Figure 6.

Inhibition of cell-free conversion of PrP-sen to PrP-res by PcTS-Fe³⁺ (A, D, and E) and PcTrS-Al³⁺ (B, D, and E) under GdnHCl-free (A, B, and D) or GdnHCl-containing conditions (E). ³⁵S-PrP-sen was incubated with unlabeled PrP-res for 2 days in the presence of the designated concentration of phthalocyanine. One-tenth of the reaction was analyzed by SDS/PAGE without PK digestion; the remainder was digested with PK. (A and B) Phosphor autoradiographic images of ³⁵S-PrP species; open and solid triangles, monoglycosylated and unglycosylated ³⁵S-PrP, respectively, without PK treatment; open and solid circles, monoglycosylated and unglycosylated ³⁵S-PrP-res, respectively, after PK digestion. (C) Immunoblot analysis of the total PrP-res in the PK-digested reaction products using mAb 3F4 [which has an epitope within the normally PK-resistant portion of PrP-res (50)] as described (51). Molecular mass markers are designated in kDa along the right side of A–C. The loss of ³⁵S-PrP in the 100 μM PcTS-Fe³⁺ lane (-PK) appeared to be due to SDS-insoluble aggregation because higher-molecular-mass ³⁵S-PrP species were visible near the top of the lane (not shown). Because this apparent aggregation was not observed with lower, but highly inhibitory, concentrations of PcTS-Fe³⁺ (e.g., 1 μM) or with PcTrS-Al³⁺ or several other inhibitory tetrapyrroles described in the text, we conclude that it was not related to inhibition. (D and E) Graphs of the quantitated ³⁵S-PrP-res products (bands marked with circles in A and B) using GdnHCl-free or GdnHCl-containing conditions, respectively. The data points show the mean ± SD of triplicate determinations.