Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster.
The properties of cuticular enzymes involved in sclerotization of Drosophila melanogaster puparium were examined. The cuticle-bound phenoloxidase from the white puparium exhibited a pH optimum of 6.5 in phosphate buffer and oxidized a variety of catecholic substrates such as 4-methylcatechol, N-beta-alanyldopamine, dopa, dopamine, N-acetyldopamine, catechol, norepinephrine, 3,4-dihydroxyphenylglycol, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid. Phenoloxidase inhibitors such as potassium cyanide and sodium fluoride inhibited the enzyme activity drastically, but phenylthiourea showed marginal inhibition only. This result, coupled with the fact that syringaldazine served as the substrate for the insoluble enzyme, confirmed that cuticular phenoloxidase is of the "laccase" type. In addition, we also examined the mode of synthesis of the sclerotizing precursor, 1,2-dehydro-N-acetyldopamine. Our results indicate that this catecholamine derivative is biosynthesized from N-acetyldopamine through the intermediate formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide as established for Sarcophaga bullata [Saul, S. and Sugumaran, M., F.E.B.S. Letters 251, 69-73 (1989)]. Accordingly, successful solubilization and fractionation of cuticular enzymes involved in the introduction of a double bond in the side chain of N-acetyldopamine indicated that they included o-diphenoloxidase, 4-alkyl-o-quinone:p-quinone methide isomerase, and N-acetyldopamine quinone methide:dehydro N-acetyldopamine isomerase and not any side chain desaturase.
Sugumaran
M
M
Department of Biology, University of Massachusetts, Boston 02125.
Giglio
L
L
Kundzicz
H
H
Saul
S
S
Semensi
V
V
D000818:Animals; D004331:Drosophila melanogaster / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D011679:Pupa / Q000201:enzymology; D013379:Substrate Specificity
D010088:Oxidoreductases; C060479:N-acetyldopamine desaturase; D014442:Monophenol Monooxygenase