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. 1995 Jul;61(7):2738–2744. doi: 10.1128/aem.61.7.2738-2744.1995

Comparison of toxin overlay and solid-phase binding assays to identify diverse CryIA(c) toxin-binding proteins in Heliothis virescens midgut.

E A Cowles 1, H Yunovitz 1, J F Charles 1, S S Gill 1
PMCID: PMC167546  PMID: 7618886

Abstract

The binding proteins, or receptors, for insecticidal Bacillus thuringiensis subsp. kurstaki delta-endotoxins are located in the brush border membranes of susceptible insect midguts. The interaction of one of these toxins, CryIA(c), with proteins isolated from Heliothis virescens larval midguts was investigated. To facilitate the identification of solubilized putative toxin-binding proteins, a solid-phase binding assay was developed and compared with toxin overlay assays. The overlay assays demonstrated that a number of proteins of 170, 140, 120, 90, 75, 60, and 50 kDa bound the radiolabeled CryIA(c) toxin. Anion-exchange fractionation allowed the separation of these proteins into three toxin binding fractions, or pools. Toxin overlay assays demonstrated that although the three pools had distinct protein profiles, similar-size proteins could be detected in these three pools. However, determination of toxin affinity by using the solid-phase binding assay showed that only one of the three pools contained high-affinity binding proteins. The Kd obtained, 0.65 nM, is similar to that of the unsolubilized brush border membrane vesicles. Thus, the solid-phase binding assay in combination with the toxin overlay assay facilitates the identification and purification of high-affinity B. thuringiensis toxin-binding proteins from the insect midgut.

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Selected References

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  1. Adang M. J., Staver M. J., Rocheleau T. A., Leighton J., Barker R. F., Thompson D. V. Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta. Gene. 1985;36(3):289–300. doi: 10.1016/0378-1119(85)90184-2. [DOI] [PubMed] [Google Scholar]
  2. Bulla L. A., Jr, Bechtel D. B., Kramer K. J., Shethna Y. I., Aronson A. I., Fitz-James P. C. Ultrastructure, physiology, and biochemistry of Bacillus thuringiensis. Crit Rev Microbiol. 1980;8(2):147–204. doi: 10.3109/10408418009081124. [DOI] [PubMed] [Google Scholar]
  3. Ferré J., Real M. D., Van Rie J., Jansens S., Peferoen M. Resistance to the Bacillus thuringiensis bioinsecticide in a field population of Plutella xylostella is due to a change in a midgut membrane receptor. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5119–5123. doi: 10.1073/pnas.88.12.5119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Garczynski S. F., Crim J. W., Adang M. J. Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis. Appl Environ Microbiol. 1991 Oct;57(10):2816–2820. doi: 10.1128/aem.57.10.2816-2820.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gill S. S., Cowles E. A., Pietrantonio P. V. The mode of action of Bacillus thuringiensis endotoxins. Annu Rev Entomol. 1992;37:615–636. doi: 10.1146/annurev.en.37.010192.003151. [DOI] [PubMed] [Google Scholar]
  6. Gould F., Martinez-Ramirez A., Anderson A., Ferre J., Silva F. J., Moar W. J. Broad-spectrum resistance to Bacillus thuringiensis toxins in Heliothis virescens. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7986–7990. doi: 10.1073/pnas.89.17.7986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hofmann C., Vanderbruggen H., Höfte H., Van Rie J., Jansens S., Van Mellaert H. Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7844–7848. doi: 10.1073/pnas.85.21.7844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Höfte H., Whiteley H. R. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev. 1989 Jun;53(2):242–255. doi: 10.1128/mr.53.2.242-255.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Knight P. J., Crickmore N., Ellar D. J. The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N. Mol Microbiol. 1994 Feb;11(3):429–436. doi: 10.1111/j.1365-2958.1994.tb00324.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Knowles B. H., Knight P. J., Ellar D. J. N-acetyl galactosamine is part of the receptor in insect gut epithelia that recognizes an insecticidal protein from Bacillus thuringiensis. Proc Biol Sci. 1991 Jul 22;245(1312):31–35. doi: 10.1098/rspb.1991.0084. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. MacIntosh S. C., Stone T. B., Jokerst R. S., Fuchs R. L. Binding of Bacillus thuringiensis proteins to a laboratory-selected line of Heliothis virescens. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8930–8933. doi: 10.1073/pnas.88.20.8930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Oddou P., Hartmann H., Geiser M. Identification and characterization of Heliothis virescens midgut membrane proteins binding Bacillus thuringiensis delta-endotoxins. Eur J Biochem. 1991 Dec 5;202(2):673–680. doi: 10.1111/j.1432-1033.1991.tb16422.x. [DOI] [PubMed] [Google Scholar]
  14. Oddou P., Hartmann H., Radecke F., Geiser M. Immunologically unrelated Heliothis sp. and Spodoptera sp. midgut membrane-proteins bind Bacillus thuringiensis CryIA(b) delta-endotoxin. Eur J Biochem. 1993 Feb 15;212(1):145–150. doi: 10.1111/j.1432-1033.1993.tb17644.x. [DOI] [PubMed] [Google Scholar]
  15. Rovati G. E., Rodbard D., Munson P. J. DESIGN: computerized optimization of experimental design for estimating Kd and Bmax in ligand binding experiments. II. Simultaneous analysis of homologous and heterologous competition curves and analysis blocking and of "multiligand" dose-response surfaces. Anal Biochem. 1990 Jan;184(1):172–183. doi: 10.1016/0003-2697(90)90030-d. [DOI] [PubMed] [Google Scholar]
  16. Sanchis V., Ellar D. J. Identification and partial purification of a Bacillus thuringiensis CryIC delta-endotoxin binding protein from Spodoptera littoralis gut membranes. FEBS Lett. 1993 Feb 1;316(3):264–268. doi: 10.1016/0014-5793(93)81305-j. [DOI] [PubMed] [Google Scholar]
  17. Sangadala S., Walters F. S., English L. H., Adang M. J. A mixture of Manduca sexta aminopeptidase and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86Rb(+)-K+ efflux in vitro. J Biol Chem. 1994 Apr 1;269(13):10088–10092. [PubMed] [Google Scholar]
  18. Tabashnik B. E., Finson N., Groeters F. R., Moar W. J., Johnson M. W., Luo K., Adang M. J. Reversal of resistance to Bacillus thuringiensis in Plutella xylostella. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4120–4124. doi: 10.1073/pnas.91.10.4120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Urdal D. L., Call S. M., Jackson J. L., Dower S. K. Affinity purification and chemical analysis of the interleukin-1 receptor. J Biol Chem. 1988 Feb 25;263(6):2870–2877. [PubMed] [Google Scholar]
  20. Vadlamudi R. K., Ji T. H., Bulla L. A., Jr A specific binding protein from Manduca sexta for the insecticidal toxin of Bacillus thuringiensis subsp. berliner. J Biol Chem. 1993 Jun 15;268(17):12334–12340. [PubMed] [Google Scholar]
  21. Van Rie J., Jansens S., Höfte H., Degheele D., Van Mellaert H. Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins. Appl Environ Microbiol. 1990 May;56(5):1378–1385. doi: 10.1128/aem.56.5.1378-1385.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Van Rie J., Jansens S., Höfte H., Degheele D., Van Mellaert H. Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects. Eur J Biochem. 1989 Dec 8;186(1-2):239–247. doi: 10.1111/j.1432-1033.1989.tb15201.x. [DOI] [PubMed] [Google Scholar]
  23. Van Rie J., McGaughey W. H., Johnson D. E., Barnett B. D., Van Mellaert H. Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis. Science. 1990 Jan 5;247(4938):72–74. doi: 10.1126/science.2294593. [DOI] [PubMed] [Google Scholar]
  24. Wolfersberger M. G. The toxicity of two Bacillus thuringiensis delta-endotoxins to gypsy moth larvae is inversely related to the affinity of binding sites on midgut brush border membranes for the toxins. Experientia. 1990 May 15;46(5):475–477. doi: 10.1007/BF01954236. [DOI] [PubMed] [Google Scholar]

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