cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation.

Abstract

Prophenoloxidase (proPO), an enzyme that is the terminal component of the so-called proPO activating system, a defense and recognition system in crustaceans and insects, has been purified and cloned from a crayfish blood cell cDNA library. The deduced amino acid sequence codes for a polypeptide with a mass of 80,732 Da, which is close to 76 kDa, the apparent mass of the purified enzyme. proPO contains two copper atoms, and two putative copper-binding sites were found in the deduced amino acid sequence. Sequence comparisons show that these putative copper-binding sites are similar to the corresponding sites in arthropod hemocyanins and also, although the sequence similarities are less extensive, similar to tyrosinases from vertebrates and microorganisms. The purified enzyme is a typical tyrosinase because it hydroxylates monophenols and oxidizes odiphenols but does not oxidize p-diphenols. If a homogeneous preparation of crayfish proPO were incubated with a homogeneous sample of the proPO activating enzyme, a serine proteinase, the cleavage of proPO by this trypsin-like enzyme was found to occur between Arg-176 and

Tarantula Hemocyanin Shows Phenoloxidase Activity

“…In the case of crustacean hemocyanins, these linker subunit types are not present: only 6-mers and 2 ϫ 6-mers are found in the hemolymph of modern arthropods (3)(4)(5). In addition, species of two large groups among arthropods, Crustacea and Insecta, have evolved prophenoloxidases, most probably by gene duplication of hemocyanins, which is indicated by the close structural relationship between phenoloxidase and hemo- cyanin (14,15), and consecutive optimization of a physiologically beneficial phenoloxidase activity.…”

Cresolase, catecholase and laccase activities in haemocytes of the red swamp crayfish

“…Thus, the presence of SDS is able to trigger the activation of proPO and hence its possible polymerisation to itself or other proteins under the denaturing conditions of SDS-PAGE. It is interesting to note that a monospecific antiserum preparation against crayfish proPO is able to bind to the 76 kDa protein (molecular mass of purified proPO), but is unable to recognise any other protein in crude extracts from crayfish haemocytes after SDS-PAGE (Aspán et al, 1995). It is not clear whether in this last report crude extracts were boiled prior to SDS-PAGE, and thus protein aggregates might have been broken up.…”

“…The deduced amino acid sequence of a cDNA cloned proPo from Pacifastacus leniusculus codes for a protein of 80 kDa (Aspán et al, 1995). This molecular mass corresponds with another PO activity band of approximately 80 kDa which stained with DAB, hydroquinone and with catechol in DMG (see Figs 2 and 4).…”

“…It was inhibited when the gel was pretreated with DETC as compared to nonpretreated controls, and its PO activity was impaired at temperatures higher than 70 C. It is possible that the 10 kDa band is a degradation product that retains some PO activity. This degradation product might only bear one of the two putative Cu-binding centers of crayfish proPO (Aspán et al, 1995), important for enzyme activity. Based on a cDNA clone of crayfish proPO (Aspán et al, 1995), a polypeptide that would contain the two putative Cu-binding centers would have a molecular mass>28 kDa.…”

“…This degradation product might only bear one of the two putative Cu-binding centers of crayfish proPO (Aspán et al, 1995), important for enzyme activity. Based on a cDNA clone of crayfish proPO (Aspán et al, 1995), a polypeptide that would contain the two putative Cu-binding centers would have a molecular mass>28 kDa.…”