Immuno cross-reactivity suggests that catecholamine biosynthesis enzymes and beta-adrenergic receptors may be related.
Turkey red blood cell, beta 1-adrenergic receptors (BARs) were prepared to electrophoretic homogeneity by affinity chromatography, size exclusion high performance liquid chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to prepare rabbit polyclonal anti-BAR antibodies. Anti-BAR activity was confirmed by immunoadsorption of [125I]cyanopindolol-labeled BAR to a protein A affinity column using the anti-BAR antibodies. BAR was compared to the catecholamine biosynthetic enzyme dopamine B-hydroxylase (DBH) by anti-BAR antibody cross-reactivity. DBH was purified from bovine adrenal medullae chromaffin vesicles by ion exchange, size exclusion, and concanavalin A-Sepharose chromatography. Final DBH specific activities were 42 +/- 4 units/mg of protein. Homogeneity was confirmed by nondenaturing polyacrylamide gel electrophoresis. Both DBH and BAR were recognized by the anti-BAR antibodies on Western transfer and immunoblotting. No interactions were observed with preimmune controls. Similar results were obtained with glycosylated and deglycosylated DBH, suggesting that the anti-BAR antibodies recognize specific portions of DBH amino acid sequence and not associated carbohydrate. DBH-cross-reactive antibodies were also purified by affinity chromatography using immobilized DBH and shown to immunoadsorb [125I]cyanopindolol-labeled BAR by protein A affinity chromatography. These results suggest that the catecholamine biosynthetic enzyme DBH and BAR may be related in structure.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000276:immunology; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004299:Dopamine beta-Hydroxylase / Q000276:immunology* / Q000302:isolation & purification; D006031:Glycosylation; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D011943:Receptors, Adrenergic, beta / Q000276:immunology* / Q000302:isolation & purification; D014422:Turkeys
D011943:Receptors, Adrenergic, beta; D004299:Dopamine beta-Hydroxylase