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  • Title: Affinity labeling in the presence of the reduced diphosphopyridine nucleotide NADH identifies peptides associated with the activities of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase.
    Author: Thomas JL, Nash WE, Crankshaw MW, Strickler RC.
    Journal: J Soc Gynecol Investig; 1994; 1(2):155-63. PubMed ID: 9419765.
    Abstract:
    OBJECTIVE: We sought to identify peptides associated with activity in the primary structure of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase (3 beta-HSD/isomerase). METHODS: Purified human placental 3 beta-HSD/isomerase was affinity-radioalkylated by 2 alpha-bromo [2'-14C]acetoxyprogesterone (2 alpha-[14C]BAP) in the presence or absence of the reduced diphosphopyridine nucleotide, NADH. NADH protected both 3 beta-HSD and isomerase from inactivation by 2 alpha-[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 2 alpha-[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 2 alpha-[14C]BAP only in the presence of NADH. CONCLUSIONS: We have previously reported that the substrate pregnenolone blocks the inactivation of 3 beta-HSD by 2 alpha-[14C]BAP through the protection of His262 in the Arg-250 peptide. Protection by NADH against the inactivation of isomerase as well as 3 beta-HSD is evidence that 2 alpha-[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 3 beta-HSD and isomerase reactions are catalyzed in this region of the enzyme protein.
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