Figure 8.

Fusion of isolated ZGs at porosome-reconstituted bilayer and GTP-induced expulsion of α-amylase. (A) Schematic diagram of the EPC9 bilayer apparatus showing the cis and trans chambers. Isolated ZGs when added to the cis chamber, fuse at the bilayers-reconstituted porosome. Addition of GTP to the cis chamber induces ZG swelling and expulsion of its contents such as α-amylase to the trans bilayers chamber. (B) Capacitance traces of the lipid bilayer from three separate experiments following reconstitution of porosomes (green arrowhead), addition of ZGs to the cis chamber (blue arrowhead), and the red arrowhead represents the 5 min time point after ZG addition. Note the small increase in membrane capacitance following porosome reconstitution, and a greater increase when ZGs fuse at the bilayers. (C) In a separate experiment, 15 min after addition of ZGs to the cis chamber, 20 μM GTP was introduced. Note the increase in capacitance, demonstrating potentiation of ZG fusion. Flickers in current trace represent current activity. (D) Immunoblot analysis of α-amylase in the trans chamber fluid at different times following exposure to ZGs and GTP. Note the undetectable levels of α-amylase even up to 15 min following ZG fusion at the bilayer. However, following exposure to GTP, significant amounts of α-amylase from within ZGs were expelled into the trans bilayers chamber. (n=6) [8].