Figure 4.
AANAT and 14-3-3 proteins form a complex in chicken retina. A, B, AANAT was coimmunoprecipitated from protein extract of dark-adapted retina (A) and FPLC fraction corresponding to peak 2 of AANAT activity (B) with rabbit anti-14-3-3 but not with normal rabbit IgG. AANAT was detected with rabbit antibody 2992, directed against chicken AANAT1–21 (1:200) (Iuvone et al., 2002). Molecular weight standards are shown on the left. These experiments were replicated twice with similar results. C, Incubation with R18 peptide causes dissociation of ∼80–100 kDa peak and reduced total enzyme activity. Protein extracts from nighttime dark-adapted retinas were incubated in the presence of 10 μm lactacystin, 20 μm R18, or scrambled peptide for 16 h at +4°C. Separation was performed on a Superdex 75 10/300 GL column. Chromatographic profiles representative of results from five separate experiments are shown.