2.1. Preparation of brush border membrane vesicles (BBMV)

Early fourth instar larvae of black cutworm were kept on ice for an hour and were then dissected to remove their midguts. BBMV were prepared from the midgut tissues using differential magnesium precipitation method given by Wolfersberger et al. (1987), in the presence of protease inhibitors [5 mg/mL pepstatin, antipain, aprotonin, and leupeptin; 1 mM PMSF; 5 mM benzamidine]. The midgut tissue was homogenized in 9 vol buffer A [300 mM mannitol; 5 mM EGTA; 17 mM Tris-HCl] using a glass Teflon homogenizer [9 up-and-down strokes at 3000 rpm]. An equal volume of 24 mM MgCl₂ solution was added to the homogenate, which was then re-homogenized. The homogenate thus obtained was left on ice for 15 min and then centrifuged at 4 °C, at 4500 rpm for 15 min. The supernatant was decanted into a fresh tube and centrifuged again at 4 °C, at 31,000g for 30 min. The supernatant obtained was discarded and the pellet was resuspended in a half volume of buffer A; the procedure was repeated with this suspension. The final pellet was resuspended in buffer A containing protease inhibitors, flash-frozen in liquid nitrogen, and stored at –85 °C.