Effects of membrane fluidity and identification of the rate-limiting step in the protein-mediated phosphatidylcholine exchange reaction

Abstract

The properties of the membrane interface play a very significant role in the regulation of the exchange reaction as catalyzed by the phosphatidylcholine (PC) exchange protein from beef liver. The reaction was studied using unilamellar vesicles of egg PC as donor membranes and multilamellar vesicles (MLV's) of dipalmitoyl PC (DPPC) or egg PC containing phosphatidylglycerol as acceptor membranes. MLV's of pure DPPC were found to be poor substrates in the exchange reaction, but could be improved significantly by the addition of DPPG. Maximum exchange occurred when 5 mol% DPPG was incorporated into the liposomes, the reaction being inhibited thereafter. Progress curves for the reaction were also shown to exhibit a biphasic characteristic, where an initial rapid period is followed by a much slower phase. It is postulated that this biphasic characteristic can be accounted for by the changing properties of the donor vesicles as they become coated with DPPC. When annealed and nonannealed MLV's of DPPC were used as acceptors, marked differences in the biphasic characteristic of the progress curves were observed. The half time of the initial rapid period of exchange using nonannealed vesicles was an order of magnitude lower than that for the annealed vesicles, indicating that the exchange protein selectively recognizes structural features present in the nonannealed vesicles. The effect of membrane fluidity was also investigated by the measurement of initial reaction velocities at 37 and 45 °C, below and above the phase transition temperature of DPPC. It was found that the desorption rate constant (k−A) for the DPPC acceptors, which represents the rate-limiting step in the exchange reaction, increases fivefold above the phase transition temperature of this lipid. It seems then that a major factor in determining the rate of the exchange reaction is the facility of extraction of a lipid from the membrane interface.