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

Radiolabeled manducin, which is the arylphorin (i.e. tyrosine-rich larval serum protein) o f the tobacco hornworm Manduca sexta was prepared by in vivo biosynthesis from [U -l4C]-L-tyrosine. In order to see whether manducin is incorporated into the cuticle o f developing pupae and participates in cuticle sclerotization, the radiolabeled protein was injected into late last larval instar larvae. Fractionation o f the sclerotized pupal cuticle into a buffer soluble, acid soluble and acid insoluble fraction shows that up to 2.6% o f 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 o f 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 m elanin-like material. The in­ corporation o f manducin into the cuticle o f pupae is also evident from im m unological studies. Relative large quantities of radiolabeled acid insoluble m elanine like material is also recovered from sclerotized cuticle after incorporation o f radiolabeled tyrosine as well as tanning substrates N-acetyldopamine and N-/?-alanyldopamine. Application o f doubly labeled [7-3H,8l4C]-N-acetyldopamine shows a high loss o f 3H in the acid insoluble fraction. It is suggested that tanning agents form lignine-like polymers and that sclerotization results from copolym erization with peptidic tyrosine residues in the cuticle. Thus, the arylphorin m anducin appears to be an important constituent o f the sclerotization system in Manduca sexta.