Grant E, Beeler T J, Taylor K M, Gable K, Roseman M A
Clinical Investigation Division, Brooke Army Medical Center, Fort Sam Houston, Texas 78234.
Biochemistry. 1992 Oct 20;31(41):9912-8. doi: 10.1021/bi00156a008.
The magainins, peptide antibiotics secreted by the frog Xenopus laevis, have previously been shown to permeabilize phospholipid vesicles. To elucidate the mechanism of permeabilization, we have conducted detailed kinetic studies of magainin 2 amide (mgn2a)-induced release of 6-carboxyfluorescein from vesicles of phosphatidylserine. The results show that dye release occurs in (at least) two stages--an initial rapid phase, with t1/2 approximately 3 s, followed by a much slower phase that approaches zero leakage rate before all the dye is released. Light-scattering studies showed that mgn2a does not cause gross changes in vesicle structure. The peptide was found to rapidly equilibrate between vesicles; this was demonstrated by determining a binding isotherm for the peptide-lipid interaction, and by showing that addition of unloaded vesicles rapidly quenches peptide-induced leakage from loaded vesicles. Transient dye release in the presence of an equilibrating peptide can be explained in two ways: (1) the peptide exists only transiently in an active form; (2) the vesicles are only transiently leaky. Preincubation of mgn2a at assay concentrations in buffer alone or with unloaded vesicles did not inactivate the peptide. Therefore, rapid leakage is probably due to transient destabilization of the vesicle upon addition of mgn2a.
爪蟾抗菌肽是由非洲爪蟾分泌的肽类抗生素，此前已证明它能使磷脂囊泡通透。为阐明通透机制，我们对爪蟾抗菌肽2酰胺（mgn2a）诱导磷脂酰丝氨酸囊泡释放6 - 羧基荧光素进行了详细的动力学研究。结果表明，染料释放至少分两个阶段进行：初始快速阶段，半衰期约为3秒，随后是一个慢得多的阶段，在所有染料释放之前泄漏速率趋近于零。光散射研究表明，mgn2a不会引起囊泡结构的总体变化。发现该肽能在囊泡之间迅速达到平衡；这通过测定肽 - 脂质相互作用的结合等温线以及表明添加未装载染料的囊泡能迅速抑制肽诱导的已装载染料囊泡的泄漏得以证明。在存在平衡肽的情况下染料的瞬时释放可以用两种方式解释：（1）肽仅以活性形式短暂存在；（2）囊泡仅短暂渗漏。在缓冲液中单独或与未装载染料的囊泡一起以测定浓度预孵育mgn2a不会使该肽失活。因此，快速泄漏可能是由于添加mgn2a后囊泡的瞬时不稳定所致。
