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Affinity Labeling in the Presence of the Reduced Diphosphopyridine Nucleotide NADH Identifies Peptides Associated With the Activities of Human Placental 3ß-Hydroxy-5- Steroid Dehydrogenase/Isomerase

Departments of Obstetrics and Gynecology and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri

William E. Nash, BS

Departments of Obstetrics and Gynecology and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri

Mark W. Crankshaw, PhD

Departments of Obstetrics and Gynecology and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri

Ronald C. Strickler, MD

Departments of Obstetrics and Gynecology and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri

OBJECTIVE: We sought to identify peptides associated with activity in the primary structure of human placental 3ß-hydroxy-⁵-steroid dehydrogenase/isomerase (3ß-HSD/isomerase).

METHODS: Purified human placental 3ß-HSD/isomerase was affinity-radioalkylated by 2- bromo[2'- ¹⁴ C]acetoxyprogesterone (2-[¹⁴C]BAP) in the presence or absence of the reduced diphosphopyridine nucleotide, NADH. NADH protected both 3ß-HSD and isomerase from inactivation by 2-[ 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 2- [¹⁴C]BAP away from the Arg-250 peptide (²⁵¹GQFYYISDDTPHQSYDNLNYTLSK²⁷⁴) and toward the Lys-135 tryptic peptide (¹³⁶EIIQNGHEEEPLENTWPAPYPHSK ¹⁵⁹). 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 2-[¹⁴C]BAP only in the presence of NADH.

CONCLUSIONS: We have previously reported that the substrate pregnenolone blocks the inac tivation of 3ß-HSD by 2-[¹⁴C]BAP through the protection of His²⁶² in the Arg-250 peptide. Protection by NADH against the inactivation of isomerase as well as 3ß-HSD is evidence that 2-[ 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 3ß-HSD and isomerase reactions are catalyzed in this region of the enzyme protein. (J Soc Gynecol Invest 1994;1:155-63)

Key Words: 3ß-hydroxy-5-steroid dehydrogenase/isomerase • 2-bromo[2'-14C]acetoxyprogesterone • NADH • tryptic peptides • affinity radioalkylation.

Journal of the Society for Gynecologic Investigation, Vol. 1, No. 2, 155-163 (1994)
DOI: 10.1177/107155769400100211


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