Isolation and characterization of the subunits of highly purified ovine follicle-stimulating hormone.

Highly purified ovine follicle-stimulating hormone (FSH) was separated into two noncovalently linked, nonidentical subunits (CX and @) by treatment with 8 M urea followed by ion exchange chromatography on DEAE-Sephadex. The individual subunits were purified by gel filtration on Sephadex G-100. This step resulted in the removal of a larger molecular weight contaminant from the /I subunit. This contaminant, designated as undissociated FSH because of its relatively high biological activity, was not dissociated by further treatment with urea, suggesting that it may consist of covalently linked subunits. Although their electrophoretic mobilities differed greatly, both subunits exhibited microheterogeneity as indicated by analytical acrylamide disc gel electrophoresis. The molecular weights of native FSH and the a! and /I subunits as determined by ultracentrifugation were 33,000, 15,500, and 18,500, respectively. The subunits contained the amino acids and carbohydrates usually found in glycoprotein hormones with the exception that methionine was not present in the ,8 subunit. Although they differed markedly in amino acid and carbohydrate content, both subunits exhibited relatively high amounts of threonine, halfcystine, glucosamine, and mannose. The amino acid composition of the a! subunit was very similar to the amino acid contents of the LY subunits of ovine and bovine luteinizing hormone. The sum of the amino acid and carbohydrate contents of the two subunits corresponded well with the amino acid and carbohydrate composition of the native FSH. Incubation of the biologically inactive CY and p subunits followed by gel filtration on Sephadex G-100 resulted in reconstituted FSH which possessed approximately 68% of the biological activity of the original FSH as measured by the augmentation bioassay. The reconstituted FSH was almost as potent as the original FSH in the ability to stimulate the prostates, seminal vesicles, and testes in hypophysectomized male rats.