Affinity radiolabeling identifies peptides associated with the isomerase activity of human type I (placental) 3beta-hydroxysteroid dehydrogenase/isomerase.

3β-Hydroxysteroid dehydrogenase and steroid Δ5→4-isomerase (3β-HSD/isomerase) were purified as a single protein from human term placenta. The affinity alkylator, 5,10-secoestr-4-yne-3,10,17-trione (secosteroid), was incubated with the purified enzyme (30/1 secosteroid/enzyme molar ratio) to produce an 80% loss of initial isomerase activity over 90 min in a time-dependent, irreversible manner. The secosteroid inactivated 3β-HSD by only 20% during the same 90 min. Incubations containing the isomerase substrate steroid, 5-androstene-3,17-dione, completely protected the isomerase activity from inactivation by the secosteroid and did not slow the inactivation of 3β-HSD. The enzyme containing covalently bound steroid was separated from unreacted secosteroid by reversed phase HPLC. Ketones on the protein-bound secosteroid were radiolabeled by reduction with sodium boro[3H]hydride (specific radioactivity 50 μCi/μmol for the transferred tritium). After removal of the unreacted sodium boro[3H]hydride, the affinity-...