Aldosterone-induced proteins: characterization using lectin-affinity chromatography.
Aldosterone-stimulated Na+ transport in toad urinary bladder is associated with the synthesis of a specific group of proteins whose induction appears to be related to the natriferic effect of the hormone. These aldosterone-induced proteins (AIPs) occur in two slightly different molecular weight classes (around 70 kDa), each class being composed of several proteins with discrete isoelectric points (range, 5.5-6.0). Because glycosylation is a common cause of such electrophoretic polymorphism and microheterogeneity, we examined whether these proteins are glycoproteins. Tunicamycin (a specific inhibitor of N-linked glycosylation) inhibited aldosterone-stimulated Na+ transport and AIP synthesis without affecting overall protein synthesis. The vast majority of epithelial cell proteins did not bind to the mannose-specific lectin, concanavalin A-sepharose. In contrast, both classes of AIPs bound to concanavalin A-sepharose, but the affinities of the higher and lower molecular weight proteins were markedly different: the former were readily eluted with 0.2 M alpha-methyl-D-mannoside alone, whereas the latter could only be eluted with 0.4 M alpha-methyl-D-mannoside in combination with high concentrations of NaCl (2.5-5.0 M). These studies indicate that 1) glycosylation is important in the natriferic response to aldosterone, 2) the AIPs are N-linked mannose-containing glycoproteins, and 3) the electrophoretic polymorphism of the AIPs is due, at least in part, to differences in glycosylation. Furthermore, concanavalin A-affinity chromatography provides a simple means for the partial purification of these putative "effectors" of the cellular action of aldosterone.
D000450:Aldosterone / Q000494:pharmacology*; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002024:Bufo marinus; D002846:Chromatography, Affinity / Q000379:methods; D005260:Female; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification; D012685:Sepharose / Q000031:analogs & derivatives; D012964:Sodium / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology; D001743:Urinary Bladder / Q000378:metabolism*
D008565:Membrane Proteins; D011506:Proteins; C028326:concanavalin A-sepharose; D014415:Tunicamycin; D000450:Aldosterone; D012685:Sepharose; D012964:Sodium