Conformationally changed cytochrome c-mediated fusion of enzyme- and substrate-containing liposomes.
The fusion between enzyme-containing liposomes and substrate-containing liposomes was studied, utilizing conformationally altered cytochrome c as fusion mediator under stress conditions. The liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and liposome aggregation and subsequent liposome fusion were induced by the addition of cytochrome c, which was partially denatured by 0.5 M guanidinium hydrochloride (GuHCl). In the presence of 0.5 M GuHCl, cytochrome c was found to have a significantly large local hydrophobicity which was determined with the aqueous two-phase partitioning method. Under these conditions, cytochrome c could efficiently bind to POPC bilayer membranes as quantitatively evaluated by immobilized liposome chromatography (ILC). The retardation of cytochrome c treated with 0, 0.5, and 1 M GuHCl on ILC could be correlated with the corresponding local hydrophobicity of cytochrome c. The enzymatic reaction triggered by liposome fusion involved the proteolytic enzyme alpha-chymotrypsin and its substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA), which were separately trapped in POPC liposomes. Addition of partially denatured cytochrome c (most likely in the molten globule state) to the mixture of enzyme- and substrate-containing liposomes resulted in the release of one of the hydrolysis products, p-nitroaniline, to the outer phase of the fused liposomes, indicating that the enzymatic reaction occurred during the liposome fusion process. Such a coupled fusion-reaction system may have specific advantages over the conventional fusion analysis and may find application as drug delivery system.
Yoshimoto
M
M
Department of Chemical Science and Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan, and Institut für Polymere, ETH-Zentrum, Universitatstrasse 6, CH-8092 Zü.
Walde
P
P
Umakoshi
H
H
Kuboi
R
R
D002463:Cell Membrane Permeability; D002863:Chromogenic Compounds / Q000737:chemistry / Q000378:metabolism; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes; D008560:Membrane Fluidity; D008561:Membrane Fusion; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation
D002863:Chromogenic Compounds; D003574:Cytochrome c Group; D008051:Lipid Bilayers; D008081:Liposomes; D009842:Oligopeptides; D010713:Phosphatidylcholines; C043307:succinyl-alanyl-alanyl-prolyl-phenylalanine-4-nitroanilide; D002918:Chymotrypsin; C028694:1-palmitoyl-2-oleoylphosphatidylcholine