Evidence that lectin-binding sites are present on the surface of isolated hypothalamic granules containing luteinizing hormone-releasing hormone

Abstract

In the present study, we wished to ascertain if lectin-binding sites are present on hypothalamic granules containing luteinizing hormone-releasing hormone (LHRH). LHRH granules (isolated from homogenates of hypothalami of adult male rats by means of hypo-osmotic shock and differential centrifugation) were subjected to affinity chromatography on columns of concanavalin A (Con A)-Sepharose. The amount of LHRH present in the unbound and bound fraction was measured by radioimmunoassay. Of the total LHRH present in the granule suspension chromatographed, 30 to 40% was bound to the columns, and this binding was not related to the binding of the bulk to the proteins. Synthetic LHRH by itself or synthetic LHRH added to the LHRH granules before chromatography did not bind to Con A-Sepharose, indicating that only LHRH contained within granules is retained on the columns. In addition, we found that the binding of LHRH granules to Con A-Sepharose has a requirement for Ca2+ and Mn2+: after equilibration of Con A-Sepharose with Ca2+ and Mn2+ (prior to the chromatography), all of the LHRH granules were bound to the columns, and this binding was prevented by EDTA. To examine the specificity of the binding of LHRH granules to Con A-Sepharose, a competing sugar (alpha-methyl-D-mannoside (alpha-MM)) was added to the LHRH granules, and the columns were equilibrated with alpha-MM prior to chromatography. Under this condition, the Ca2+- and Mn2+-dependent binding of LHRH granules to Con A-Sepharose was inhibited. In addition to Con A-Sepharose, we observed that LHRH granules bind to wheat germ agglutinin-Sepharose but not to Sepharose which does not contain a lectin. These findings are indicative that lectin-binding sites, carbohydrate in nature, are present on the surface of isolated LHRH granules. We propose that such binding sites may play a role in the release of LHRH from hypothalamic neurons.