The prion protein (PrP) is a Cu 2؉ binding cell surface glycoprotein that can misfold into a ␤-sheet-rich conformation to cause prion diseases.

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The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. The cellular prion protein PrP c is a cell surface glycoprotein with large ␣-helical content that is attached to the plasma membrane by a glycosylphosphatidylinositol anchor. This cellular form of the protein binds copper, and it is argued that this copper binding to PrP c is intimately linked to the normal cellular function of the protein either in copper transport, sequestration, or antioxidant activity (1-3). The native cellular protein may undergo a conformational transformation to a proteaseresistant species known as a prion (PrP Sc ) with high ␤-sheet content and a tendency to aggregate. This transformed species is the putative proteinaceous infectious agent of transmissible spongiform encephalopathies or prion diseases (4). Prion diseases are characterized by a metal imbalance in the brain that occurs simultaneously to conversion of PrP c to PrPSc . The metal imbalance is associated with the loss of copper binding from PrP c on conversion to PrP Sc (5, 6). This raises important questions concerning the role of copper and other metals in prion diseases. Loss of protein function as a result of impaired copper binding in diseased PrPSc could have serious implications for disease progression.As a result, knowledge of copper binding to PrP is important in considering both the normal function of PrP c and its influence in prion diseases.Although it is accepted that PrP c is a Cu(II) binding protein, there are many facets of the interaction of Cu(II) and PrP c that are disputed. These include the exact number of binding sites and their relative affinity for Cu(II). Initial studies focused on a fragment of the protein termed the octameric repeat region because it contains four repeats of an octamer. This region contains histidine and so was a likely candidate for the binding site. These early studies (7, 8) suggested Cu(II) binds to this region with a micromolar affinity that was little more specific than histidine on its own. Further studies with larger fragments also suggested that Cu(II) binds PrP c , but again the affinity seen was similarly a low micromolar association (9). A study of the effect of PrP c expression on Cu(II) uptake into cells suggested that the affinity must at least be in the nanomolar range (1). Nanomolar affinities for the octameric repeat region have rarely been suggested (10). Only one publication has suggested that the affinity of Cu(II) for PrP is in the femtomolar range (11). Although such a high affinity implies that PrP is a very specific Cu(II)-binding protein, this finding has never been reproduced. Therefore, there is currently no...

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The human NUDT12 Nudix hydrolase has been expressed in insect cells from a baculovirus vector as a His-tagged recombinant protein. In vitro, it efficiently hydrolyses NAD(P)H to NMNH and AMP (2',5'-ADP), and diadenosine diphosphate to AMP. It also has activity towards NAD(P)(+), ADP-ribose and diadenosine triphosphate. K (m) values for NADH, NADPH and NAD(+) are 11, 16 and 190 microM and k (cat) values are 11, 16 and 10.5 s(-1) respectively. Thus, like other NADH diphosphatases of the Nudix family, NUDT12 has a marked substrate preference for the reduced nicotinamide nucleotides. Optimal activity was supported by 50 microM Mn(2+) ions in vitro, with 3-fold lower activity at 0.4 mM Mg(2+). Expression of NUDT12 as a C-terminal fusion to green fluorescent protein revealed that it was targeted to peroxisomes by the C-terminal tripeptide PNL acting as a novel type 1 peroxisomal targeting signal. Deletion of PNL resulted in diffuse cellular fluorescence. In addition, C-terminal, but not N-terminal, fusions with or without the PNL signal accumulated in large, unidentified cytoplasmic structures. NUDT12 may act to regulate the concentration of peroxisomal nicotinamide nucleotide cofactors required for oxidative metabolism in this organelle.