A mixture of Manduca sexta aminopeptidase and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86Rb(+)-K+ efflux in vitro.
CryIA(c) delta-endotoxin, a member of the CryI family of Bacillus thuringiensis insecticidal proteins, specifically recognizes and binds with high affinity to target proteins in the midgut of susceptible insects. Protein blots of Manduca sexta brush-border membranes probed with 125I-CryIA(c) identify a major binding protein of 120 kDa and a minor binding protein of 65 kDa. Monoclonal antibodies were raised against the 120-kDa toxin binding protein. Using isoelectric focusing and monoclonal antibodies (2B3, 8G1, and 12B8) 120- and 65-kDa brush-border proteins were isolated. Labeled CryIA(c) and monoclonal antibodies probed to blots of the affinity-selected proteins recognized the 120- and 65-kDa proteins. When reconstituted into phospholipid vesicles, antibody-selected proteins increased toxin binding (35%) and enhanced toxin-induced 86Rb+ release up to 1000-fold. The 120-kDa protein was identified as aminopeptidase N (EC 3.4.11.2). A CryIA(c)-sensitive phosphatase was also present in the 120/65-kDa protein mixture. These findings provide the first identification of B. thuringiensis toxin binding proteins, although confirmation is needed in vivo.
Sangadala
S
S
Department of Entomology, University of Georgia, Athens 30602-2603.
Walters
F S
FS
English
L H
LH
Adang
M J
MJ
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D001413:Bacillus thuringiensis / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001427:Bacterial Toxins; D001665:Binding Sites; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006460:Hemolysin Proteins; D007525:Isoelectric Focusing; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008871:Microvilli / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology*; D009865:Oocytes / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D012413:Rubidium / Q000378:metabolism*; D013552:Swine
D000911:Antibodies, Monoclonal; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011480:Protease Inhibitors; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010744:Phosphoric Monoester Hydrolases; D000626:Aminopeptidases; D012413:Rubidium; D011188:Potassium