title: A new mechanism for the control of phenoloxidase activity: inhibition and complex formation with quinone isomerase.
abstract: Insect phenoloxidases participate in three physiologically important processes, viz., cuticular hardening (sclerotization), defense reactions (immune reaction), and wound healing. Arrest or even delay of any of these processes compromises the survival of insects. Since the products of phenoloxidase action, viz., quinones, are cytotoxic, uncontrolled phenoloxidase action is deleterious to the insects. Therefore, the activity of this important enzyme has to be finely controlled. A novel inhibition of insect phenoloxidases, which serves as a new regulatory mechanism for control of its activity, is described. The activity of phenoloxidases isolated from both Sarcophaga bullata and Manduca sexta is drastically inhibited by quinone isomerase (isolated from Calliphora), an enzyme that utilizes the phenoloxidase-generated 4-alkylquinones. In turn, phenoloxidase reciprocated the inhibition of isomerase. By forming a complex and controlling each other's activity, these two enzymes seem to regulate the levels of endogenously quinones. In support of this contention, an endogenous complex consisting of phenoloxidase, quinone isomerase, and quinone methide isomerase was characterized from the insect, Calliphora. This sclerotinogenic complex was isolated and purified by borate extraction of the larval cuticle, ammonium sulfate precipitation, and Sepharose 6B column chromatography. The complex exhibited a molecular mass of about 620-680 kDa, as judged by size-exclusion chromatography on Sepharose 6B and HPLC and did not even enter 3% polyacrylamide gel during electrophoresis. The phenoloxidase activity of the complex exhibited a wide substrate specificity. Incubation of the complex with N-acetyldopamine rapidly generated N-acetylnorepinephrine, dehydro-N-acetyldopamine, and its dimers. In addition, transient accumulation of N-acetyldopamine quinone was also observed. These results confirm the presence of phenoloxidase, quinone isomerase, and quinone methide isomerase in the complex. Attempts to dissociate the complex with even trace amounts of SDS ended in the total loss of quinone isomerase activity. The complex does not seems to be made up of stoichiometric amounts of individual enzymes as the ratio of phenoloxidase to quinone isomerase varied from preparation to preparation. It is proposed that the complex formation between sequential enzymes of sclerotinogenic pathway is advantageous for the organism to effectively channel various reactive intermediates during cuticular hardening.
authors.affiliation: Department of Biology, University of Massachusetts, Boston, Massachusetts 02125, USA. manickam.sugumaran@umb.edu
authors.forename: M
authors.initials: M
authors.lastname: Sugumaran
authors.forename: K
authors.initials: K
authors.lastname: Nellaiappan
authors.forename: K
authors.initials: K
authors.lastname: Valivittan
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004298:Dopamine / Q000031:analogs & derivatives / Q000378:metabolism; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D018402:Manduca / Q000201:enzymology*; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011809:Quinones / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D019476:Insect Proteins; D009097:Multienzyme Complexes; D011809:Quinones; C047663:cuticle proteins, insects; D014442:Monophenol Monooxygenase; D019746:Intramolecular Oxidoreductases; C061418:N-acetyldopamine-quinone isomerase; C057265:quinone methide isomerase; D010100:Oxygen; D004298:Dopamine