We previously showed that bovine apolipoprotein A-II (apoA-II) had antimicrobial activity against Escherichia coli and the yeast Saccharomyces cerevisiae in PBS. We have characterized here the active domain of apoA-II using synthetic peptides. A peptide corresponding to C-terminal residues Leu49-Thr76 exhibited significant antimicrobial activity against E. coli in PBS, but not against S. cerevisiae. Experiments using amino-acid-substituted peptides indicated that the residues Phe52-Phe53-Lys54-Lys55 are required for the activity. Peptide Leu49-Thr76 induced the release of calcein trapped inside the vesicles whose lipid composition resembles that of E. coli membrane, suggesting that peptide Leu49-Thr76 can destabilize the E. coli membrane. CD measurements showed that the α-helicity of peptide Leu49-Thr76 increased from 3.5 to 36% by addition of the vesicles. When E. coli cells were incubated with peptide Leu49-Thr76, some proteins were released to the external medium, probably owing to membrane destabilization caused by the peptide. In electron micrographs of E. coli cells treated with peptide Leu49-Thr76, transparent nucleoids and granulated cytoplasm were observed. Amino acid substitutions, Phe52Phe53 → AlaAla (Phe52,53 → Ala) in peptide Leu49-Thr76 caused the loss of antimicrobial activity against E. coli, although protein-releasing activity was retained. Electron micrographs of the cells treated with peptide Leu49-Thr76(Phe52,53 → Ala) revealed morphological change only at the nucleoids. Therefore peptide Leu49-Thr76 appears to primarily target the cytoplasm rather than the membrane of E. coli cells.

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