Fusion of phospholipid vesicles induced by α-lactalbumin and lysozyme

Abstract

$\alpha$-Lactalbumin ($\alpha$-LA) is found to induce fusion of phosphatidylserine/phosphatidylethanolamine (1:1) vesicles at low pH. The fusogenic behavior and the binding to phospholipid vesicles of $\alpha$-LA are studied at a wide range of conditions. The initial rate of $\alpha$-LA-induced fusion increases with decreasing pH. The binding of $\alpha$-LA to phospholipid vesicles also increases with increasing acidity below pH 6, but there is practically no binding at pH 6 and pH 7. By determining the amount of $\alpha$-LA which had been incubated with the $\alpha$-LA at acidic pH (pH 2, 3 and 4), released after neutralization of the vesicles to pH 7, the binding reaction is found to be partially irreversible. The amount of irreversively bound protein also increases with the decreasing pH. As the ionic strength is increased from 0.1 to 0.5, the initial rate of fusion value decreases gradually, but it approaches a fnite value for each $\alpha$-LA concentration. A segment of $\alpha$-LA is found to be resistant to the proteolytic digestion when initially incubated with the vesicles at low pH. Hydrophobic labeling with dansyl chloride renders support that this fragment indeed penetrates into the hydrophobic interior of bilayer. The molecular weight of this segment as determined by SDS-PAGE is found to be approximately 4000 for each pH value studied. The amino acid composition of this segment was determined, and from this the sequence of $\alpha$-LA fragment, which appears to be inserted into the bilayer, is deduced. This is the segment from 80 (Phe) to 108 (Lys) of $\alpha$-LA. Since both the N-terminal and C-terminal of this vesicle-bound $\alpha$-LA are accessible to the externally added proteolytic enzymes, it is concluded that a loop of the polypeptide segment goes into the bilayer. The $\alpha$-LA segment which penetrates into PS/PE (1:1) vesicle bilayer under acidic condition was photoactively labeled with 3-(trifluoromethyl)-3-(m-(125I)iodophenyl)diazirin...