The role of catecholamina-beta-alanyl ligase in Ceratitis capitata and other insects

PéREZ,ET AL (AND QUESADA-ALLUé, L.A.)

Salvador-Bahia-Brazil

Congreso; 6th meeting of thr working group on fruit flies of the western hemisphere y otro; 2006

Working group on fruit flies of the western hemisphere

We have demonstrated the activity of catecholamine-â-alanyl ligase (CBAL), the enzyme synthesizing N-â-alanyldopamine (NBAD) in the epidermis and in the brain of Ceratitis capitata and other dipterans like Drosophila melanogaster and Haematobia irritans as well as of other insects. NBAD is the main cross-linking precursor of the brown insect cuticles. The epidermal enzyme is induced by 20-OH-ecdysone at the time of pupariation and of adult ecdysis whereas the nervous system enzyme appears to be constitutive. The properties and requirements required to in vitro synthesize NBAD are almost identical for both tissues and the enzymatic activity in different insects appeared to be similar. For comparison, activity values at puparium peak sclerotization are 45.2 pmoles/min.mg for D. melanogaster and 23.11 pmoles/min.mg for the medfly. The data on in vitro synthesis agree well with the synthesis in vivo. The identity of NBAD and other products were confirmed by HPLC and TLC. The CBAL from C. capitata and other insects showed peculiar characteristics like a broad specificity of substrate and being extremely sensitive to temperature and purification steps. The enzyme is codified by the gene in the locus niger. The C. capitata melanic mutant niger1 lacks enzymatic activity both in epidermis and brain and was found to be phenotypically equivalent to the mutant ebony from D. melanogaster. The gene niger has not been cloned yet and therefore the studies of gene expression were carried out in ebony. The mutant ebony4 was cloned and sequenced and the expression studies were performed in W.T. and mutants. Our results showed that different regulation of CBAL is required in epidermis and brain and that other previously unknown substances might be synthesized in addition to NBAD. Other preliminary results are shown and a tentative model of CBAL regulation is proposed.