CDNA CLONING OF PROPHENOLOXIDASE FROM THE FRESH-WATER CRAYFISH PACIFASTACUS-LENIUSCULUS AND ITS ACTIVATION

Prophenoloxidase (proPO), an enzyme that is the terminal component of the so-called proPO activating system, a defense and recognition syste m in crustaceans and insects, has been purified and cloned from a cray fish blood cell cDNA library. The deduced amino acid sequence codes fo r a polypeptide with a mass of 80,732 Da, which is close to 76 kDa, th e apparent mass of the purified enzyme, proPO contains two copper atom s, and two putative copper-binding sites were found in the deduced ami no acid sequence, Sequence comparisons show that these putative copper -binding sites are similar to the corresponding sites in arthropod hem ocyanins and also, although the sequence similarities are less extensi ve, similar to tyrosinases from vertebrates and microorganisms. The pu rified enzyme is a typical tyrosinase because it hydroxylates monophen ols and oxidizes o-diphenols but does not oxidize p-diphenols. If a ho mogeneous preparation of crayfish proPO were incubated with a homogene ous sample of the proPO activating enzyme, a serine proteinase, the cl eavage of proPO by this trypsin-like enzyme was found to occur between Arg-176 and Thr-177.