Incorporation of Phosphoproteins and Phenol-Bound Proteins into the Cuticle of Periplaneta americana

The incorporation of phosphoproteins and phenol-bound proteins into the cuticle of Periplaneta americana was analyzed. Radioactive monosodium phosphate was injected into newly ecdysed cockroaches. Incorporation into haemolymph proteins as well as tanned cuticle was observed. Phosphoproteins were observed to be combined with the cuticle to a greater extent than monosodium phosphate. This suggests that phosphate is being incorporated into the cuticle more readily than monosodium phosphate. The relevance of these observations is discussed in light of phosphate being a possible crosslinking agent between chitin and cuticle protein. Binding of dopamine metabolites to cuticle proteins was investigated. Specific metabolites studied were N-acetyldopamine-3-0-sulphate and dopamine-3-0-sulphate. In an in vitro experiment, these metabolites were readily bound to cuticular, structural proteins while they remained unchanged when incubated with soluble cuticular proteins. The presence of sulphatase activity was also evidenced in the structural proteins but not in the soluble proteins. The binding of N-acetyldopamine to lysyl-lysine was accomplished in vitro. Binding was observed with and without newly ecdysed cuticle homogenate. The products of the binding were separated wia P-2 polyacrylamide gel chromatography, paper chromatography and high voltage electrophoresis. Absorbancy at 280 nm, as well as the reaction with ninhydrin were monitored for each fraction. A metabolite (or metabolites) was separated which was both UV positive and ninhydrin positive. This metabolite was analyzed by NMR and no aromatic protons were observed thus indicating complete substitution on the ring. These data support the existing theory that suggests protein binding to the sclerotization agent occurs on the ring