Sensillum-lymph proteins from antennal olfactory hairs of the moth Antheraea polyphemus (Saturniidae)

Abstract

The soluble proteins in the antennae of the male and female saturniid moth Antheraea polyphenus were analyzed by isoelectric focusing in miniature ultrathin-layer polyacrylamide gels and by microgradient PAGE (μPAGE). Different preparations of sensillum lymph from male antennal olfactory hairs (isolated sensillum-lymph droplets, rinsing solution from isolated hairs) and homogenates of male and female antennae were compared. The pheromone-binding protein, PBP, and the pheromone-degrading sensillar esterase, SE, were detected only in male antennal preparations. Isolated sensillum lymph exhibited two proteins in Brilliant Blue staining: (1) the PBP appeared as a double band with a relative molecular mass of M r < 14,400 (α-lactalbumin); (2) an oligopeptide was detected as a faint band at a very small molecular mass ( M r ⪡ 14,000 ). The PBP in isolated sensillum lymph was identical to the PBP in the rinsing solution of isolated hairs and in male branch homogenates. Purified PBP from branch homogenates showed a third slightly smaller band. In reducing SDS-μPAGE, the denatured PBP from all preparations showed only one band at a M r of 14,400. The isoelectric point (pI) of the PBP was 4.7. The PBP appeared to be a non-glycosylated protein. Glycoproteins were present in the hemolymph in different patterns in both sexes. The concentration of PBP in sensillum-lymph droplets was ≈ 10 mM, corresponding to a total amount of ≈9 μg/55 μl sensillum lymph in one antenna. A third protein in the sensillum lymph, the sensillar esterase, was only visible by enzyme staining and had a M r of 55,000 and a pI of 3.0. The integumental esterases found in male and female antennae had a M r of 65,000, pI 5.85, and M r 90,000, pI 5.0. From body scales of both sexes, esterases in the range of M r 100,000 were solubilized by 0.01% Triton buffer and could be demonstrated in μPAGE.