The phenoloxidase system responsible for the sclerotization of cockroach ootheca is found to be present as an inactive form in the left colleterial gland of Periplaneta americana. The supernatant fraction obtained by centrifugation of the milky white secretions contained the inactive phenoloxidase which required both sodium dodecyl sulfate (SDS) and the insoluble sediment for exhibitingenzymeactivity. Bovineserum albumin could replace the sediment in the activation process. Proteins separated from the supernatant fraction by molecular sieve chromatography on Sephadex C-25 did not require either albumin or the sediment, but required SDS for exhibiting the phenoloxidase activity. Among the detergents tested, SDS (anionic) and cetylpyridinium chloride (cationic) activated the phenoloxidase, but CHAPS (mitterionic) or nonionic detergents failed to activate the enzyme. The activation caused by SDS occurred well below the critical micellar concentration of SDS indicating that SDS i s causing the activation by binding to the protein and altering its conformation. Chloroform-methanol extracts of vestibulum or right gland could replace SDS confirming the presence of endogenous activator@) of phenoloxidase system. A variety of exogenously added lipids could activate the latent enzyme, among which linoleate, oleate, laurate, linolenate, phosphatidylethanolamine, and phosphatidylglycerol proved to be the effective activators of the latent phenoloxidase.
Partially purified phenoloxidase was found to be extremely labile and lost its activity on a) freezing and thawing, b) dialysis, and c) heating for 10 min at 55Β°C. It exhibited a pH optimum of 7 and was inhibited drastically by phenylthiourea and diethyldithiocarbamate. It readily oxidized a number of o-diphenols such as 3,4-dihydroxybenzylalcohol, 3,4-dihydroxyphenethyl alcohol, catechol, N-acetyldopamine, N-acetylnorepinephrine, dopa, dopamine, etc., but failed to oxidize both 3,4-dihydroxybenzoic acid and 3,4-dihydroxybenzaldehyde. It neither converted the typical laccase substrate syringaldazine to its quinone methide product, nor oxidized the p-diphenols, hydroquinone and methylhydroquinorie. Therefore, the enzyme participating in the quinone tanning Acknowledgments: This research was supported by grants from National Institutes of Health (R01-AI-14753) and University of Massachusetts at Boston (BRSG, Ed. Needs, and Fac. Dev.).