Purification and characterization of insect toxins derived from the mite, Pyemotes tritici

Abstract

M. D. Tomalski, R. Kutney, W. A. Bruce, M. R. Brown, spM. S. Blum and J. Travis. Purification and characterization of insect toxins derived from the mite, Pyemotes tritici. Toxicon 27, 1151–1167, 1989.—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 is 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 dose 50 of TxP-I ( pd 50) in wax moth larvae is ca. 500 μg/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.