, 4°C), the lower organic phase was collected in a Schott Duran glass tube (Hermann Kröpke GmbH, Berlin, Germany). The upper inorganic phase was extracted again by addition of one milliliter of chloroform, vortexed for one minute and centrifuged. The lower phase was collected and pooled with the first. If required, the re-extraction was repeated once more. The organic solvent was removed under a gentle stream of Nitrogen by using a thermostatically controlled heating block equipped with a multiple probe evaporator (Liebisch Thermochem-Metallblock-Thermostat/Multiplex-Ventil-Depot, Gebr.Liebisch GmbH & Co., Bielefeld, Germany).

3.11 Quantitation of phospholipids

The total phospholipid content of phospholipid containing samples was determined by measurement of the phospholipid phosphorus according to Rouser (Rouser, 1966). First, the lipids were extracted after the method of Bligh & Dyer (see 3.10). After evaporation of the organic solvent (see 3.10), 400 µl perchloric (72% (w/w)) acid was added and the samples were boiled for 1.5 h at 180°C in a thermostatically controlled heating block (Blockthermostat BT­200, Kleinfeld Labortechnik, Hannover, Germany). After the solutions had cooled down, four [page 33↓]milliliters of Molybdate reagent (0.22% (w/v) ammonium molybdate, 0.25 M sulfuric acid) and 500 µl of 10% (w/v) ascorbic acid were added with mixing and incubated for ten minutes in a boiling water bath. The tubes were allowed to cool. The absorbance was read at 812 nm versus a blank sample. The phospholipid content of IIMV, proteoliposomes, liposomes and phospholipid containing suspensions were calculated from appropriately generated Na2HPO4 standard curves.

3.12 Detergent determination

Triton X-100 absorbance at 275 nm was used to check the removal of detergent during reconstitution as described by Hrafnsdottir and Menon (Hrafnsdottir and Menon, 2000). To this end, 150 µl of sample were mixed with 600 µl of methanol and 300 µl of chloroform and vortexed. The suspension was centrifuged (15,000 g, 15 min, 15°C) to remove the precipitated protein and the absorbance at 275 nm was measured. To determine the Triton X-100 concentration, identically treated detergent standards were assayed.

3.13  The measurement of the purity of isolated IIMV

To test the degree of contamination of the IIMV preparation with outer membrane fractions the activity of PLA2, an outer membrane marker, was measured. To this end, five nanomol of head group labeled N-Rh-PE were diluted into one milliliter chloroform. Eleven microliter of this suspension were dried under a gentle stream of nitrogen, resuspended in five microliters of 50 mM Tris (pH 8), 20 mM calcium chloride, 0.2% Triton X-100. Subsequently, five microliters of the respective fraction were added and the incubation mixture was allowed to react for one hour at 37°C. After incubation, 22 µl of chloroform/methanol (1:1) were added, vortexed and centrifuged at 1,000 g for two minutes. Thin layer chromatography (TLC) of the lower phase was carried out with chloroform:methanol:water (65:25:4) on silica 60 plates. The spots were analyzed using a VD 40 Hitachi 3-Chip analyzing system (Desaga GmbH, Wiesloch Germany). PLA2 activity was detected by spots corresponding to the [page 34↓]lyso derivative of N-Rh-PE. The analysis was performed with the standard accessory software (ProViDoc 3.04).

Figure 4: Representative experiment of detection of the PLA 2 activity in IIMV and outer membrane vesicles (OMV) derived from E.coli . The white arrow indicates the lyso derivatives of the PLA 2 activity. The blue arrow indicates the non-cleaved N-Rh-PE molecules by PLA 2 .

In fractions of the outer membrane, a high degree of Phospholipase A2 activity was detected. The appearance of lyso N-Rh-PE (indicated with the white arrow in Figure 4) in the fraction of IIMV was below the detection level. Thus, IIMV are not or less contaminated with outer membrane fragments.

3.14  Kinetic analysis

The experimental data were fitted to a theoretical time course using a three-compartment model (Figure 5). This kinetic model describes transbilayer movement as well as the transfer of phospholipid analogues between the outer leaflet of the membrane vesicle and BSA (Marx, et al., 2000).

The outward and inward movements of phospholipid analogues are described by the rate constants k+1 and k-1, respectively. The movement of the analogues from the IIMV to BSA is characterized by the rate constant k+2 (extraction of the analogues by BSA) and k -2 for the movement of analogues back from BSA to the vesicle membrane. Due to the excess of BSA used, the exchange process described by k-2 did not contribute to the kinetics and the values for this time constant were very small (typically 10-12 s-1).


[page 35↓]

Figure 5 : Model of the transbilayer movement of phospholipid (analogues) across membranes and extraction of fluorescent NBD-lipid analogues by BSA. The rate constants k -1 and k +1 indicate the inward and outward movement of phospholipid analogues across the membrane, respectively. The extraction of fluorescent analogues is described by k +2 and the movement from BSA to the membrane is indicated by k -2 .

and are the concentrations of analogue in the outer and inner leaflet of the IIMV. At the time of BSA addition (t = 0s), the transmembrane distribution is at steady state, i.e.,

(1)

The concentration of analogue transferred to BSA is taken to be zero at the time of addition of BSA.

The model is represented by the following system of differential equations:

(2)

(3)

(4)

(5)

(6)


[page 36↓]

For further details see (Marx, et al., 2000). Fitting was performed by least-square minimization.


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