MOLECULAR-CLONING OF INSECT PRO-PHENOL OXIDASE - A COPPER-CONTAINING PROTEIN HOMOLOGOUS TO ARTHROPOD HEMOCYANIN

Pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-d opa:oxygen oxidoreductase, EC 1.14.18.1)] is present in the hemolymph plasma of the silkworm Bombyx mori. Pro-PO is a heterodimeric protein synthesized by hemocytes, A specific serine proteinase activates both subunits through a limited proteolysis. The amino acid sequences of bo th subunits were deduced from their respective cDNAs; amino acid seque nce homology between the subunits was 51%. The deduced amino acid sequ ences revealed domains highly homologous to the copper-binding site se quences (copper-bidding sites A and B) of arthropod hemocyanins. The o verall sequence homology between silkworm pro-PO and arthropod hemocya nins ranged from 29 to 39%, Phenol oxidases from prokaryotes, fungi, a nd vertebrates have sequences homologous to only the copper-binding si te B of arthropod hemocyanins, Thus, silkworm pro-PO DNA described her e appears distinctive and more closely related to arthropod hemocyanin s, The pro-PO-activating serine proteinase was shown to hydrolyze pept ide bonds at the carboxyl side of arginine in the sequence -Asn-49-Arg -50-Phe-51-Gly-52- of both subunits. Amino groups of N termini of both submits were indicated to be N-acetylated. The cDNAs of both pro-PO s ubmits lacked signal peptide sequences, This result supports our conte ntion that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins.