Abstract
Abstract: :
Purpose: To identify the binding proteins of α, ß, γ tubulin subunits in rods and cones outer segments. Methods: Bovine and frog rods outer segments (ROS) were used for immunoprecipitation and mass spectrometry analysis. Immunoprecipitation materials were obtained in conventional way using anti-tubulin α, anti-tubulin ß, and anti-tubulin γ. For mass spectrometry, each material was loaded to 2D-PAGE. After silver staining the gels, the stained spots were excised and in-gel digested. The extracted peptides from the gel spots were analyzed by ion-trap mass spectrometry by using MS/MS method. Results: Some interested proteins were identified by mass spectrometry. A microtubule associated protein (Tau) was co-precipitated with tubulin ß and γ. Actuary, immunocytochemistry revealed that Tau were localized in membranous region in outer segment. An annexin II was also co-precipitated with tubulin ß and γ. Anti-tubulin α derived a guanylate cyclase from ROS. We confirmed the co-precipitated material that was precipitated with anti-tubulin α containing the guanylate cyclase by western blotting assay. Conclusion: Some proteins bound with tubulin subunits in ROS. These results showed that the tubulin subunits were co-localized with Tau and/or annexin II and/or guanylate cyclase in membranous region in outer segment. Physiological role of this co-localization in the outer segment was not obvious at present, but it is conceivable that these proteins may be involved in formation of the outer segment and regulation of peripheral membrane proteins.
Keywords: photoreceptors • immunohistochemistry • microscopy: electron microscopy