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.

Fusion Domains Guide the Oriented Insertion of Light-Driven Proton Pumps into Liposomes

“…4 C) (20,21). The fluorescent moieties of the PR constructs are only accessible to proteinase K in the extravesicular space, whereas the lipid bilayer acts as a diffusion barrier and prevents inward-facing moieties from digestion by proteinase K (22,23). The analysis of the fluorescence before and after digestion on an SDS-PAGE gel revealed that a vast majority of proteins were inserted in the predefined orientation.…”

Surfactant Assemblies and their Various Possible Roles for the Origin(S) of Life

“…In contrary, the permeability for small neutral molecules (like water, ammonia, urea, methanol) is high. A modulation of the vesicle membrane permeability can be achieved in different ways, for example by varying the vesicle shell composition, e. g. by simply varying the chain length of the lipids (Paula et al, 1996), by adding small amounts (at sub-solubilizing concentrations) of a micelle-forming surfactant (Treyer et al, 2002;Yoshimoto et al, 2004), thereby forming more permeable mixed vesicles. If the micelle-forming surfactant is in excess, however, the vesicles will be transformed into (mixed) micelles, accompanied by a complete release of the originally trapped aqueous content.…”

Organic Nano-Compartments as Biomimetic Reactors and Protocells

“…phatidylcholine (PC) bilayers at sublytic concentrations [77], or ethanol [89] that causes the interdigitation of the two layers of a lipid bilayer [effectively causing the thinning of the bilayer (Fig. (6C)), as well as a significant increase in surface area], can enable the supply of encapsulated enzymes [77,90,91]. The membrane transport can occur in any direction, which depends on the direction of molecular gradients across the membrane.…”