Characterization of a new phenoloxidase inhibitor from the cuticle of Manduca sexta

Melanin, the phenolic biopolymer that serves as a skin- and hair pigment-pr otecting agent against harmful solar radiation and a free radical trap, is biosynthesized in animals mainly by the action of tyrosinase also known as phenoloxidase. Regulation of tyrosinase and hence melanogenesis is vital fo r all animals. In this report, we present the isolation and characterizatio n of a new, heat-labile glycoprotein inhibitor of phenoloxidase from the la rvae of Manduca sexta. The inhibitor was isolated from the live larval cuti cle by buffer extraction and purified to homogeneity employing ammonium sul fate precipitation, dialysis, and concanavalin A-Sepharose chromatography. It migrated with a molecular weight of 380,000 on SDS-PAGE gels and inhibit ed the activity of insect and plant as well as fungal phenoloxidases. Inhib itor formed a tight complex with phenoloxidases, which resisted dissociatio n even by 1% Triton X-100 or SDS. Selective inhibition of phenoloxidase, wh ile acting on certain but not all different substrates, was observed. The p hysiological importance of this newly discovered high-molecular-weight phen oloxidase inhibitor is discussed. (C) Academic Press.