OBJECTIVE:We sought to identify peptides associated with activity in the primary structure of human placental 3β-hydroxy-Δ⁵-steroid dehydrogenase/isomerase (3β-HSD/isomerase).METHODS:Purified human placental 3β-HSD/isomerase was affinity-radioalkylated by 2α- bromo[2'-¹⁴ C]acetoxyprogesterone (2α-[¹⁴C]BAP) in the presence or absence of the reduced diphosphopyridine nucleotide, NADH. NADH protected both 3β-HSD and isomerase from inactivation by 2α-[¹⁴ C]BAP. Tryptic peptides of unprotected and NADH-protected radioalkyl ated enzyme were purified by high-pressure liquid chromatography. The amino acid sequence of each radiolabeled peptide was determined and localized within the cDNA-derived primary struc ture of the enzyme.RESULTS:According to the sequence analyses, NADH shifted radioalkylation by 2α- [¹⁴C]BAP away from the Arg-250 peptide (²⁵¹GQFYYISDDTPHQSYDNLNYTLSK²⁷⁴) and toward the Lys-135 tryptic peptide (¹³⁶EIIQNGHEEEPLENTWPAPYPHSK¹⁵⁹). Based on amino acid analysis to quantitate radioactivity incorporated per nmol peptide, NADH decreased the radiolabeling of His²⁶² in the Arg-250 peptide by 8.2-fold. His¹⁴² in the Lys-135 peptide was radiolabeled by 2α-[¹⁴C]BAP only in the presence of NADH.CONCLUSIONS: We have previously reported that the substrate pregnenolone blocks the inac tivation of 3β-HSD by 2α-[¹⁴C]BAP through the protection of His²⁶² in the Arg-250 peptide. Protection by NADH against the inactivation of isomerase as well as 3β-HSD is evidence that 2α-[¹⁴C]BAP binds at the active sites of both enzyme activities. Because the same Arg-250 peptide has been affinity-alkylated in studies that targeted each of the two activities, we propose that the 3β-HSD and isomerase reactions are catalyzed in this region of the enzyme protein. (J Soc Gynecol Invest 1994;1:155-63)