Purification of testicular anti-Müllerian hormone allowing direct visualization of the pure glycoprotein and determination of yield and purification factor.
An improved method is described for the purification of anti-Müllerian hormone from incubation medium of bovine fetal testes, using RIA to optimize the yield at different steps. Proteins present in incubation medium are precipitated by ammonium sulphate at 30-45% saturation and subjected to ion-exchange chromatography on DEAE-Sepharose. The bulk of the hormone is eluted from the ion-exchanger by a 0.12 M concentration of NaCl. Purification is achieved by immunochromatography on a monoclonal antibody: usually, addition of extraneous protein to the eluting buffer is required, but can be dispensed with if a second, cumulative, immunochromatography is performed by pooling the eluates from the first procedure. AMH obtained by this procedure has been studied using Coomassie blue and immunoblotting, and compared with fucose-labelled AMH, which is recognized by the same monoclonal antibodies as the carrier of the anti-Müllerian biological activity. In the absence of reducing conditions, several multimers, from 145 000 to 235 000 in molecular weight, are present. Reduction of disulphide bond linkages results in the disappearance of the multimeric forms, and the appearance of a 72 000 monomer. Equivalence between RIA and weight units has been established in one experiment: one AMH unit corresponds to 15 micrograms of protein. Pure AMH isolated by this procedure is highly bioactive at a concentration of 200 mU/ml.
D000818:Animals; D054304:Anti-Mullerian Hormone; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006023:Glycoproteins; D006131:Growth Inhibitors; D008297:Male; D008722:Methods; D008970:Molecular Weight; D011247:Pregnancy; D011863:Radioimmunoassay; D013735:Testicular Hormones / Q000302:isolation & purification*; D013737:Testis / Q000032:analysis*
D006023:Glycoproteins; D006131:Growth Inhibitors; D013735:Testicular Hormones; D054304:Anti-Mullerian Hormone