Figure 5

SeHAS inactivated by heat, oxidation or NEM modification does not mediate CB release from extrusion liposomes. A. Heat inactivation. CB-containing liposomes were treated at 30°C as in Fig 2 except that before addition to liposomes, purified SeHAS was either treated at 47°C for 5 min to inactivate the enzyme (black triangles) or left untreated on ice (black circles). FL quenching was monitored as in Fig 2. B. Oxidative inactivation. Purified oxidized SeHAS was prepared by omitting DTT in the elution buffer. Oxidized SeHAS was then either left oxidized (black triangles) or was reduced by treatment with 1 mM DTT for 1 hr at 4°C (black circles) and then added to extrusion liposomes and FL quenching monitored as in Fig 2. The response with reduced and rescued SeHAS is essentially identical to the untreated enzyme in A. C. NEM modification. Purified SeHAS was untreated (black circles) or treated (black triangles) for 15 min at 4°C with 6 mM NEM, which inhibits SeHAS activity (24); DTT (12 mM) was then added to react with the remaining NEM. SeHAS was added to CB-liposomes to a final concentration of 200 nM and FL changes were monitored as in Fig 2. CB-liposomes without SeHAS were also incubated in the same way with NEM and DTT as a control (black squares) to assess possible leakage.