Kim J, Kim H
Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology, Seoul.
Arch Biochem Biophys. 1989 Oct;274(1):100-8. doi: 10.1016/0003-9861(89)90420-7.
The lysozyme-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles as studied at a wide range of pH is found to correlate well with the binding of this protein to the vesicles. An identical 6000 molecular weight segment of lysozyme at the N-terminal region is found to be protected from tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride, which is partitioned into the bilayer. These results suggest the penetration of one segment of lysozyme into the bilayer. Photoactivated labeling of the membrane-penetrating segment of lysozyme with 3-(trifluoromethyl)-3-([125I]iodophenyl)diazirine ([125I]TID) and subsequent identification of the labeled residues by Edman degradation and gamma-ray counting indicate that four amino acids from the N-terminal are located outside the hydrophobic core of the bilayer. Although treatment of the membrane-embedded segment with aminopeptidase failed to cleave any amino acids from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into the bilayer at acidic pH. A helical wheel diagram shows that the labeling is done mainly on one surface of the alpha-helix. The penetration kinetics as studied by time-dependent [125I]TID labeling coincide with the fusion kinetics, strongly suggesting that the penetration of the lysozyme segment into the vesicles is the cause of the fusion.
在广泛的pH范围内研究发现，溶菌酶诱导的磷脂酰丝氨酸/磷脂酰乙醇胺囊泡融合与该蛋白质与囊泡的结合密切相关。当在几个pH值下最初与囊泡一起孵育时，发现溶菌酶N端区域相同的6000分子量片段可免受胰蛋白酶消化。只有这个片段被丹磺酰氯标记，丹磺酰氯被分配到双层中。这些结果表明溶菌酶的一个片段渗透到了双层中。用3-(三氟甲基)-3-([125I]碘苯基)重氮甲烷([125I]TID)对溶菌酶的膜穿透片段进行光活化标记，随后通过埃德曼降解和γ射线计数鉴定标记的残基，结果表明N端的四个氨基酸位于双层疏水核心之外。尽管用氨肽酶处理膜嵌入片段未能从N端切割任何氨基酸，但似乎在酸性pH下，溶菌酶片段靠近N端的一个环会渗透到双层中。螺旋轮图显示标记主要在α螺旋的一个表面上进行。通过时间依赖性[125I]TID标记研究的渗透动力学与融合动力学一致，强烈表明溶菌酶片段渗透到囊泡中是融合的原因。
