The copper catalysis from the oxidation of catechol to em o /em -quinone is mediated through the formation of a PrP23C231CCu2+Ccatechol complex, which favours the transfer of electrons from catechol to PrP23C231CCu2+ and the formation of PrP23C231CCuO2+ from your resulting PrP23C231CCu+ and O2. in the presence of various bivalent metallic ions. Collectively, these results indicate the copper bound to prion protein undergoes catalytic cycling in the presence of catecholamines and causes the oxidation of the protein. and suggests that its function relates to copper homoeostasis or to copper-dependent enzymatic functions [1,2]. Because Cu2+ stimulates PrPC endocytosis, it has been suggested that PrPC plays a role in shuttling Cu2+ from your synaptic space to the cell interior [3,4]. However, another study showed that cuproenzyme activity was not influenced by the degree of PrP (prion protein) manifestation in brain cells [5]. The authors suggested that PrPC might act as a reversible sink or a carrier of the metallic ion [5]. In addition, a recent study indicated SCH 23390 HCl the manifestation of PrP improved copper binding to cells, but did not impact copper uptake, antioxidant enzyme activities or glutathione levels [6]. Another suggestion is definitely that PrP might be a stress sensor for copper and might be able to initiate, following copper binding, a signal transduction process for increasing cell defences by acting on the antioxidant systems [6]. An enzymatic part for copper-bound PrP was also proposed as it exhibited SOD (superoxide dismutase) activity, protecting synaptic areas from oxidative stress [7C10]. Our earlier study indicated that copper ions bound to an N-terminal portion of PrP (PrP23-98) catalysed oxidations of L-ascorbate and dopamine [11]. The last two studies have shown the PrP-bound copper undergoes redox cycling in the presence of electron donors, such as superoxide ions (O2?), dopamine and L-ascorbate. PrPC consists of SCH 23390 HCl a C-terminal website, residues 126C231, that has a globular collapse composed of three -helices and two short -strands [12,13]. Under Cu2+-free conditions, the N-terminal portion of the adult PrPC, residues 23C125 of the primary translation product, is largely unstructured [13C15]. Residues 60C91 consist of an octa-peptide sequence, PHGGGWGQ, which is definitely repeated four instances. Several studies have shown that this unstructured region selectively binds Cu2+ over additional bivalent metallic ion varieties [16C24]. This octapeptide repeat region binds four Cu2+ ions co-operatively with identical co-ordination geometry [20,24C26]. The affinity of this copper binding is in the femtomolar to micromolar range [21,23]. In addition, the fifth Cu2+-binding site centred at His-96 and His-111 has also been observed [22,23,26]. In fact, affinity-purified PrPC preparations from mouse and human brain have been shown to bind three and approx. seven copper atoms respectively [10,27]. Moreover, PrPC from cultured cells was found to bind one to four copper atoms, depending on the availability of copper in the tradition medium [10]. In the light of these results, it is probable that native PrP binds copper [10,27], remain largely unknown. In the present study, we have investigated catecholamine-induced oxidation of a recombinant mouse PrP23C231 to which copper offers previously loaded. The results possess indicated that in the presence of catecholamine, copper-bound PrP23C231 undergoes carbonylation on its own part, and partly prospects to its dimerization and fragmentation. EXPERIMENTAL Antibodies and reagents PrP A (rabbit polyclonal anti-prion protein A antibody) realizing epitope 228C244 of bovine PrP (epitope 216C232 of mouse PrP) was purchased from Cosmo Bio Co. (Tokyo, SCH 23390 HCl Japan). Mouse monoclonal antibody SAF 32 realizing epitope 78C91 of hamster PrP, mouse monoclonal antibody SAF 8G8 realizing epitope 95C110 of human being PrP, mouse monoclonal antibody SAF Splenopentin Acetate 70 realizing epitope 142C160 of hamster PrP and mouse SCH 23390 HCl monoclonal antibody SAF 84 realizing epitope 160C170 of hamster PrP were purchased from Cayman Chemical Co. (Ann Arbor, MI, U.S.A.). These antibodies show cross-reactivity to mouse PrP as explained in the product info. Horseradish-peroxidase-conjugated anti-mouse IgG secondary antibody was purchased from Chemicon International (Temecula, CA, U.S.A.), and horseradish-peroxidase-conjugated anti-rabbit IgG secondary antibody was from Cappel Study Products (Durham, NC, U.S.A.). Plasmid pUC 19 was purchased from Takara Bio (Tokyo, Japan). Plasmid pET-39b SCH 23390 HCl (+) and S-proteinCagarose were purchased from Novagen (Madison, WI, U.S.A.). BSA, catecholamines, rabbit polyclonal anti-dinitrophenyl antibody and Protein GCSepharose 4B were purchased from SigmaCAldrich (St. Louis, MO, U.S.A.). BenzamidineCSepharose 6B, SP Sepharose, ECL? (enhanced chemiluminescence) Western blotting detection reagents and Hybond ECL? nitrocellulose membrane were purchased from Amersham Biosciences (Piscataway, NJ, U.S.A.). EnterokinaseMax was purchased from Invitrogen (Carlsbad, CA, U.S.A.). SOD and catalase were purchased from Alexis Biochemicals (San Diego, CA, U.S.A.) and Nacalai Tesque (Kyoto, Japan) respectively. Xanthine oxidase and xanthine were purchased from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals were purchased from Nacalai Tesque, unless specified otherwise. Distilled water was purified by passage through a Milli-Q Academic A10 system (Millipore, Bedford, MA, U.S.A.). The resistance.