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Activation and fragmentation of Bacillus thuringiensis delta-endotoxin by high concentrations of proteolytic enzymes.
Can J Microbiol. 1999 Oct; 45(10):816-25.CJ

Abstract

Commercial enzymes and insect gut juice at various concentrations were used to digest Bacillus thuringiensis subsp. sotto Cry1Aa protoxin and examine the fragmentation pattern and effect on insecticidal activity. Trypsin at both high (5 mg/mL) and low (0.05 mg/mL) concentrations converted protoxin to toxin with no difference in insecticidal activity against Bombyx mori larvae. In both cases, the toxin protein had an apparent M(r) of 58.4 kDa (SDS-PAGE). Active toxin of identical M(r) was also produced with low concentrations of Pronase and subtilisin, but at high concentration, it was degraded into two protease-resistant fragments of apparent M(r) 31.8 and 29.6 kDa, and exhibited no insecticidal activity. Sequencing data established the primary cleavage site to be in domain II, the receptor-binding region of the toxin, in an exposed loop between two beta-sheet strands. Fragmentation was not observed, however, when the digests were analyzed by native protein techniques, but rather the toxin molecule appeared to be intact. The amount of activated toxin produced by Choristoneura fumiferana gut juice was markedly reduced when the gut-juice concentration was increased from 1 to 50% and correlated with a loss in insecticidal activity. However, no lower M(r) protease-resistant fragments were evident in the SDS-PAGE of these digests.

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Authors+Show Affiliations

Pang AS
Canadian Forest Service, Great Lakes Forestry Centre, Sault Ste. Marie, ON, Canada. apang@nrcan.gc.ca
Gringorten JL
No affiliation info available
Bai C
No affiliation info available

MeSH

Amino Acid SequenceAnimalsBacillus thuringiensisBacillus thuringiensis ToxinsBacterial ProteinsBacterial ToxinsBiological AssayEndopeptidasesEndotoxinsHemolysin ProteinsMothsPeptide FragmentsPronaseSubtilisinTrypsin

Pub Type(s)

Journal Article

Language

eng

PubMed ID

10907418

Citation

Pang, A S., et al. "Activation and Fragmentation of Bacillus Thuringiensis Delta-endotoxin By High Concentrations of Proteolytic Enzymes." Canadian Journal of Microbiology, vol. 45, no. 10, 1999, pp. 816-25.
Pang AS, Gringorten JL, Bai C. Activation and fragmentation of Bacillus thuringiensis delta-endotoxin by high concentrations of proteolytic enzymes. Can J Microbiol. 1999;45(10):816-25.
Pang, A. S., Gringorten, J. L., & Bai, C. (1999). Activation and fragmentation of Bacillus thuringiensis delta-endotoxin by high concentrations of proteolytic enzymes. Canadian Journal of Microbiology, 45(10), 816-25.
Pang AS, Gringorten JL, Bai C. Activation and Fragmentation of Bacillus Thuringiensis Delta-endotoxin By High Concentrations of Proteolytic Enzymes. Can J Microbiol. 1999;45(10):816-25. PubMed PMID: 10907418.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Activation and fragmentation of Bacillus thuringiensis delta-endotoxin by high concentrations of proteolytic enzymes. AU - Pang,A S, AU - Gringorten,J L, AU - Bai,C, PY - 2000/7/25/pubmed PY - 2000/9/2/medline PY - 2000/7/25/entrez SP - 816 EP - 25 JF - Canadian journal of microbiology JO - Can J Microbiol VL - 45 IS - 10 N2 - Commercial enzymes and insect gut juice at various concentrations were used to digest Bacillus thuringiensis subsp. sotto Cry1Aa protoxin and examine the fragmentation pattern and effect on insecticidal activity. Trypsin at both high (5 mg/mL) and low (0.05 mg/mL) concentrations converted protoxin to toxin with no difference in insecticidal activity against Bombyx mori larvae. In both cases, the toxin protein had an apparent M(r) of 58.4 kDa (SDS-PAGE). Active toxin of identical M(r) was also produced with low concentrations of Pronase and subtilisin, but at high concentration, it was degraded into two protease-resistant fragments of apparent M(r) 31.8 and 29.6 kDa, and exhibited no insecticidal activity. Sequencing data established the primary cleavage site to be in domain II, the receptor-binding region of the toxin, in an exposed loop between two beta-sheet strands. Fragmentation was not observed, however, when the digests were analyzed by native protein techniques, but rather the toxin molecule appeared to be intact. The amount of activated toxin produced by Choristoneura fumiferana gut juice was markedly reduced when the gut-juice concentration was increased from 1 to 50% and correlated with a loss in insecticidal activity. However, no lower M(r) protease-resistant fragments were evident in the SDS-PAGE of these digests. SN - 0008-4166 UR - https://www.unboundmedicine.com/medline/citation/10907418/Activation_and_fragmentation_of_Bacillus_thuringiensis_delta_endotoxin_by_high_concentrations_of_proteolytic_enzymes_ L2 - https://cdnsciencepub.com/doi/10.1139/w99-086?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -