Incorporation of Radiolabeled Tyrosine, N-Acetyldopamine, N-β-Alanyldopamine, and the Arylphorin Manducin into the Sclerotized Cuticle of Tobacco Hornworm (Manduca sexta) Pupae

Abstract

Abstract Radiolabeled manducin, which is the arylphorin (i.e. tyrosine-rich larval serum protein) of the tobacco hornworm Manduca sexta was prepared by in vivo biosynthesis from [U-14C]-ʟ-tyrosine. In order to see whether manducin is incorporated into the cuticle of developing pupae and participates in cuticle sclerotization, the radiolabeled protein was injected into late last larval instar larvae. Fractionation of the sclerotized pupal cuticle into a buffer soluble, acid soluble and acid insoluble fraction shows that up to 2.6% of peptidic tyrosine residues end up in the acid soluble portion. Another 0.5% are recovered from the acid insoluble fraction by combustion. Only 30% of peptidic tyrosine residues of manducin incorporated into the acid soluble fraction are recovered as tyrosine. The presence of radioactivity in the acid insoluble fraction suggests that peptidic tyrosine residues are transformed partly into melanin-like material. The in­corporation of manducin into the cuticle of pupae is also evident from immunological studies. Relative large quantities of radiolabeled acid insoluble melanine like material is also recovered from sclerotized cuticle after incorporation of radiolabeled tyrosine as well as tanning substrates N-acetyldopamine and N-β-alanyldopamine. Application of doubly labeled [7-3H,8-14C]-N-acetyldopamine shows a high loss of 3H in the acid insoluble fraction. It is suggested that tanning agents form lignine-like polymers and that sclerotization results from copolymerization with peptidic tyrosine residues in the cuticle. Thus, the arylphorin manducin appears to be an important constituent of the sclerotization system in Manduca sexta.