Comp. Biochem. Physiol.Vol. 102B,No. 3, pp. 605-610, 1992 Printed in Great Britain
0305-0491/92 $5.00+ 0.00 © 1992Pergamon Press Ltd
ISOLATION A N D PARTIAL CHARACTERIZATION OF BINDING PROTEINS FOR IMMOBILIZED DELTA ENDOTOXIN FROM SOLUBILIZED BRUSH BORDER MEMBRANE VESICLES OF THE SILKWORM, BOMBYX MORI, A N D THE COMMON CUTWORM, SPODOPTERA LITURA LESLIE S. INDRASITHand HIDETAKAHORI* Laboratory of Biochemistry and Genetic Engineering, Tsukuba Laboratories of Research and Development, Kubota Corporation, Koyodai, Ryugasaki, Ibaraki 301, Japan (Received 28 October 1991)
Brush border membrane vesicles (BBMV) were prepared from the Bombyx mori, and Spodoptera litura, midguts. BBMV was solubilized. 2. Activated delta endotoxin from Bacillus thuringiensis were immobilized. 3. Solubilized BBMV proteins were applied to the toxin column and the proteins bound were analyzed by SDS-PAGE. 4. In the case of B. mori M r = 220,000, 150,000 and 130,000 and in the case of S. litura 160,000 bands were detected. 5. The bindings were inhibited by N-acetyl galactosamine and mannose. 6. The binding proteins applied onto a Con A column and eluted by 0.1 M methyl-or-glucose,suggesting that hybrid type sugar sidechain may involve in the interaction. Abstract--1.
INTRODUCTION
Insecticidal crystal proteins (ICPs) from Bacillus thuringiensis subsp, kurstaki HD-1 (Btk HD-I) are digested by proteinases in larval gut juice and truncated toxin protein with a molecular size of 60-70,000 was found to attack midgut epithelial cells (Tojo and Aizawa, 1983; Lilley et al., 1980). The activated toxin may then disrupt the Na ÷ and/or K ÷ ion balance of the midgut epithelial cells (Himeno, 1988) or it may make a hole on the plasma membrane through which ion or various molecules can pass (Knowles and Ellar, 1986). Precise insecticidal mechanism of ICP, however, still remains to be resolved. Ellar and his colleagues revealed the characteristics of the plasma membrane receptor for the ICP from B. thuringiensis subsp. Israelensis. ICPs bound to the liposomes containing phosphatidyl choline, sphingomyelin, phosphatidyl ethanol amine, and phosphatidyl serine (Thomas et al., 1983). This may explain the mechanism of cytolytic toxin, i.e. Cyt A protein of Israelensis, against larvae. For the toxin specific to insect midgut cells, however, different binding mechanisms should be necessary. Knowles et al. (1984) showed that the toxicity of the CrylA
*To whom all correspondence should be addressed. Abbreviations--Me-Man:methyl
0305-0491/92 $5.00+ 0.00 © 1992Pergamon Press Ltd
ISOLATION A N D PARTIAL CHARACTERIZATION OF BINDING PROTEINS FOR IMMOBILIZED DELTA ENDOTOXIN FROM SOLUBILIZED BRUSH BORDER MEMBRANE VESICLES OF THE SILKWORM, BOMBYX MORI, A N D THE COMMON CUTWORM, SPODOPTERA LITURA LESLIE S. INDRASITHand HIDETAKAHORI* Laboratory of Biochemistry and Genetic Engineering, Tsukuba Laboratories of Research and Development, Kubota Corporation, Koyodai, Ryugasaki, Ibaraki 301, Japan (Received 28 October 1991)
Brush border membrane vesicles (BBMV) were prepared from the Bombyx mori, and Spodoptera litura, midguts. BBMV was solubilized. 2. Activated delta endotoxin from Bacillus thuringiensis were immobilized. 3. Solubilized BBMV proteins were applied to the toxin column and the proteins bound were analyzed by SDS-PAGE. 4. In the case of B. mori M r = 220,000, 150,000 and 130,000 and in the case of S. litura 160,000 bands were detected. 5. The bindings were inhibited by N-acetyl galactosamine and mannose. 6. The binding proteins applied onto a Con A column and eluted by 0.1 M methyl-or-glucose,suggesting that hybrid type sugar sidechain may involve in the interaction. Abstract--1.
INTRODUCTION
Insecticidal crystal proteins (ICPs) from Bacillus thuringiensis subsp, kurstaki HD-1 (Btk HD-I) are digested by proteinases in larval gut juice and truncated toxin protein with a molecular size of 60-70,000 was found to attack midgut epithelial cells (Tojo and Aizawa, 1983; Lilley et al., 1980). The activated toxin may then disrupt the Na ÷ and/or K ÷ ion balance of the midgut epithelial cells (Himeno, 1988) or it may make a hole on the plasma membrane through which ion or various molecules can pass (Knowles and Ellar, 1986). Precise insecticidal mechanism of ICP, however, still remains to be resolved. Ellar and his colleagues revealed the characteristics of the plasma membrane receptor for the ICP from B. thuringiensis subsp. Israelensis. ICPs bound to the liposomes containing phosphatidyl choline, sphingomyelin, phosphatidyl ethanol amine, and phosphatidyl serine (Thomas et al., 1983). This may explain the mechanism of cytolytic toxin, i.e. Cyt A protein of Israelensis, against larvae. For the toxin specific to insect midgut cells, however, different binding mechanisms should be necessary. Knowles et al. (1984) showed that the toxicity of the CrylA
*To whom all correspondence should be addressed. Abbreviations--Me-Man:methyl
