11/20/2022 0 Comments Reference tracker zorato![]() ![]() 3A) incubated with either RyR1 (upper panel) or RyR2 (lower panel) preparations. B, GST pull-down assays using GST, GST-FKBP12, and GST-RyR constructs 4, 4A, 4B, 5, and 6 (see Fig. A, Coomassiestained gel of enriched RyR1 (lane A) and RyR2 (lane B) prepared by fast protein liquid chromatography or sucrose density centrifugation, respectively. The expected molecular masses were: control construct (2346 –2507), 19 kDa construct 1, 44 kDa construct 2, 43 kDa construct 3, 33 kDa and FKBP12, 12 kDa. Lanes J, K, L, and M show the experimental control (with Ab2142 omitted). Lane I is a positive control showing the immunoprecipitation of FKBP12. Lanes E, F, G, and H are the immunoprecipitated proteins, corresponding to the control construct (2346 –2507) and constructs 1, 2, and 3, respectively. Lane A is a control construct (RyR2 2346 –2507) lanes B, C, and D are constructs 1, 2, and 3, respectively, and lane N is FKBP12, a positive control protein known to bind RyR1. B, autoradiographs of methionine-labeled constructs 1, 2, and 3 (lanes A–D) and after incubation with RyR1, immunoprecipitation using Ab2142 (specific for RyR1 N terminus) and 15% SDS-PAGE (lanes E–H). A, coordinates of overlapping RyR2 constructs 1, 2, and 3 (6) and the position of the Ab34 epitope. Immunoprecipitation by RyR1 of recombinant constructs expressed in vitro. Lanes E and F are the silver-stained gels corresponding to lanes C and D, and these show a pattern of bands very similar to each other. Lane C shows an Ab34-immunoreactive band of 20 –25 kDa (arrow) specifically eluted from the RyR column, which was absent from lane D (the control column eluate). This digest was either incubated with RyR immobilized onto an agarose support or with a control agarose column. Lane B shows a trypsin digest after 30 min. In lane A is a 15-g undigested enriched RyR1 preparation. D, lanes A–D show a Western blot using Ab34 and a 15% gel lanes E and F show a silver-stained gel corresponding to lanes C and D. C, Coomassie-stained 5.5% gel of the enriched RyR1 preparation prepared by sucrose density gradient centrifugation. At the times shown, samples were taken and subjected to SDS-PAGE, transferred to polyvinylidene difluoride membrane, and analyzed by Western blot. B, time course of RyR1 tryptic digestion monitored using Abs 2142, 34, and 2160. A, sequence coordinates of the site-directed antibodies 2142, 34, and 2160 raised to the N terminus, central domain, and C terminus of RyR1, respectively. This region may constitute a specific subdomain participating in RyR-RyR interaction.Įpitope-specific antibody mapping of trypsinized RyR. ![]() These results identify a region at the center of the linear RyR (residues 2540-3207 of human RyR2) which is able to bind to the RyR oligomer. ![]() In GST pull-down assays, these same three fragments captured RyR2, and two of them retained RyR1. In silico analysis of secondary structure showed evidence of coil regions in two of these RyR fragment sequences, which might explain these data. The binding at high NaCl concentration suggested involvement of a hydrophobic interaction. Binding was greatest at 50-150 mm NaCl for two GST-RyR constructs, and a third GST-RyR construct demonstrated maximum binding between 150 and 450 mm NaCl. Three GST-RyR fusion proteins demonstrated specific binding, dependent upon ionic strength. To refine the binding regions, smaller RyR fragments were expressed as glutathione S-transferase (GST) fusion proteins, and their binding to RyR was monitored using a "sandwich" enzyme-linked immunosorbent assay. Three overlapping RyR fragments encompassing this epitope, expressed using an in vitro mammalian expression system, were immunoprecipitated by RyR. Using epitope-specific antibodies, we identified a RyR tryptic fragment that specifically bound the intact immobilized RyR. In this study, we report experiments designed to identify the region(s) of the RyR molecule, participating in this reciprocal interaction. RyR interoligomeric association has also been inferred from observations of simultaneous channel gating during multi-RyR channel recordings in lipid bilayers. Specific interactions between adjacent ryanodine receptor (RyR) molecules to form ordered two-dimensional arrays in the membrane have been demonstrated using electron microscopy both in situ, in tissues and cells, and in vitro, with the purified protein. ![]()
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