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  • Title: Isolation of histidyl peptides of the steroid-binding site of human placental estradiol 17 beta-dehydrogenase.
    Author: Murdock GL, Warren JC.
    Journal: Steroids; 1982 Feb; 39(2):165-79. PubMed ID: 6951320.
    Abstract:
    Human placental estradiol 17 beta-dehydrogenase (E.C. 1.1.1.62) was inactivated at pH 6.3 by 3-bromo [2'-14C] acetoxy-1,3,5(10) estratrien-17-one, a know substrate. The affinity-alkylated enzyme was then hydrolyzed by trypsin. Radioactive peptides were initially isolated by gel filtration and identified according to which residue was alkylated. Tryptic peptides containing radioactive 3-carboxymethylhistidyl residues were further purified by cation-exchange chromatography. The population of these peptides varied, depending upon the conditions of enzyme inactivation. With 60 microM 3-bromo[2'-14]acetoxy-1,3,5 (10) estratrien -17-one four major peptides (a,b,c,d) each containing radioactive 3-carboxymethylhistidine, were eluted from the cation-exchange column. The alkylation of all of these peptides was completely suppressed when the enzyme was inactivated in the presence of excess estradiol-17 beta. The presence of equimolar NADPH during incubation greatly enhanced the alkylation of all four peptides. In the presence of NADPH, estradiol-17 beta most significantly decreased the formation of peptide d. Peptide d was the only peptide identified when the concentration of the alkylating steroid was lowered to 6 microM, a value approaching the Km. These observations indicate that peptide d is a histidyl-bearing peptide from the steroid-binding site which proximates the steroid A-ring. They further suggest that with the affinity labeling steroid at higher concentrations other nonspecific, hydrophobic sites on the enzyme are occupied and labeled.
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