Post-translational Import of the Prion Protein into the Endoplasmic Reticulum Interferes with Cell Viability

Abstract

Ferredoxin:NADP+ oxidoreductase (ferredoxin: NADP+ reductase, EC 1.18.1.2) was shown to form a ternary complex with its substrates ferredoxin (Fd) and NADP(H), but the ternary complex was less stable than the separate binary complexes. & for oxidized binary Fd-ferredoxin NADP+ reductase complex was less than 50 nM; increased with NADP+ concen-tration, approaching 0.5-0.6 PM when the flavoprotein was saturated with NADP+ K(NADP+) also increased from about 14 WM to about 310 PM, on addition of excess Fd. The changes in K,, were consistent with negative cooperativity between the associations of Fd and NADP+ and with our unpublished observations which suggest that product dissociation is rate-limiting in the reaction mechanism. Similar interference in binding was observed in more reduced states; NADPH released much ferredoxin:NADP+ reductase from Fd-Sepharose whether the proteins were initially oxidized or re-duced. Complexation between Fd and ferredoxin: NADP+ reductase was found to shield each center from paramagnetic probes; charge specificity suggested that the active sites of Fd and ferredoxin:NADP+ reductase were, respectively, negatively and positively charged.