Kinetics and mechanism of transfer of reduced and carboxymethylated apolipoprotein A-II between phospholipid vesicles

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Abstract
The transfer of 14C-labeled, reduced and carboxymethylated human apolipoprotein A-II (RCM-AII) between small unilamellar vesicles (SUV) has been investigated. Ion-exchange chromatography was used for rapid separation of negatively charged egg phosphatidylcholine (PC)/dicetyl phosphate donor SUV containing bound 14C-labeled RCM-AII from neutral egg PC acceptor SUV present in 10-fold molar excess. The kinetics of 14C-labeled RCM-AII transfer in incubations of up to 12 h at 37.degree. C are consistent with the existence of fast, slow, and apparently "nontransferrable" pools of SUV-associated apolipoprotein; the transfers from these pools occur on the time scales of seconds or less, hours, and days/weeks, respectively. For donor SUV (0.15 mg of phospholipid/mL reaction mixture) containing about 15 RCM-AII molecules per vesicle, the sizes of the fast, slow, and nontransferrable pools are 13, 69, and 18%, respectively. The transfer of RCM-AII from the slow kinetic pool follows first-order kinetics, and the half-time (t1/2) is about 3 h. The different kinetic pools of SUV-associated RCM-AII probably reflect apoprotein in different conformations of the SUV surface. Increasing the number of RCM-AII per donor SUV enlarges the size of the fast pool and increases the t1/2 of transfer from the slow pool. In contrast, raising the incubation temperature reduces the t1/2 of slow transfer. The t1/2 of RCM-AII transfer from the slow kinetic pool is inversely proportional to the acceptor/donor SUV ratio which suggests that the transfer of apoprotein molecules in this kinetic pool is mediated by SUV collisions. The transition state probably involves the desorption of some segments of the transferring apolipoprotein molecule from the surface of the donor SUV and the adsorption of these segments to the acceptor SUV to form a transient, ternary complex.