Cuticle from adult cyclorrhaphid dlies and mosquitoes are crosslinked at loci bridging proteins and chitin. Partial acid hydrolysates from populations labelled with L[ring14C] tyrosine and 3H amino acids can be fragmented with NBS and UV photolysis. After resolution by HPLC and chromatography on hydrophobic supports, isolates are O-dealkylated by transfer to thetin and repurified. The unblocked materials are then divided into phenolic and nonphenolic classes by affinity chromatography and examined for crosslinking. Bridging is detected by nonequivalence of N- and C-termini and the presence of doubly labelled conjugates on cation exchangers. Bridged peptide(s) sequence is elucidated by Edman and Stark procedures and the alkali-labile aryl component purified separately. Following acid hydrolysis, basic groups are citraconylated and separated by HPLC. Peaks are viewed by NMR, Ir, UV, and chemical probes before and after deblocking of citraconyl at pH 2. The chitin-bound component described previously will be isolated from soft abdominal adult cuticle (Sarcophagid) and proximal amino acids compared with those described for larval and pupal material. Soluble intermediates of higher molecular weight will be prepared by administration of sublethal doses of diflubensuron to head-ligated flies and presumptive sequence homology compared with life stages investigated in the previous award. The point of attack of fungal enzymes degrading sclerotized cuticle will be investigated further by analysis of the products released from labelled integument. Sclerotization is completely dependent on water extrusion and reorientation of cuticle polymers. The movement of water between bound and unbound
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