Incorporation of Lysozyme into Liposomes

To define a model for enzyme latency in vitro, the capture was studied of egg white lysozyme by liposomes (phospholipid spherules in the smectic mesophase). Gel filtration resolved liposomes formed in the absence of enzyme from free enzyme when both were chromatographed immediately after mixing. In contrast, if liposomes were swollen in the presence of lysozyme, subsequent chromatography disclosed two peaks of enzyme activity: one associated with liposomes, the other emerging as free Iysozyme. The activity of lysozyme associated with liposomes was “latent”; i.e. there was little or no activity on substrate (Micrococcus Zysodeikticus cell walls) unless the spherules were disrupted by Triton X-100, amphotericin B, or nystatin. Lysozyme unassociated with lipid was as available to substrate in the absence of detergent or polyenes as in their presence. Charge-induced associations were not crucial for enzyme capture since both positively (lecithin-stearylamine-cholesterol) and negatively charged liposomes (lecithin-dicetylphosphate-cholesterol) captured lysozyme, in latent form. As a measure of their integrity, liposomes could also be shown to capture a marker molecule (glucose), the bulk of which was released together with lysozyme from liposomes by detergent or polyenes. As the aqueous interspaces of positively charged liposomes were increased by incremental incorporation of stearylamine, capture of both glucose and lysozyme was increased proportionally. For these and other reasons it was considered likely that the liposomes had captured lysozyme in the interspaces between lipid lamellae.