Mapping of antigenic domains on phytochrome from etiolated Avena sativa L. by immunoblot analysis of proteolytically derived peptides.
Several monoclonal antibodies to phytochrome that interact with putative functionally important domains have been previously identified. The locations of some of these domains are determined here by epitope mapping experiments that utilize immunoblot analyses of proteolytically degraded phytochrome. Seven independent epitopes are identified. An epitope that is recognized by monoclonal antibody Oat-25 is confirmed to be wholly located near the N terminus of phytochrome. This domain undergoes a conformational change when phytochrome is interconverted between its red- and far-red-absorbing forms and is recognized by Oat-25 better in the red-absorbing form. A second domain that also undergoes a photointerconvertible conformation change and that contains the epitope for Oat-16 is localized near the site of chromophore attachment, which is about 36 kDa from the N terminus. A third domain, which contains the most highly conserved epitope on phytochrome that has so far been identified, is recognized by Pea-25 and is located about 85 kDa from the N terminus. Other epitopes and their approximate distances from the N terminus are those recognized by Oat-22 (36 kDa), Oat-13 (65 kDa), and Oat-8 and Oat-28 (70-75 kDa). Even though epitopes for Oat-16 and Oat-22, as well as for Oat-8 and Oat-28, are close together, competitive binding assays indicate that they are different. Immunoblot analyses also indicate that the epitope for Oat-28 is further from the N terminus of phytochrome than is that for Oat-8.
Pratt
L H
LH
Botany Department, University of Georgia, Athens 30602.
Cordonnier
M M
MM
Lagarias
J C
JC
D000911:Antibodies, Monoclonal; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D015151:Immunoblotting; D010446:Peptide Fragments / Q000032:analysis*; D010447:Peptide Hydrolases; D010449:Peptide Mapping; D010834:Phytochrome / Q000032:analysis / Q000276:immunology*; D010940:Plant Proteins / Q000276:immunology*; D013329:Structure-Activity Relationship; D013381:Subtilisins; D014357:Trypsin
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D010940:Plant Proteins; D010834:Phytochrome; D010447:Peptide Hydrolases; D013381:Subtilisins; D014357:Trypsin