title: Enhancement of apparent substrate selectivity of proteinase K encapsulated in liposomes through a cholate-induced alteration of the bilayer permeability.
abstract: Proteinase K-containing liposomes with highly selective membrane permeability properties were prepared. The selectivity obtained was with respect to the two substrate molecules added to the external aqueous phase of the liposomes: acetyl-L-Ala-Ala-Ala-p-nitroanilide (Ac-AAA-pNA) and succinyl-L-Ala-Ala-Ala-p-nitroanilide (Suc-AAA-pNA). The liposome-forming lipid used was POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and modulation of the membrane permeability was achieved using the detergent cholate. Proteinase K-containing mixed liposomes (PKCL) were prepared by adding cholate to preformed proteinase K-containing POPC liposomes (PKL) at a defined effective cholate/POPC molar ratio in the liposomal bilayer membrane R(e). Proteinase K was kept inside PKCL with a negligible amount of leakage into the bulk aqueous phase at R(e) < or = 0.30. At higher R(e), leakage of proteinase K was pronounced, even under conditions where POPC/cholate mixed liposomes seemed to be still intact (0.30 < R(e) < or = 0.39). At R(e) < or = 0.30, the reactivity of proteinase K in the PKCL measured with the externally added substrate Ac-AAA-pNA increased with increasing R(e), while the reactivity measured with Suc-AAA-pNA remained low, regardless of the R(e) value. This showed that externally added Ac-AAA-pNA molecules permeated the liposomal membrane more easily than Suc-AAA-pNA by modulating the membrane with cholate. Consequently, Ac-AAA-pNA was hydrolyzed in PKCL with considerably higher apparent substrate selectivity in comparison with the cases of proteinase K in PKL and free proteinase K (without liposomal encapsulation). The results obtained clearly demonstrate that the prepared PKCL can be utilized as a kind of nano-scaled bioreactor system which can take up a particular target substrate with high apparent substrate selectively from the external phase of the liposomes. Inside the liposomes, the target substrate is then converted into the corresponding products.
authors.affiliation: Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube, 755-8611 Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Yoshimoto
authors.forename: Shaoqing
authors.initials: S
authors.lastname: Wang
authors.forename: Kimitoshi
authors.initials: K
authors.lastname: Fukunaga
authors.forename: Mike
authors.initials: M
authors.lastname: Treyer
authors.forename: Peter
authors.initials: P
authors.lastname: Walde
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Kuboi
authors.forename: Katsumi
authors.initials: K
authors.lastname: Nakao
mesh_terms: D020355:Cholates / Q000737:chemistry*; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D004058:Diffusion; D019286:Endopeptidase K / Q000737:chemistry*; D004789:Enzyme Activation; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D010539:Permeability; D010713:Phosphatidylcholines / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D020355:Cholates; D020099:Coated Materials, Biocompatible; D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D019286:Endopeptidase K; C028694:1-palmitoyl-2-oleoylphosphatidylcholine