Purification and characterization of insect toxins derived from the mite, Pyemotes tritici.
Three low mol. wt proteins which have contracting-paralyzing activity in insects were isolated from extracts of the straw itch mite, Pyemotes tritici. One of these toxins, referred to as TxP-I, was purified to apparent homogeneity using the following sequence: ion-exchange, affinity, hydroxyapatite and reverse-phase chromatography. The other two toxins, referred to as TxP-II, remained as a mixture. Peptide mapping and immunoblot analysis suggest that TxP-I and TxP-II are probably isoproteins. The apparent mol. wt of native TxP-I and of the two components of TxP-II were 27,000, 28,000 and 29,000, respectively. The apparent mol. wt of the toxins after reductive carboxamidomethylation increased to 38,000, 41,000 and 43,000, respectively. The amino acid composition of TxP-I indicates a high content of Cys (8 mole%). Therefore, several disulfide bonds may impart a very compact tertiary structure to this protein which, upon denaturation, unfolds and increases its Stoke's radius resulting in retarded mobility on a polyacrylamide gel. The N-terminal sequence of TxP-I is not homologous with any other protein for which the sequence is known. The paralysis dose50 of TxP-I (PD50) in wax moth larvae is ca. 500 micrograms/kg and it is not toxic to mice at a dose of 50 mg/kg. A polyclonal antibody, raised against TxP-I, reacted with both TxP-I and TxP-II. The antibody neutralized the rapid, muscle-contracting paralysis of these toxins. Using this antibody and immunocytochemistry, we found the toxins localized in posterior glands which appear to be connected with the stylet through a series of ducts. We conclude that TxP-I and TxP-II are part of a complex mixture of neurotoxins which P. tritici utilizes to capture prey.
Tomalski
M D
MD
Department of Entomology, University of Georgia, Athens 30602.
Kutney
R
R
Bruce
W A
WA
Brown
M R
MR
Blum
M S
MS
Travis
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001180:Arthropod Venoms / Q000032:analysis* / Q000276:immunology; D015153:Blotting, Western; D008925:Mites / Q000032:analysis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009498:Neurotoxins / Q000032:analysis* / Q000276:immunology; D010449:Peptide Mapping
D000596:Amino Acids; D001180:Arthropod Venoms; D009498:Neurotoxins