Enhanced purification of Mullerian inhibiting substance by lectin affinity chromatography.
Mullerian inhibiting substance (MIS), a secreted testicular product responsible for regression of the Mullerian ducts in the male mammalian embryo, was purified 7000 fold, exploiting the glycoprotein nature of this important fetal regressor to achieve enhanced purification. The present procedure employs media incubation of newborn calf testis, passage through DEAE Bio-Gel A and CM Bio-Gel A and sequential lectin affinity chromatography on wheat germ lectin (WGL)-Sepharose 6MB and concanavalin A (Con A)-Sepharose 4B. Strongly bioactive MIS was released from both lectin columns in the bound glycoprotein fraction only after elution with lectin-specific sugar. Carbohydrate analysis of the highly purified glycoprotein fraction eluted from Con A indicated the presence of both N-acetyl glucosamine and mannose, as would be expected from its sequential lectin affinity, as well as of galactose, galactosamine and N-acetyl neuraminic acid. Electrophoresis of this fraction on polyacrylamide-SDS gels showed an identical band pattern after staining with either Coomassie blue or periodic acid-Schiff reagent, further indicating that MIS is a glycoprotein.
D000818:Animals; D054304:Anti-Mullerian Hormone; D002417:Cattle; D002846:Chromatography, Affinity; D003208:Concanavalin A; D006023:Glycoproteins / Q000032:analysis / Q000302:isolation & purification*; D006131:Growth Inhibitors; D008297:Male; D009095:Mullerian Ducts / Q000033:anatomy & histology*; D013735:Testicular Hormones / Q000032:analysis / Q000302:isolation & purification*; D013737:Testis / Q000032:analysis*
D006023:Glycoproteins; D006131:Growth Inhibitors; D013735:Testicular Hormones; D003208:Concanavalin A; D054304:Anti-Mullerian Hormone