| Автор | Thomas, James, L. |
| Автор | Nash, William, E. |
| Автор | Crankshaw, Mark, W. |
| Автор | Strickler, Ronald, C. |
| Дата выпуска | 1994 |
| dc.description | OBJECTIVE:We sought to identify peptides associated with activity in the primary structure of human placental 3β-hydroxy-Δ<sup>5</sup>-steroid dehydrogenase/isomerase (3β-HSD/isomerase).METHODS:Purified human placental 3β-HSD/isomerase was affinity-radioalkylated by 2α- bromo[2'-<sup> 14</sup> C]acetoxyprogesterone (2α-[<sup>14</sup>C]BAP) in the presence or absence of the reduced diphosphopyridine nucleotide, NADH. NADH protected both 3β-HSD and isomerase from inactivation by 2α-[<sup> 14</sup> 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- [<sup>14</sup>C]BAP away from the Arg-250 peptide (<sup>251</sup>GQFYYISDDTPHQSYDNLNYTLSK<sup>274</sup>) and toward the Lys-135 tryptic peptide (<sup>136</sup>EIIQNGHEEEPLENTWPAPYPHSK<sup> 159</sup>). Based on amino acid analysis to quantitate radioactivity incorporated per nmol peptide, NADH decreased the radiolabeling of His<sup>262</sup> in the Arg-250 peptide by 8.2-fold. His<sup>142</sup> in the Lys-135 peptide was radiolabeled by 2α-[<sup>14</sup>C]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-[<sup>14</sup>C]BAP through the protection of His<sup>262</sup> in the Arg-250 peptide. Protection by NADH against the inactivation of isomerase as well as 3β-HSD is evidence that 2α-[<sup> 14</sup>C]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) |
| Издатель | Sage Publications |
| Тема | 3β-hydroxy-Δ5-steroid dehydrogenase/isomerase |
| Тема | 2α-bromo[2'-14C]acetoxyprogesterone |
| Тема | NADH |
| Тема | tryptic peptides |
| Тема | affinity radioalkylation. |
| Название | Affinity Labeling in the Presence of the Reduced Diphosphopyridine Nucleotide NADH Identifies Peptides Associated With the Activities of Human Placental 3β-Hydroxy-Δ5- Steroid Dehydrogenase/Isomerase |
| Тип | Other |
| DOI | 10.1177/107155769400100211 |
| Print ISSN | 1071-5576 |
| Журнал | Journal of the Society for Gynecologic Investigation |
| Том | 1 |
| Первая страница | 155 |
| Последняя страница | 163 |
| Аффилиация | Thomas, James, L., Departments of Obstetrics and Gynecology and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri |
| Аффилиация | Nash, William, E., Departments of Obstetrics and Gynecology and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri |
| Аффилиация | Crankshaw, Mark, W., Departments of Obstetrics and Gynecology and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri |
| Аффилиация | Strickler, Ronald, C., Departments of Obstetrics and Gynecology and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri |
| Выпуск | 2 |
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