FURTHER CHARACTERIZATION OF BT-R-1, THE CADHERIN-LIKE RECEPTOR FOR CRY1AB TOXIN IN TOBACCO HORNWORM (MANDUCA-SEXTA) MIDGUTS

BT-R-1, the Manduca sexta midgut receptor for the crystal toxin Cry1Ab produced by Bacillus thuringiensis ssp, berliner, was partly purified by gel filtration from M, sexta brush border membrane vesicles in the presence of the detergent CHAPS, Fractions containing BT-R-1 were tes ted for their stability against degradation as indicated by retention of Cry1Ab binding on ligand blots, At 4 degrees C and pH 7.4 in the pr esence of Ca2+, BT-R-1 was stable for up to 48 h but a 65% loss of bin ding was observed after 100 h, Under the same conditions, no loss of b inding was observed in the presence of EGTA after 100 h, Cry1Ab bindin g decreased markedly as pH increased from 6 to 10 for incubations of 2 4 h at 4 degrees C, Increasing the temperature of incubation from 4 to 37 degrees C also decreased Cry1Ab binding, Neither metal ions nor fr ee sulfhydryl groups are involved in Cry1AB binding to BT-R-1. A tryps in-like, metal-ion-dependent proteolytic activity co-eluted with BT-R- 1 during gel filtration, This endoproteolytic activity was unaltered b y the addition of Cry1Ab, BT-R-1 did not co-elute with peaks of aminop eptidase, alkaline phosphatase, alpha-glucosidase, beta-glucosidase an d beta-galactosidase activities, When BT-R-1 in the gel filtration fra ction was further purified on a Mono Q anion exchange column, ;partial separation of the trypsin-like activity from BT-R-1 was observed, BT- R-1 could be removed from the appropriate Mono Q fraction by immunopre cipitation with only a slight decrease in this activity, These results demonstrate that there is no copurification of BT-R-1 and these enzym es and that BT-R-1 is unlikely to form complexes with them, Binding of Cry1Aa and Cry1Ac to BT-R-1 in gel filtration fractions is similar to that of Cry1Ab, indicating that BT-R-1 may be the high-affinity recep tor for the Cry1A toxins. Binding of Cry1Ab to a 120 kDa protein has n ot been observed in this study, (C) 1997 Elsevier Science Ltd.