Dopamine beta-hydroxylase and other glycoproteins from the soluble content and the membranes of adrenal chromaffin granules: isolation and carbohydrate analysis.
Chromogranin A and two other proteins (A1 and A2) of the soluble proteins of bovine chromaffin granules were isolated by extraction from polyacrylamide gels after electrophoresis. The carbohydrate content of these proteins was 5%, with galactose, N-acetylgalactosamine, and sialic acid as the main sugars. Membranes of chromaffin granules were solubilized with sodium dodecyl sulphate (SDS) and three glycoproteins were isolated by sequential affinity chromatography on Concanavalin A (Con A) and wheat germ lectin (WGL) Sepharose columns. Two glycoproteins, designated GP II and III, were found to have a high carbohydrate content of about 30%. Mannose, galactose, N-acetylgalactosamine, and sialic acid were the main sugars. In addition membrane-bound dopamine beta-hydroxylase was isolated by this procedure. No significant differences between the carbohydrate composition of the membrane-bound and the soluble enzyme were revealed. It was shown that all four subunits of dopamine beta-hydroxylase possess carbohydrate chains with an affinity for Con A. The isolation methods established in this study will be useful for immunological studies on these glycoproteins.
D000313:Adrenal Medulla / Q000032:analysis* / Q000201:enzymology; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002837:Chromaffin Granules / Q000032:analysis* / Q000201:enzymology; D002838:Chromaffin System / Q000032:analysis*; D002846:Chromatography, Affinity; D004299:Dopamine beta-Hydroxylase / Q000302:isolation & purification*; D006023:Glycoproteins / Q000302:isolation & purification*; D007425:Intracellular Membranes / Q000032:analysis*; D008565:Membrane Proteins / Q000302:isolation & purification*
D002241:Carbohydrates; D006023:Glycoproteins; D008565:Membrane Proteins; D004299:Dopamine beta-Hydroxylase