[page 37↓]

4  Results

One goal in this study was to characterize the transbilayer distribution and transbilayer movement of fluorescence labeled phospholipid analogues in the inner membrane of E.coli. The phospholipid flip-flop in native and reconstituted E.coli inner membranes was analyzed using the BSA back-exchange stopped-flow assay developed by Marx et al. (Marx, et al., 2000) in combination with the tool of reconstitution of membrane proteins (Hrafnsdottir and Menon, 2000;Hrafnsdottir, et al., 1997;Menon, et al., 2000). Earlier studies on the translocation process of phospholipids could not demonstrate a clear requirement of proteins and/or lacked a sufficient time resolution (Hrafnsdottir and Menon, 2000;Hrafnsdottir, et al., 1997;Huijbregts, et al., 1996;Huijbregts, et al., 1998). Therefore, the transmembrane distribution and movement of fluorescent short-chain phospholipid analogues in IIMV were characterized by using the highly time resolving stopped-flow technique (chapter 4.2 and 4.3). To extend the progress in characterization of the putative flippase, triton extracts (TE) of the inner membrane of E.coli were reconstituted (4.3). The transbilayer movement of different fluorescent phospholipid analogues in proteoliposomes derived from IIMV of E.coli was characterized using the BSA back-exchange and the dithionite assay (see chapter 4.3, 4.4 and 4.5). Furthermore, strong evidence were found for an involvement of protein in this translocation process as shown by protease treatment and reconstitution experiments (4.6). Moreover, the flippase activity could be recovered from fractions after chromatographic separation of solubilized IIMV (4.7).


[page 38↓]

4.1  Incorporation of fluorescent phospholipid analogues into IIMV

In aqueous solution, the NBD-labeled phospholipid analogues are mainly organized in micelles where their fluorescence is self-quenched. Upon adding acceptor membranes, such as IIMV or pure phospholipid vesicles, the fluorescence increases, since the fluorescent labeled phospholipid molecules spontaneously incorporate into IIMV and thus, micelles are dissolved. Incorporation of fluorescent lipids into membranes can therefore be directly monitored by fluorescence increase.

Figure 6 Kinetics of incorporation of NBD-labeled phospholipid analogues into IIMV membranes. Two molpercent of M-C6-NBD-PE, M-C6-NBD-PC or M-C6-NBD-PG in 10mM HPS, with respect to the total lipid content of the IIMV, was added to IIMV (12.5µM final phospholipid concentration), and the kinetics of membrane incorporation of the analogues were monitored. At time point zero, the IIMV were added to the label suspension. The value of 100% corresponds to incorporation of all fluorescent labeled analogues. All experiments were performed at room temperature. Due to the low time resolution, the initial fluorescence increase upon mixing of IIMV with lipid analogues could not be adequately resolved.

To analyze the incorporation of fluorescent lipid analogues into inner membrane vesicle from E.coli, an aliquot of IIMV was mixed with 10 mM HPS containing two molpercent of M-C6-NBD-PC, -PE or M-C6-NBD-PG in a quartz cuvette and the resulting increase of fluorescence intensity was monitored using a fluorescence spectrometer (see 3.4).


[page 39↓]

As evident from Figure 6, an initial rapid phase of intercalation of analogues into IIMV was observed. After ten minutes, about 90% of M-C6-NBD-PE and -PC were incorporated. However, the final plateau was reached within 30 min. Essentially all of the PE and PC analogues were intercalated into the IIMV. Moreover, the kinetics of incorporation of the fluorescent analogues of PE and PC were very similar. In contrast to these phospholipids analogues, M-C6-NBD-PG was much faster incorporated into IIMV. A stable plateau of fluorescence intensity was reached within ten minutes i.e., all of the NBD-labeled PG was incorporated within this time.

To verify, whether all analogues were incorporated into IIMV after a final plateau was attained, an extra aliquot of vesicles was added. After addition of an extra aliquot of vesicles no further fluorescence increase was observed (data not shown), indicating that all of the analogues were incorporated into the IIMV.


[page 40↓]

4.2  Transbilayer movement of fluorescent phospholipid analogues across IIMV membranes

For the measurement of the transbilayer movement of M-C6-NBD-PE, M-C6-NBD-PC and M-C6-NBD-PG across the IIMV membranes, stopped-flow method was used taking advantage of the fact that short-chain lipid analogues can be extracted from the membrane by BSA (BSA back-exchange), and that the quantum yield of analogues bound to BSA is different from that of membrane incorporated analogues.

Figure 7 : Kinetics of extraction of M-C6-NBD-PE from IIMV by BSA. An aliquot (25 µl) of IIMV (2.89 mg protein/µmol phospholipid) were incubated with two milliliters of a buffered suspension of M-C6-NBD-PE (two molpercent of the lipid content) for 30 min at room temperature. Subsequently, the labeled IIMV were rapidly mixed with an equal volume of 4% (w/v) BSA in a stopped-flow accessory. The fluorescence decay was recorded with a time resolution of 0.2 s. The kinetics were normalized as follows: The initial fluorescence intensity (before BSA extraction) was set to one, the intensity after 300 s to zero. The curve represents the average of five measurements (A). The solid yellow line represents the fit obtained by fitting the data to the three-compartment model. The dashed blue line was obtained by fitting the data to a monoexponential function. The residuals of the model fit (B) according to the three-compartment model ( 3.14 ) and the monoexponential fit (C) are depicted.

After labeling of IIMV with max. two molpercent of fluorescent analogues for 30 min at room temperature, vesicles were rapidly mixed with 2% (w/v) BSA (final concentration) in 10 mM HPS by stopped-flow. The time dependent decrease of fluorescence intensity resulting from back-exchange of analogues by BSA was monitored.


[page 41↓]

Table 1 : Half-times of transbilayer movement of NBD-labeled phospholipids across the membrane of IIMV and of extraction of analogues from the outer leaflet.

analogue

outward movement [s]

inward movement [s]

extraction [s]

[PL i ] t=0 [%]

M-C6-NBD-PE

58

160

4.6

26.6

 

48

210

2.5

18.6

M-C6-NBD-PC

17

100

1.1

14.7

 

63

242

2.7

20.7

M-C6-NBD-PG

64

55

3.3

53.8

 

65

97

4.4

40.1

P-C6-NBD-PS

52

136

15.5

27.7

 

65

196

14.9

24.9

[PLi]t=0 refers to the amount of analogues at time point of BSA addition

As shown in Figure 7 (displayed for M-C6-NBD-PE), the fluorescence emission intensity was found to decay in two distinct phases. After 300 s no change of fluorescence was observed, suggesting that all phospholipid analogues were extracted and bound to BSA (see below). Therefore, the kinetics were normalized as follows: the initial fluorescence intensity (before BSA extraction) was set to one, the intensity after 300 s to zero. The fast initial decrease of fluorescence intensity reflects the extraction of phospholipid analogues localized in the outer leaflet of the vesicles. Furthermore, the second slower phase is caused by extraction of M-C6-NBD-PE translocated from the lumenal leaflet to the outer leaflet. To determine the characteristic half-times of the two phases, the data were fitted as described in 3.14 to the three-compartment model (Figure 7A, yellow line). Additionally, a monoexponential fit was performed (Figure 7A, blue dashed line) to compare the parameter based on the three-compartment model with a simple monoexponential process. The residuals - the differences between measured and fitted values - clearly showed that a monoexponential function did not fit the recorded data (Figure 7C). However, the data could be well fitted by a model process yielding four rate constants (Figure 7A, and Figure 7B). Based on the three-compartment model (see 3.14), the half-times of flip-flop, extraction of analogues as well as their initial transbilayer distribution were estimated (Table 1). The transbilayer dynamics and distribution of M-C6-NBD-PE and M-C6-NBD-PC were found to be very similar (Table 1).


[page 42↓]

The same results were observed for a fluorescent analogue of PS, which contains an elongated fatty acid on the sn-1 position. Despite of the palmitic acid (16 carbon atoms) instead of myristic acid, the slightly enhanced hydrophobicity of this analogue had no influence of the characteristic transbilayer movement of P-C6-NBD-PS. Moreover, the half-times of flip-flop and the transbilayer distribution of P-C6-NBD-PS were almost identical to those found for M-C6-NBD-PE (Table 1).

Figure 8 : Time dependence of extraction of M-C6-NBD-PE at room temperature. An aliquot of IIMV was mixed with buffer containing two molpercent of M-C6-NBD-PE of the total lipid content and the mix was immediately transferred to the stopped-flow device. Subsequently, the IIMV/label suspension was rapidly mixed with an equal volume of 4% (w/v) BSA in a stopped-flow accessory and the fluorescence decay was recorded with a time resolution of 0.5 s at the indicated time points. The kinetics were normalized as described before, and the data were fitted to the three-compartment model. The transbilayer distributions were elucidated from the fits and are displayed as function of incubation time of the analogues with IIMV. The open symbols refer to the concentration of analogues in the inner leaflet, the closed symbols to the concentration of analogues in the outer leaflet of the IIMV. The solid lines display simple one-phase regressions.

While data in Table 1 refer to measurements on IIMV samples labeled for 30 min with NBD-lipids, experiments were carried out to test the efficiency of labeling and the influence of long term incubation of the IIMV with fluorescent lipid analogues. An aliquot of IIMV was mixed with buffer containing two molpercent of the appropriate fluorescent lipid analogue with respect to the total lipid concentration of IIMV. This mixture was rapidly transferred to the stopped-[page 43↓]flow device. This procedure took less than 90 s. Subsequently, the stopped-flow BSA back-exchange assay was performed immediately as described above. Briefly, at time points 0, 5 min, 10 min, 15 min, 20 min, 30 min, 60 min and 90 min the IIMV-NBD-lipid suspension and 4% (w/v) BSA were mixed in the stopped-flow chamber and extraction kinetics were monitored. The kinetics were fitted according to the three-compartment model (see 3.14), and the resulting transbilayer distributions of analogues were displayed vs. incubation time of analogues (Figure 8 – shown for M-C6-NBD-PE). Up to 15 min after addition of analogues to IIMV, the distribution of the fluorescent lipids and the half-times of transbilayer movement were dependent on the incubation time. After 20 min, no changes in distribution and movement were found, when performing stopped-flow measurements on IIMV preparations labeled up to 90 min (Figure 8). This indicates that after 20 min of labeling, the analogues were equilibrated between the two leaflets of the bilayer, consistent with a rapid flip-flop of the phospholipid analogues.

The kinetics displayed in Figure 8 were fitted to a simple first order function. The resulting half-times were ti 1/2 ~1 min and to 1/2 ~1.95 min for the inner and outer leaflet, respectively. These data are in agreement with the half-times calculated from the stopped-flow BSA back-exchange assay stated in Table 1.

To verify whether all analogues were extracted by BSA, an aliquot of IIMV was labeled with two molpercent of M-C6-NBD-PE or M-C6-NBD-PC for 30 min, then the vesicles were incubated with 2% (w/v) BSA for 300 s at room temperature and the resulting NBD-fluorescence intensity was measured. The fluorescence intensity was the same as that found, when an equal amount of analogues in aqueous suspension was incubated with 2% (w/v) BSA (Figure 9B, C - only shown for M-C6-NBD-PE) and about 55% of that seen, when the analogues were all membrane integrated (compare Figure 9B with A).


[page 44↓]

Figure 9 : Complete extraction of M-C6-NBD-PE from IIMV by BSA. (A) IIMV (60 µM final phospholipid concentration) were labeled with two mol% M-C6-NBD-PE for 30 min, (B) subsequently, the analogues were extracted by 2% (w/v) BSA in 10 mM HPS for 300 s. Longer incubation (>300 s) did not change the fluorescence intensity. (C) BSA (2% (w/v))-bound M-C6-NBD-PE (two mol% of total phospholipid content of IIMV) in the absence of IIMV. (D) An aliquot of IIMV was added to BSA-bound M-C6-NBD-PE (two mol% of total phospholipid content of IIMV). (E) IIMV (equal to the amount used in (A) and (D)) in two ml 10 mM HPS. (F) 2% (w/v) BSA in two milliliters 10 mM HPS. The error bars indicate the standard deviation of two experiments. The experiments were performed at room temperature.

Our data clearly show that all PE or PC analogues were completely extracted by BSA after 300 s. When an aliquot of IIMV was added to the sample containing the BSA-bound analogues no changes in fluorescence occurred due to the presence of unlabeled IIMV (Figure 9D). This is consistent with the assumption that k-2 (transfer of analogues from BSA to membranes) contributes very little to our analysis (Figure 5).


[page 45↓]

4.3  Transbilayer movement of fluorescent phospholipid analogues across the membrane of reconstituted proteoliposomes derived from IIMV

To analyze the relevance of proteins for phospholipid flip-flop, proteoliposomes containing detergent solubilized protein extract from inner membrane vesicles and two molpercent M-C6-NBD-PE were reconstituted as described in chapter 3.3. By this procedure ~50-70% of the protein and ~60-85% of the phospholipids from the reconstitution mixture were recovered in the resulting proteoliposomes. Essentially all of the initial Triton X-100 (>99.9%) was removed (data not shown).

Figure 10 : Stopped-flow kinetics of extraction of M-C6-NBD-PE from reconstituted IIMV by BSA. IIMV-derived proteoliposomes were labeled with two molpercent of the fluorescent phospholipid analogues during the reconstitution as described in 3.3 . In brief, equal volumes of proteoliposomes and 4% (w/v) BSA were mixed in the stopped-flow apparatus and the fluorescence decrease was recorded at room temperature (A). The curve represents the average of five separate kinetic traces. Kinetics were corrected for scattering. The solid yellow line represents the fit of the experimental data to the three-compartment model shown Figure 5 . The dashed blue line was obtained by a monoexponential fit of the data. The residuals for the fit of the data to the three-compartment model (B) and of a monoexponential fit (C) of the extraction kinetics are displayed.

The proteoliposomes were rapidly mixed with BSA (2% (w/v) final) by stopped-flow, and the time-dependent decrease of fluorescence intensity caused by BSA back-exchange of the analogues was monitored.


[page 46↓]

Figure 10A displays the kinetics of extraction of M-C6-NBD-PE from IIMV derived reconstituted proteoliposomes. As with IIMV, a similar bi-phasic decline of the fluorescence intensity on adding BSA to the M-C6-NBD-PE-labeled proteoliposomes was found (Figure 10A). Again, the BSA back extraction of M-C6-NBD-PE could not be fitted by a monoexponential function (blue dashed line in Figure 10A and panel C). Therefore, the kinetics were analyzed by the three-compartment model (yellow solid line in Figure 10A and panel B). As evident from Figure 10B, this model fitted the back extraction kinetics excellently. From fitting of the experimental data to the model, it was determined that transbilayer movement of NBD-phospholipids in proteoliposomes was very similar to that seen in IIMV. The respective rate constants were in the same order as those found for IIMV (data not shown). From the analysis of five independent experiments (n=5) a half-time for the outward movement of M-C6-NBD-PE of tk+1= 29 s, for the inward movement tk-1= 70 s and a half-time of extraction tk+2= 5 s was estimated (Table 2). The relative amount of analogues residing at the lumenal side at time point zero was [PLi]t=0= 29%. The rate constant of the backward movement of M-C6-NBD-PE from BSA to the proteoliposomes given by the fit was very small (k –2= 10-12s-1) and is therefore negligible.

Table 2 : Half-times of transbilayer movement of NBD-labeled phospholipids across the membrane of reconstituted IIMV-derived proteoliposomes and extraction of analogues from the outer leaflet

analogue

outward movement [s]

inward movement [s]

extraction

[s]

[PLi] t=0 [%]

M-C6-NBD-PE (n=5)

28.7 ± 2.2

69.5 ± 3.7

5.16 ± 0.73

29.2

M-C6-NBD-PG (n=3)

119.3 ± 7.0

69.7 ± 2.8

4.2 ± 0.07

63.1

[PLi]t=0 refers to the amount of analogues at time point of BSA addition

In Figure 11 a representative experiment for the extraction of M-C6-NBD-PE from pure lipid vesicles (liposomes) made from ePC is shown. The liposomes (black line in Figure 11 A) were labeled with M-C6-NBD-PE during the reconstitution and subsequently, incubated with 4% (w/v) buffered BSA in a stopped-flow device. The fluorescence intensity decayed with a half-time of about eight seconds. The fluorescence intensity plateau leveled off at ~80% of the initial


[page 47↓]

Figure 11: Extraction kinetics of pure ePC-vesicles. Control vesicles were prepared in parallel to proteoliposomes but without bacterial protein as described in 3.3 . (A) Pre-labeled ePC-vesicles (black line) were prepared by adding two molpercent M-C6-NBD-PE during the reconstitution procedure, whereas post-labeled ePC-vesicles (red line) were incubated with two molpercent of the same analogue prior the fluorescence measurement for 30 min. The stopped-flow back-exchange assay was performed as described for proteoliposomes (see Text and Figure 10 ). In panels B and C the residuals resulting from monoexponential fits from pre-labeled and post-labeled ePC-vesicles, respectively, are shown.

fluorescence intensity. This is consistent with the extraction of ~50% of analogues from IIMV (compare to Figure 9). As evident from the residuals displayed in Figure 11B, the extraction kinetics of protein-free liposomes (Figure 11 A) could be fitted to a monoexponential function. The residuals yielded from this fit showed no time dependence.

For comparison, the BSA back-extraction kinetics of pure lipid liposomes labeled with the same amount of M-C6-NBD-PE after reconstitution (red line) is shown in Figure 11. The post-labeled liposomes exhibited a similar monoexponential fluorescence intensity decline, resulting in a final intensity plateau of ~50% of the initial fluorescence emission intensity. This value corresponds to the complete extraction of the fluorescence analogues from the vesicles. Furthermore, it can be concluded that no flip-flop of phospholipid analogues occurred across the vesicle membrane in the absence of bacterial proteins. These results are consistent with the interpretation that the initial phase corresponds to extraction of the analogues from the outer leaflet, while the second slower phase corresponds to the transbilayer movement.


[page 48↓]

Figure 12 : Stopped-flow-kinetics of extraction of M-C6-NBD-PG from reconstituted IIMV by BSA. IIMV-derived proteoliposomes were labeled with two molpercent of the fluorescent phospholipid analogues during the reconstitution as described in 3.3 . Equal volumes of proteoliposomes and 4% (w/v) BSA were mixed in the stopped-flow apparatus, and the fluorescence decrease was recorded at room temperature. A typical fluorescence kinetics of extraction of the fluorescent PG analogue is shown. The fluorescence intensity did not reach a stable plateau after 300 s of BSA incubation. In the inset, representative extraction kinetics of M-C6-NBD-PG with an elongated incubation time to 30 min is shown. Essentially all PG analogues were extracted by BSA after 30 min. The experiments were carried out at room temperature.

In order to characterize the membrane translocation of an analogue of the second major phospholipid in the inner membrane of E.coli - PG - identical experiments as for M-C6-NBD-PE were performed as described above for M-C6-NBD-PE.

As for the fluorescent PE analogue, the fluorescence decay was essentially a bi-phasic process. As already concluded by the above given arguments, the initial decrease of fluorescence intensity reflects the extraction of phospholipid analogues localized in the outer leaflet of the vesicles. The second slower phase was caused by extraction of M-C6-NBD-PG translocated from the lumenal leaflet to the outer leaflet. The half-times of transbilayer movement of M-C6-NBD-PG across the membrane of proteoliposomes were 119 s and 70 s for the outward and inward movement, respectively (Table 2).

The rate of outward movement of the fluorescent PG analogue is about four times slower than that of PE analogues. As deduced from the model, the lumenal concentration of M-C6-NBD-PG at time point zero was about two times higher [page 49↓](63.1%, see Table 2) as for PE analogues. The extraction of M-C6-NBD-PG was not finished after 300 s (Figure 12). However, after 300 s more than 90% of the PG analogues were extracted by BSA. As evident from Figure 12A only after 30 min essentially all of the PG analogues were extracted. This is consistent with the findings for the initial distribution of the PG analogue.


[page 50↓]

4.4  Effect of proteins on the transbilayer movement of phospholipids

4.4.1  Extraction of M-C6-NBD-PE from IIMV membranes

To analyze the mechanism of phospholipid flip-flop further, series of proteoliposomes from IIMV containing different amounts of detergent solubilized protein extract and two molpercent M-C6-NBD-PE were reconstituted as described in chapter 3.3. The reconstituted proteoliposomes were rapidly mixed with a BSA solution (4% (w/v) in 10 mM HPS) by stopped-flow, and the time dependent decrease of fluorescence caused by back-exchange of analogues by BSA was monitored.

Figure 13 : Effect of protein concentration on flip-flop and extraction of M-C6-NBD-PE. (A) The proteoliposomes were prepared with 4.5 mM ePC, increasing amounts of the TE and labeled with M-C6-NBD-PE during the reconstitution procedure. Subsequently, the labeled proteoliposomes made from IIMV were analyzed by stopped-flow assay as described before. The kinetics represent the mean of at least six records corresponding to pure ePC-liposomes (trace a), and to proteoliposomes with 40 µg protein/ml (trace b), 100 µg protein/ml (trace c) and 180 µg protein/ml (trace d). (B) The final amplitudes of fluorescence decrease (see (A)) of two independent experiments with increasing amounts of proteins are shown. The error bars depict the standard deviation. The letters a-d next to each data point correspond to the traces (for one of the two experiments) in panel A.

At high protein content (180 µg/ml, Figure 13A trace d), it was observed that the fluorescence decreased finally to about 55% of the initial value. Based on quantum yields, this indicates that most of the analogues (≥90%) were extracted from the proteoliposomes to BSA. At lower protein content, removal of analogues [page 51↓]was not complete, possibly indicating that a fraction of the proteoliposomes lacked the putative flippase protein (Figure 13 traces b and c).

For vesicles reconstituted in the absence of protein extract but in the presence of Triton X-100, only the rapid initial phase of fluorescence decline but no further (slow) decrease (Figure 13A) was observed. The fluorescence in the liposomes samples reached a final plateau between 80 and 85%, consistent with the removal of about 50% of the analogues by BSA (see also Figure 9).

Figure 14: Protein dependence of transmembrane movement and extraction of M-C6-NBD-PE. Proteoliposomes containing fluorescent phospholipid analogues and increasing amounts of TE were assayed by stopped-flow as described in the text. The percentage of fluorescence decrease of six independent experiments is depicted.

The amplitude of the extraction of M-C6-NBD-PE was proportional to the protein/phospholipid ratio in the range 0-100 µg/µmol. As evident from Figure 13B the amplitude of fluorescence reduction leveled off at a plateau, possibly indicating that at a protein/phospholipid ratio of ~100 µg/µmol almost every vesicle was equipped with at least one flippase and therefore, no further substantial increase in fluorescence intensity was observed.

In Figure 14 a composite of data obtained from a number of experiments using IIMV derived proteoliposomes reconstituted with different protein concentration is summarized. The inflection point of the dose response plot displayed in Figure 14 lays at a similar protein to phospholipid ratio as that [page 52↓]depicted in Figure 13B. This indicates that at this concentration (100 µg/ml) of proteins the majority of vesicles are equipped with proteins, which facilitate the rapid transbilayer movement of phospholipid analogues. Taken together, these data strongly indicate that proteins are involved in the rapid transbilayer movement of phospholipids across proteoliposome bilayer.

4.4.2  Reduction of M-C6-NBD-PE in IIMV-derived membranes by dithionite

To verify the results of the BSA back-exchange assay, an alternative assay was established to analyze the transbilayer movement of M-C6-NBD-PE across IIMV derived proteoliposomes. In the past, the dithionite assay has been successfully used for investigations of transmembrane movement and distribution of fluorescent lipid analogues (McIntyre and Sleight, 1991).

Figure 15: Stopped-flow kinetics of fluorescence quenching of M-C6-NBD-PE from reconstituted IIMV by dithionite. IIMV-derived proteoliposomes were labeled with one molpercent of the fluorescent phospholipid analogues during the reconstitution as described in 3.3 . Equal volumes of proteoliposomes and 20 mM freshly prepared dithionite were mixed in the stopped-flow apparatus, and the fluorescence decrease was recorded at room temperature. A typical fluorescence kinetics of fluorescence reduction of the fluorescent PE analogue is shown. The protein content of the reconstituted proteoliposomes was 150 µg/ml.

Dithionite quenches the NBD fluorescence irreversibly by a chemical reaction (see 3.6 and Figure 3; Figure 15). Similar to the BSA back-extraction assay, when adding dithionite to a fluorescent labeled vesicle suspension, only the [page 53↓]fluorescent molecules residing on the outer leaflet will be accessible by dithionite. Subsequently, only on these molecules the fluorescence will be quenched.

To analyze the transbilayer movement of M-C6-NBD-PE in dependence of the protein content, series of proteoliposomes with different amounts of bacterial proteins were created, as described in chapter 4.4.1 (see also 3.3). The proteoliposomes were rapidly mixed with a freshly prepared dithionite solution (20 mM) by stopped-flow, and the decrease of fluorescence was monitored for 300s. In Figure 15 an example of a typical fluorescence reduction kinetics by dithionite is shown. As evident from Figure 15, the fluorescence intensity decays below 50% of the initial fluorescence intensity. Therefore, fluorescent PE analogues must have moved from the exoplasmic leaflet to the cytosolic leaflet and became accessible to dithionite (see discussion). Hence, the amplitudes of the dithionite fluorescence kinetics are useful for analyzing the ability of protein-mediated phospholipid flip-flop. The resulting amplitudes of fluorescence intensity were determined and are summarized in Figure 16.

Similar to the BSA back-extraction assay, the amplitudes of the final fluorescence intensity decreased with increasing amounts of bacterial protein reconstituted into proteoliposomes. Analyzing liposomes, i.e., pure lipid vesicles prepared in the presence of detergent but without proteins, the fluorescence intensity decayed to ~50% of the initial value, reflecting that only the outer leaflet populating fluorescent phospholipid analogues were reduced by dithionite (Figure 16), and no flip-flop occurred. With increasing quantity of proteins in the proteoliposomes the amplitudes of fluorescence decreased in the range 0-80 µg/ml protein. No consistent decrease in amplitude was observed, when proteoliposomes were prepared at a protein content greater than ~80 µg/ml.

The kinetic measurements, which form the basis of the data displayed in Figure 16, were analyzed by the three-compartment model (3.14). A precondition of this analysis is the impermeability of the reducing reagent dithionite. Moreover, the original three-compartment model was modified with respect to the absence of the fourth rate constant k-2 (movement of the analogue from BSA to the vesicle).


[page 54↓]

Figure 16: Protein dependence of transmembrane transport and extraction of M-C6-NBD-PE by the dithionite assay. Proteoliposomes containing one molpercent M-C6-NBD-PE and increasing amounts of TE were assayed by stopped-flow dithionite assay as described in 3.6 . Each data point represents the mean of five dithionite kinetics. The percentage of fluorescence decrease of three independent experiments is summarized. The dotted line indicates the linear range and the plateau of protein content-dependent fluorescence intensity amplitudes.

Subsequently, the dithionite kinetics were fitted with three parameters: k-1, k+1, and k+2; describing the outward movement, inward movement and fluorescence reduction by dithionite, respectively.

The results are presented in Table 3. The half-times of flip-flop of M-C6-NBD-PE were very similar to those found with the BSA back-extraction assay (Table 2). The half-times of outward movement were faster than for the inward movement of the analogue, similarly to half-times estimated from the BSA back-exchange assay. Interestingly, half-times determined from the dithionite assay were slower than those found by the BSA back-extraction assay. One possible explanation for this is that the proteoliposomes were slightly leaky for dithionite. Thus, the estimation of the rate constants for the transbilayer movement led to a small overestimation of the corresponding half-times.


[page 55↓]

Table 3: Half-times of transbilayer movement of M-C6-NBD-PE across E.coli -derived proteoliposomes determined by the dithionite assay. Three independent experiments (n=3) were analyzed by the modified three-compartment model.

protein content [µg/ml]

outward movement [s]

inward movement [s]

dithionite reduction [s]

fluorescence amplitude [%]

76

47.9 ± 6

135 ± 20.1

4.18 ± 0.55

68.0

87

40.6 ± 2.7

149.6 ± 18.9

5.96 ± 0.40

76.3

169

36.3 ± 6.1

156 ± 49.8

5.76 ± 0.56

79.7

However, the findings with the alternative dithionite stopped-flow assay further support the hypothesis that the transmembrane movement and the resulting distribution of the fluorescent PE analogue is fast and mediated by bacterial proteins.


[page 56↓]

4.5  Effect of the chain length of fluorescent phospholipid analogues on the transbilayer movement across IIMV-derived membranes

So far, short-chain fluorescent phospholipid analogues were used to study the transbilayer movement of phospholipids across IIMV membranes, taking advantage of the rapid and quantitative extractability of such analogues by BSA. Although the phospholipid analogues used, have structural features of endogenous phospholipids, it has to be taken into account, that these analogues possibly do not adequately mimic the endogenous phospholipids. It is conceivable that the particular structure of the fluorescent analogue may lead to different results than those obtained with natural phospholipids. To address whether long-chain or head-group labeled phospholipid analogues behave similarly to the so-far used short-chain fluorescent phospholipids, in terms of transbilayer movement and initial distribution between the two leaflets, experiments were carried out with reconstituted (proteo)liposomes containing the long-chain fluorescent analogue palmitoyl-dodecan-NBD-PE (P-C12-NBD-PE) or the head-group labeled N- NBD-dipalmitoyl- PE (N-DP-NBD-PE). Since BSA is not able to extract both long-chain phospholipid analogues efficiently and fast enough to get adequately rate constants for model analysis due to strong hydrophobicity of the longer fatty acids, a combined dithionite stopped-flow assay was established. Aliquots of IIMV were reconstituted and symmetrically labeled as described in chapter 3.3. Afterwards, equal volumes of labeled (proteo)liposomes and dithionite solution (10 mM final concentration) were mixed by stopped-flow, and the decrease of fluorescence intensities was monitored (chapter 3.6 ).

In Figure 17 kinetics of fluorescence reduction of long-chain fluorescent analogues due to chemical quenching by dithionite are displayed. Analyzing proteoliposomes labeled with either P-C12-NBD-PE or N-DP-NBD-PE, the fluorescence intensity decayed in two distinct phases (solid blue and black line in Figure 17). This was in agreement with the findings of the BSA back-extraction of NBD-labeled short-chain phospholipid analogues (chapter 4.2; 4.3). The rapid first phase reflects the fluorescence quenching of NBD-molecules that initially resided in the outer leaflet of the membrane of proteoliposomes and were therefore immediately accessible for dithionite. Phospholipid analogues residing


[page 57↓]

Figure 17 : Influence of the chain length of analogues on the rapid transbilayer movement of fluorescent phospholipid analogues across the membrane of proteoliposomes derived from IIMV. The proteoliposomes were labeled with 0.5 mol% of the respective analogue during the reconstitution procedure (see 3.3 ). Equal volumes of the vesicle suspension and 20 mM dithionite were mixed in the stopped-flow accessory, and the fluorescence decrease was monitored at room temperature. The fluorescence traces represent the average of three separate kinetics. Each kinetics was normalized to the maximum fluorescence intensity.

at the lumenal leaflet of the membrane became accessible for dithionite quenching, only after they were translocated to the exoplasmic leaflet by the putative flippase, resulting in a second slower phase of fluorescence decrease (Figure 17). In the time course of the experiment (300 s), the fluorescence decreased to ~20% of the initial fluorescence intensity for the P-C12-NBD-PE and to ~30% for the head-group labeled phospholipid analogues used in this approach (Figure 17). As evident from Figure 17, the initial phase of fluorescence reduction of N-DP-NBD-PE was slower than for P-C12-NBD-PE. This indicates that the reduction of head-group labeled phospholipid analogues was possibly restricted due to steric limitations of accessibility of the NBD-group by dithionite (see discussion). Consequently, the quantitative analysis of the transbilayer movement of the fluorescent head-group labeled phospholipid analogues led only to a rough estimate (see below). Nevertheless, N-DP-NBD-PE underwent a transbilayer movement across the proteoliposome membrane. The comparison of the fluorescence traces of protein-free phospholipid vesicles (dotted blue line in Figure 17) and the corresponding proteoliposomes revealed that the presence of [page 58↓]bacterial proteins resulted to an enhanced reduction of fluorescence. From this, it can be concluded that long-chain, head-group labeled analogues moved from the inner leaflet to the outer leaflet of proteoliposomes facilitated by proteins and thereby became accessible for dithionite.

As evident from the fluorescence trace, the transbilayer movement of P-C12-NBD-PE (solid black line Figure 17) in proteoliposomes was very similar to that found for the short-chain phospholipid analogue M-C6-NBD-PE (compare Figure 10 and Figure 13). For protein-free liposomes (dotted lines Figure 17), which were reconstituted in parallel with proteoliposomes but in the absence of bacterial proteins, the fluorescence was quenched to more than 50% of the initial fluorescence intensity. This indicates a symmetrical distribution of the fluorescent analogues between the vesicle leaflets and the inability to cross the membrane due to the strong polar head-group. Nevertheless, a permeation of dithionite under the experimental conditions used in this assay was apparent (see below).

The quantitative analysis of transbilayer movement of long-chain and head-group labeled phospholipid analogues across the membrane of the (proteo)liposomes yielded differing rate constants as those calculated from BSA back-exchange assays (data not shown). As evident from Figure 17, the traces of the control liposomes (without bacterial proteins) did not reach a final plateau of fluorescence in the time course of the experiment. These traces followed a bi-exponential function but not a monoexponential course of fluorescence decrease. This indicates a small penetration of dithionite into the vesicles at room temperature. Although the leakage was small, the permeation of dithionite was not negligible for the estimation of rate constants by fitting. At lower temperatures (15°C), the penetration effect was eliminated (data not shown), but it can not be excluded that the decreased temperature affects the transbilayer movement of the analogues. However, as evident from Figure 17 the long-chain phospholipid analogues undergo a transbilayer movement in the presence of bacterial proteins (from 70% up to 80% fluorescence reduction on proteoliposomes compared to only ~50% - 60% on pure liposome samples).


[page 59↓]

4.6  Protein modifying treatment of reconstituted proteoliposomes

The reconstitution experiments (4.3; 4.4) revealed strong evidences that proteins involved in the flip-flop of fluorescent phospholipid analogues. To prove these findings, the influence of protein modifying substances was investigated.

Figure 18 : Kinetics of the extraction of M-C6-NBD-PE from reconstituted IIMV by BSA prior to and after proteinase K treatment. Reconstituted IIMV-derived proteoliposomes were labeled with two molpercent M-C6-NBD-PE during the reconstitution (see chapter 3.3 ), and a stock was treated with proteinase K (1 mg/ml) for 0 min (black trace), 30 min (red trace) and 60 min (blue trace). Each aliquot was incubated with three mM (final) PMSF for five minutes to terminate the reaction prior to analysis by stopped-flow BSA back-exchange. Traces were corrected for scattering. For comparison, the extraction kinetics measured with protein-free liposomes (gray line) is shown.

A stock of reconstituted IIMV-derived proteoliposomes labeled with two molpercent of M-C6-NBD-PE during the reconstitution was treated with proteinase K (1 mg/ml) for 0 min, 30 min and 60 min prior to analysis by stopped-flow back-exchange. The traces were corrected for scattering (see Material and Methods).

Figure 18 shows the kinetics of the extraction of M-C6-NBD-PE from reconstituted IIMV by BSA prior to and after proteinase K treatment. For comparison, the extraction kinetics measured with protein-free liposomes is shown (gray line). The amount of extractable analogues decreased dramatically after 30 min or 60 min proteinase K treatment as revealed by comparison with kinetics of untreated proteoliposomes (black line) and protein-free liposomes. [page 60↓]This indicates that the proteolysis eliminated protein-mediated flip-flop activity in a large fraction of the proteoliposome population after 30 min of proteinase K treatment (red line). The pool of transport-active vesicles was eliminated by the treatment with proteinase K after 60 min (blue line).


[page 61↓]

4.7  Ion exchange chromatography (IEC) with Triton extracts derived from IIMV of E.coli

In the preceding experiments (see chapter 4.4, 4.3, 4.6), it has been shown that proteins were involved in the transbilayer movement of fluorescent phospholipid analogues across the IIMV membrane (see discussion). In an attempt to identify the protein(s) harboring flippase activity, ion-exchange columns were used to yield protein fractions with flippase activity. 1 ml Hi Trap Q HP columns (Amersham-Pharmacia Biotech), strong anion exchangers, were utilized to separate the detergent solubilized proteins derived from E.coli into two fractions (flow-through and eluate) as described in chapter 3.7. After dialysis of the flow-through and the eluate proteins at room temperature, an aliquot of each fraction was reconstituted, and the resulting proteoliposomes were assayed for transport activity with the dithionite approach at 22°C. In parallel, proteoliposomes were generated from an aliquot of the total extract, i.e. from the TE, which was the starting material for the chromatographic separation of the bacterial proteins. Furthermore, ePC-vesicles were created in the absence of proteins (see chapter 3.3 for the reconstitution procedure).

4.7.1  Efficiency of the separation of proteins from E.coli with IEC

To test whether the IEC is a suitable tool for the separation of proteins from E.coli inner membranes a chromatography experiment using an anion exchange column (3.7) for fractionation was performed (see also 4.7). The resulting flow-through and eluate were analyzed by SDS-PAGE (15%) as described in chapter 3.8 .

As evident from Figure 19, the experimental conditions, used for the fractionation of bacterial proteins derived from IIMV of E.coli with Q-Sepharose, were suitable to separate bacterial proteins by their charges. The comparison of QF (flow-through) and QE (eluate) showed striking qualitative differences in the abundance of proteins in the fractions. From the polyacrylamide gel, it was evident that more proteins were present in the eluate compared to the flow-through. Data from quantitative protein determination proved these observations. [page 62↓]Commonly, about two thirds of the initial protein used for separation were bound to the Q-Sepharose column (data not shown). Moreover, a number of prominent protein bands

Figure 19 : Qualitative analysis of proteins derived from TE of IIMV. A representative polyacrylamide gel is shown. 2.9 mg protein from solubilized IIMV (TE) were applied to an anion exchange column. The resulting flow-through (QF) and eluate (QE) were analyzed by a 15% SDS-PAGE.

in QF were not present in QE and vice versa. Furthermore, several bands in QF were less intensive (less protein) than in QE, which implies that proteins with similar molecular masses but different charges were separated. In summary, it can be concluded from the (qualitative) gel data and quantitative protein determinations that the proteins from the inner membrane of E.coli were successfully separated by their charge(s) using a strong anion exchange column.

4.7.2  Enrichment of flippase activity of inner membrane proteins of E.coli by anion exchange chromatography (AEC)

To analyze the phospholipid transport activity, the resulting fractions (flow-through and eluate) obtained after AEC (3.7, 4.7.1) were reconstituted in the presence of 0.5mol% M-C6-NBD-PE of the total phospholipid content as described in chapter 3.3. In parallel, proteoliposomes from the total TE (which were originally applied to the column) were prepared (3.7). Additionally, protein-[page 63↓]free ePC-vesicles were generated in the presence of Triton X-100 only. In order to get comparable results, the applied total amount of protein for reconstitution was similar in the different samples.

Proteoliposomes and liposomes were analyzed by the dithionite assay in cuvette experiments ( 3.6 ). Upon addition of dithionite to a suspension of liposomes, the fluorescence rapidly decreased to approximately 50% of the initial fluorescence and remained at this level (Figure 20 gray line) until 1% (w/v) Triton X-100 was added. After detergent addition, the fluorescence immediately declined to zero (data not shown). Thus, a pool of fluorescent analogues protected against dithionite reduction existed that only became accessible after membrane disruption by detergent. These data confirmed that dithionite was not permeable or the dithionite penetration was negligible, and the amount of dithionite used was sufficient to reduce all M-C6-NBD-PE present in the sample.

Figure 20 : Comparison of flippase activity of reconstituted protein fractions after IEC. An aliquot TE (1.74mg protein) was applied to an anion exchange (Q-Sepharose) resin and the resulting flow-through (QF) and eluate (QE) were dialyzed. Fractions were taken for reconstitution to generate proteoliposomes with equal protein contents. Subsequently, the accessibility of M-C6-NBD-PE of proteoliposomes and liposomes was analyzed by the dithionite assay. 100 µl of each sample were diluted into 1.9 ml HPS in a cuvette, the fluorescence was recorded for 30 s. Then 10 mM (final) dithionite in 40 mM TRIS pH 8 was added and the resulting decay of fluorescence intensity was recorded for 600 s for each vesicle preparation. The protein concentration of the reconstituted proteoliposomes were 23.2µg/ml, 23.9µg/ml and 14.9µg/ml for pTE, pQF and pQE, respectively.

When assaying proteoliposomes, the fluorescence intensities decreased to less than 50% of the initial value (Figure 20). These data indicated that fluorescent [page 64↓]labeled PE, which originally resided in the inner leaflet of the proteoliposome membrane, crossed the membrane during the assay and subsequently, became accessible for dithionite. The fluorescence reduction of proteoliposomes prepared from the total protein extract (TE) and treated with dithionite was ~71%. The extent of fluorescence reduction of proteoliposomes generated from flow-through (pQF) and the eluate (pQE) of the anion exchange resin was approximately 61% (Figure 20), indicated a possibly flippase activity.

The proteoliposomes were prepared with approximately the same amounts of proteins (23.2 µg/ml, 23.9 µg/ml and 14.9 µg/ml for pTE, pQF and pQE respectively). The fluorescence traces shown in Figure 20 implicate different putative flippase activities in the proteoliposomes derived from both chromatographic fractions compared to proteoliposomes prepared from TE. The different activities were possibly due to altered protein patterns reconstituted into vesicles. As evident from Figure 20, the fluorescence traces monitored for pQF and pQE showed no differences neither in shape nor in the final fluorescence plateau, despite the two analyzed proteoliposomes classes derived from IEC fractions contained different subsets of proteins (Figure 19). This indicates that the ability of both vesicle populations to facilitate transmembrane movement of fluorescent phospholipid analogues was similar and therefore, no substantial separation of the putative flippase protein(s) under these particular conditions occurred.

Table 4: Partial purification of phospholipid flippase activity from E.coli inner membrane TE. The data are representative for five independent experiments.

fraction

activity A [%]

specific activity A S [%*µmol*µg -1 ]

protein/phospholipid ratio [µg/µmol]

TE

19.48

2.44

8.0

QF

9.36

1.11

8.4

QE

8.95

1.99

5.1

Moreover, the flippase activities found for pQF and pQE is less than that found for pTE, indicating no enrichment of flippase activity in the reconstituted fractions of anion exchange chromatography. Based on the data revealed from fluorescence measurements, the specific activities of the QF-, QE- and TE-derived [page 65↓]proteoliposomes were estimated, i.e. calculations of transport activities relative to the protein/phospholipid ratios as described in chapter 3.7. The results are shown in Table 4.

Indeed, the specific activity of phospholipid transport of the TE derived proteoliposomes was higher than those found for the column fractions derived proteoliposomes. Nevertheless, an approximately two-fold enrichment of flippase activity was detected in QE compared to the QF derived vesicles.

Figure 21 : Comparison of flippase activities of flow-through and eluate from Q-Sepharose column and TE. An aliquot of TE was applied to an anion exchange (Q-Sepharose) resin, and the resulting flow-through (QF) and eluate (QE) were dialyzed. Proteoliposomes with equal protein contents and 0.5 mol% M-C6-NBD-PE were reconstituted. Additionally, respective amounts of the originally TE applied to the column, were taken to obtain proteoliposomes (pTE) with the same protein concentration compared to the pQF and pQE and proteoliposomes with two times higher concentration of protein (2xpTE) The protein concentrations of the proteoliposomes were 48.2 µg/ml, 50.9 µg/ml, 47.1 µg/ml and 93.2 µg/ml for pTE, pQF, pQE and 2xpTE, respectively. Subsequently, aliquots of proteoliposomes and control liposomes were analyzed by the dithionite assay (see legend of Figure 20 ).

When increasing amounts of TE were reconstituted into proteoliposomes, the fluorescence reduction was greater than that seen for protein-free liposomes. The final fluorescence plateau depended on the protein/phospholipid ratio as revealed from dithionite and BSA back-extraction experiments (see chapter 4.4.1 and 4.4.2 ). To test, whether the protein content reconstituted from IEC column fractions and TE is sufficient for facilitated phospholipid flip-flop, a number of experiments with varying protein concentrations in the proteoliposomes from QF, [page 66↓]QE and TE were performed. In Figure 21 a representative experiment is displayed. The separated column fraction proteins and TE, respectively, were reconstituted into proteoliposomes containing 0.5 mol% M-C6-NBD-PE with final protein concentrations of 48.2 µg/ml, 50.9 µg/ml and 47.1 µg/ml for pTE, pQF and pQE respectively. Additionally, fluorescent labeled proteoliposomes derived from TE with an approximately doubled protein amount (93.2 µg/ml, referred to 2xpTE) were generated.

Table 5: Partial purification of phospholipid flippase activity from E.coli inner membrane TE. The data are representative for two independent experiments and were calculated from data shown in Figure 21.

fraction

fluorescence reduction [%]

activity A [%]

specific activity A S [%*µmol*µg -1 ]

protein/phospholipid ratio [µg/µmol]

TE

78.4

34.60

3.42

10.13

2xTE

84.6

40.81

1.69

24.21

QF

71.4

27.69

2.38

11.65

QE

72.0

28.27

2.15

13.16

As evident from Figure 21, the fluorescence reduction by dithionite strongly depended on the protein concentration (for comparison see also Figure 20). With increasing protein content in the proteoliposomes, an increasing number of fluorescent PE-analogues were reduced to non fluorescent species. The fluorescence traces monitored for pQF and pQE showed no differences compared to each other, and the amount of reduced analogues was lower compared to pTE (see Table 5). Moreover, proteoliposomes containing a twofold higher amount of proteins (2xpTE) showed an enhanced fluorescence reduction by dithionite (78.4% and 84.6% for pTE and 2xpTE respectively). The specific activity of 2xpTE decreased compared to pTE.

The specific activities of pQF and pQE calculated from the fluorescence measurements shown in Figure 21 were lower compared to pTE but higher than estimated for 2xpTE. Based on enhanced but similar protein contents in the proteoliposomes, no enrichment of phospholipid transport activity was detected (see 5.3). Interestingly, an enriched specific activity of phospholipid flippase [page 67↓]activity could not be exclusively attributed to pQF or pQE as evident from ten independent chromatographic experiments (data not shown).

4.7.3  Successive fractionation of solubilized proteins from IIMV with anion exchange chromatography

To examine the effect of ionic strength on protein separation with anion exchange resin, different elution buffers with varying salt concentrations were tested.

Figure 22:Flippase activities of Q-Sepharose column fractions, liposomes and TE after successive elution. An aliquot of TE was applied to an anion exchange (Q-Sepharose) resin and the resulting flow-through (QF) and eluates (QE4, QE) were dialyzed. Proteoliposomes with equal protein contents and 0.5 mol% M-C6-NBD-PE were generated. For comparison a respective amount of the originally TE applied to the column was reconstituted (pTE) to create proteoliposomes with the same protein concentration compared to the fractions. The protein concentration of the proteoliposomes were 22.8 µg/ml, 15.3 µg/ml, 14.4 µg/ml and 23.5 µg/ml for pTE, pQF, pQE4 and pQE, respectively. Subsequently, aliquots of proteoliposomes and control liposomes were analyzed by the dithionite assay. To this end, 100 µl of sample were diluted in 1.9 ml 10 mM HPS and the fluorescence decrease by reduction with 10 mM dithionite was monitored for 300 s using the stopped-flow accessory. All experiments were carried out at room temperature.

For this reason, aliquots of TE were loaded onto a 1 ml Hi Trap Q HP column (Amersham-Pharmacia Biotech) and passed over the column equilibrated with buffer B. Subsequently, the column was washed with buffer A and the wash and flow-through were pooled. Bound proteins were first eluted with two [page 68↓]milliliters modified buffer D* (buffer D with 0.25 M NaCl instead of 1 M NaCl) and finally eluted with two milliliter buffer D to yield two distinct elution fractions. The column fractions were dialyzed against 10 mM HPS (pH 7.5) containing 0.2% Triton X-100 for 1.5 h at room temperature.

Aliquots of the flow-through and the eluates were reconstituted into ePC vesicles as described in 3.3 and 3.7 to generate M-C6-NBD-PE (0.5 mol%) containing proteoliposomes with approximately similar concentrations of protein. The fluorescence measurements were performed as outlined in chapter 3.5.2.

In Figure 22 a representative experiment is displayed. The final fluorescence intensity of the control liposomes (pure lipid vesicles) leveled off at approximately 51% of the initial fluorescence (Figure 21, gray line). The fluorescence reduction of the proteoliposome samples amounted to more than 50%, indicating that short-chain phospholipid analogues of PE redistributed from the lumenal side of the membrane and became accessible to dithionite quenching. As evident from Figure 22, the flippase activity was different depending on the samples probed. The final fluorescence reduction measured for pQF (59.5% of initial intensity) was less than those monitored for pQE4, pQE and pTE (64.1%, 62.3% and 65.4% respectively), indicating slightly different flippase activities in the vesicles due to differences in the protein composition of the proteoliposomes.

Table 6 : Flippase activity in fractions of anion exchange chromatography. The data are representative for three independent experiments. pTE refers to proteoliposomes prepared from TE of IIMV. pQF, pQE4 and pQE designated for proteoliposomes reconstituted from AEC separated proteins of the flow-through and eluates, respectively.

 

fluorescence reduction [%]

activity A [%]

specific activity A S [%*µmol*µg -1 ]

protein/phospholipid ratio [µg/µmol]

pTE

65.4

16.66

2.03

8.19

pQF

59.5

10.75

2.20

4.89

pQE4

64.1

15.42

1.92

8.04

pQE

62.3

13.55

1.89

7.16

Based on these measurements, the specific activities were calculated to normalize the phospholipid flippase activity to the protein content in the proteoliposomes (3.7). The specific activity of QF derived proteoliposomes is greater than those for all other samples analyzed, although the protein content in [page 69↓]this fraction is lower compared to TE, QE4 and QE (Table 6). Nevertheless, the flippase activity was not significantly enriched in pQF after elution with varying ionic strength.


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