An assay was developed to detect tritium-labeled ovine FSH β-subunit ([3H]oFSHβ) secreted from primary ovine pituitary cultures. This procedure used affinity-enriched antibodies raised against reduced and carbamylmethylated oFSHβ (RCM-oFSHβ) in a two-cycle immunoextraction procedure. A discrete species with an apparent rhol wt of 21,000 was detected in sodium dodecyl sulfate electrophoretic patterns of immunoextracts from culture medium. This species was identified as RCM- [3H]oFSHβ by its comigration with highly purified RCM oFSHβ, its reduction in culture media after cultures were treated with 17β-estradiol, which normally decreases radioimmunoassayable oFSH; and its displacement from the extracting antibodies by excess unlabeled RCM-oFSHβ. The assay was used in a pulse-chase study to determine that [3H]oFSHβ is secreted within 1-2 h of its synthesis. Prior treatment of cultures with 17β-estradiol did not change this timing of secretion.
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