PROENZYME OF MANDUCA-SEXTA PHENOL OXIDASE - PURIFICATION, ACTIVATION,SUBSTRATE-SPECIFICITY OF THE ACTIVE ENZYME, AND MOLECULAR-CLONING

Phenol oxidase (PO) was isolated as a proenzyme (pro-phenol oxidase, p ro-PO) from the hemolymph of Manduca sexta larvae and purified to homo geneity. Pro-PO exhibits a M(r) of 130,000 on gel filtration and two b ands with an apparent M(r) of approximate to 100,000 on SDS/PAGE, as w ell as size-exclusion HPLC. Activation of pro-PO was achieved either b y specific proteolysis by a cuticular protease or by the detergent cet ylpyridinium chloride at a concentration below the critical micellar c oncentration. A cDNA clone for M. sexta pro-PO was obtained from a lar val hemocyte cDNA library. The clone encodes a polypeptide of approxim ate to 80,000 Da that contains two copper-binding sites and shows high sequence similarity to POs, hemocyanins, and storage proteins of arth ropods. The M. sexta pro-PO, together with other arthropod pre-POs, co ntains a short stretch of amino acids with sequence similarity to the thiol ester region of alpha-macroglobulins and complement proteins C3 and C4.