Isolation, partial characterization, and localization of the A and B proteins from the tubular accessory gland of male Tenebrio molitor

In its fully differentiated state, the tubular accessory gland of the male mealworm beetle, Tenebrio molitor, synthesizes five groups of proteins (A, B, C, D₁ and D₂) which are easily distinguished from one another on two-dimensional pI-SDS polyacrylamide gels (Black et al., 1982: Devl Biol. 94, 106-115). In the present work, the A and B proteins have been isolated by preparative gel electrophoresis and Amicon ultrafiltration. The isolation procedure provided two fractions of interest: one contained a mixture of A and B proteins (A/B) and the other consisted of only B proteins. The major proteins in the A class have a molecular weight of 17,900 while those of the B class are 19,000 daltons in size. Antibodies have been produced to the A/B mixture and to the B fraction. Ouchterlony immunodiffusion and straight line immunoelectrophoresis show that the A and B proteins share common immunological characteistics. The proteins from the tubular accessory gland were displayed on one dimensional SDS gels and electrophoretically blotted onto nitrocellulose paper. The antibodies to the A/B mixture recognize A and B bands on these gels. In addition, these antibodies show affinity for C proteins and another band of lower molecular weight. Using the anti-A/B with techniques of immunodiffusion, straight line immunoelectrophoresis, and immunoblotting, we have identified the A and B protein in extracts of soluble proteins in the spermatophore. Furthermore, the A/B proteins have been localized by immunohistochemical techniques within the apical portions of the secretory cells of the tubular gland and also in the lumen of the spermatophore.