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. 2005 Mar 22;387(Pt 1):247–255. doi: 10.1042/BJ20041561

Figure 1. Fragmentation and dimerization of the copper-loaded PrP23–231 in the presence of catecholamine.

Figure 1

(A) Western blot immunoassay for copper-loaded PrP23–231. Mixtures containing 50 mM Mes (pH 7.5) and 1 μM copper-loaded PrP23–231 (containing 5.2 μM copper) were incubated with or without (Nil) the indicated concentrations of dopamine, adrenaline or noradrenaline at 37 °C for 30 min. As a control, copper-loaded PrP23–231 was kept on ice was used (Cont). PrP was detected by SDS/PAGE, followed by immunoblotting with antibody SAF 84. Molecular masses in kDa are indicated on the left. The positions of the fragments at 17–19 kDa and the dimer at 46 kDa are marked by arrowhead* and arrowhead** respectively. (B) Quantification of the Western blot data. Relative chemiluminescence intensities of the bands of the fragments (A, arrowhead*) were determined using ATTO Densitograph Software Library. Because the copper-loaded PrP23–231 preparation contained a trace of the 17–19 kDa fragments, their signal was taken as 100% for expression of intensities of the test samples. Data are means±S.D. (n=3). (C) and (D) Time course of formation of 17–19 kDa fragments and dimer (46 kDa). A mixture containing 50 mM Mes (pH 7.5) and 1 μM copper-loaded PrP23–231 (containing 5.2 μM copper) was incubated without (○) or with 10 μM dopamine (●). PrP was detected by SDS/PAGE followed by immunoblotting with antibody SAF 84. Signals of 17–19 kDa fragments (C) and dimer (46 kDa) (D) were determined using ATTO Densitograph Software Library and plotted as a percentage of the control (zero time). Data are means±S.D. (n=3). (E) Release of copper from copper-loaded PrP23–231 by reducing substances. Copper-loaded PrP23–231 (1 μM, containing 5.2 μM copper) was incubated with dopamine, L-ascorbate or glutathione at 10 μM, mixtures were centrifuged immediately (open bars) or after 30 min (hatched bars) in ultrafiltration cups, and copper concentrations of the filtrates were measured by atomic absorption spectrophotometry. The measured concentrations were corrected for the copper concentration of the buffer used. Data are means±S.D. (n=3). *Significant difference (P≤0.01) from the experiment with no additions (Nil).