Flip-Flop of Phospholipids in Proteoliposomes Reconstituted from Detergent Extract of Chloroplast Membranes: Kinetics and Phospholipid Specificity
Figure 5
Biogenic membrane flippase activity in intact chloroplasts.
(A) The level of translocation of NBD-PC increases with an increase in the amount of protein in the proteoliposome reconstituted with intact chloroplast (trace a – Liposome, b – 30 µl TE, c- 60 µl TE) (P<0.001) (B) Linear relationship between the amounts of TE to PPR in the reconstituted vesicles. (C) The extent of dithionite reduction of NBD-PC depends on protein to PL ratio (PPR). Flippase activity increases proportionately with an increase in PPR and saturates at a PPR∼400 mg/mmol. Above this value the percentage of fluorescence quenching remains unchanged for proteoliposomes. Kinetics of NBD-PC flipping - The calculation of half-life time was performed using the equation F(t) = F0−[A1 exp(−k1t)+A2 exp(−k2t)] where, F(t) is the fluorescence as a function of time and F0 is the fluorescence intensity at time = 0 s (i.e. initial fluorescence of the vesicles), k1 and k2 are the rate constants for the first (fast) and second (slow) phases respectively. A1 and A2 are the amplitudes of the fast and slow phases respectively. (D) The first phase half-life time remained constant at ∼0.6 min±0.05 min which represents the quenching of fluorescently labeled outer leaflet lipids. (E) The second phase half-life time decreased with increase in PPR suggesting that the kinetics of flipping increased and stabilized at a PPR of ∼400 mg/mmol. All the experiments were carried out 3 independent times.