Francis B R, Bulla L A
Department of Molecular Biology, University of Wyoming, Laramie 82071, USA.
Insect Biochem Mol Biol. 1997 Jun;27(6):541-50. doi: 10.1016/s0965-1748(97)00029-5.
BT-R1, 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-R1 were tested 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 presence of Ca2+, BT-R1 was stable for up to 48 h but a 65% loss of binding was observed after 100 h. Under the same conditions, no loss of binding was observed in the presence of EGTA after 100 h. Cry1Ab binding decreased markedly as pH increased from 6 to 10 for incubations of 24 h at 4 degrees C. Increasing the temperature of incubation from 4 to 37 degrees C also decreased Cry1Ab binding. Neither metal ions nor free sulfhydryl groups are involved in Cry1Ab binding to BT-R1. A trypsin-like, metal-ion-dependent proteolytic activity co-eluted with BT-R1 during gel filtration. This endoproteolytic activity was unaltered by the addition of Cry1Ab. BT-R1 did not co-elute with peaks of aminopeptidase, alkaline phosphatase, alpha-glucosidase, beta-glucosidase and beta-galactosidase activities. When BT-R1 in the gel filtration fraction was further purified on a Mono Q anion exchange column, partial separation of the trypsin-like activity from BT-R1 was observed. BT-R1 could be removed from the appropriate Mono Q fraction by immunoprecipitation with only a slight decrease in this activity. These results demonstrate that there is no copurification of BT-R1 and these enzymes and that BT-R1 is unlikely to form complexes with them. Binding of Cry1Aa and Cry1Ac to BT-R1 in gel filtration fractions is similar to that of Cry1Ab, indicating that BT-R1 may be the high-affinity receptor for the Cry1A toxins. Binding of Cry1Ab to a 120 kDa protein has not been observed in this study.
BT-R1是烟草天蛾中肠对苏云金芽孢杆菌亚种berliner产生的晶体毒素Cry1Ab的受体，在去污剂CHAPS存在的情况下，通过凝胶过滤从烟草天蛾刷状缘膜囊泡中部分纯化得到。通过配体印迹上Cry1Ab结合的保留情况来检测含有BT-R1的组分对降解的稳定性。在4℃、pH 7.4且存在Ca2+的条件下，BT-R1在长达48小时内保持稳定，但100小时后观察到结合能力丧失65%。在相同条件下，100小时后在EGTA存在的情况下未观察到结合能力丧失。在4℃孵育24小时时，随着pH从6增加到10，Cry1Ab结合显著降低。将孵育温度从4℃提高到37℃也会降低Cry1Ab结合。金属离子和游离巯基均不参与Cry1Ab与BT-R1的结合。在凝胶过滤过程中，一种类胰蛋白酶的、金属离子依赖性的蛋白水解活性与BT-R1共洗脱。添加Cry1Ab不会改变这种内切蛋白水解活性。BT-R1不会与氨肽酶、碱性磷酸酶、α-葡萄糖苷酶、β-葡萄糖苷酶和β-半乳糖苷酶活性峰共洗脱。当凝胶过滤组分中的BT-R1在Mono Q阴离子交换柱上进一步纯化时，观察到类胰蛋白酶活性与BT-R1部分分离。通过免疫沉淀可以从合适的Mono Q组分中去除BT-R1，而这种活性仅略有下降。这些结果表明BT-R1与这些酶不会共纯化，并且BT-R1不太可能与它们形成复合物。凝胶过滤组分中Cry1Aa和Cry1Ac与BT-R1的结合与Cry1Ab相似，表明BT-R1可能是Cry1A毒素的高亲和力受体。在本研究中未观察到Cry1Ab与120 kDa蛋白的结合。
