| Autor: |
Thomas, James L., Nash, William E., Crankshaw, Mark W., Strickler, Ronald C. |
| Zdroj: |
Reproductive Sciences; April 1994, Vol. 1 Issue: 2 p155-163, 9p |
| Abstrakt: |
Objective: We sought to identify peptides associated with activity in the primary structure of human placental 3-ß-heydrroxyo-?5dehydrogenase/isomerase (3ß-HSD/isomerase). Methods: Purified human placental 3ß-HSD/isomerase was affinity-radioalkylated by 2a-bromo[2]-14C]aceotoxyprogesterone (2a]-[14C]BAP) in the presence or absence of the reduced diphosphopyridine nucleotide, NADH. NADH protected both 3ß-HSD and isomerase from inactivation by 2a]-[14C]BAP). Tryptic peptides of unprotected and NADH-protected radioalkylated 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 structure of the enzyme. Results: According to the sequence analyses, NADH shifted radioalkylation by 2a-[ C] BAP away from the Arg-250 peptide ( GQFYYISDDTPHQSYDNLNYTLSK ) 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 His in the Arg-250 peptide by 8.2-fold. His142in the Lys-135 peptide was radiolabeled by 2a-[ 14C]BAP only in the presence of NADH. Conclusions: We have previously reported that the substrate pregnenolone blocks the inactivation of 3ß-HSD by 2a-[ 14C]BAP through the protection of His262in the Arg-250 peptide. Protection by NADH against the inactivation of isomerase as well as 3ß-HSD is evidence that 2a-[ 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. |
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