Affinity radiolabeling identifies peptides and amino acids associated with substrate binding in human placental 3 beta-hydroxy-delta(5)-steroid dehydrogenase.
J Biol Chem
; 268(25): 18507-12, 1993 Sep 05.
Article
em En
| MEDLINE
| ID: mdl-8360152
ABSTRACT
Purified human placental 3 beta-hydroxy-delta(5)-steroid dehydrogenase (3 beta-HSD) was affinity radiolabeled by 2 alpha-bromo[2'-14C]acetoxyprogesterone (2 alpha-BAP) in the presence or absence of 3 beta-HSD substrate, pregnenolone. The substrate steroid substantially protects 3 beta-HSD activity from inactivation by 2 alpha-BAP. Tryptic peptides of unprotected and substrate-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. According to the percent total radioactivity associated with each of four radiolabeled peaks separated by high pressure liquid chromatography, two peptides were protected by substrate from affinity radioalkylation by 2 alpha-BAP. The first, 251GQFYYISDDTPHQSYDNLNYTLSK274, was produced by tryptic cleavage at Arg-250 and Lys-274 (the Arg-250 peptide) and contained radiolabeled His262. The second, 176NGGTLYTCALR186, was produced by tryptic cleavage at Lys-175 and Arg-186 (the Lys-175 peptide) and contained radiolabeled Cys183. Based on amino acid analysis to quantitate radioactivity incorporated per nmol of peptide, substrate steroid decreased the radiolabeling of His262 in the Arg-250 peptide by 3.6-fold and decreased the radiolabeling of Cys183 in the Lys-175 peptide by 3.7-fold. Three minor radiolabeled peptides (the NH2-terminal, Arg-71, and Arg-196 tryptic peptides) were also identified in the primary structure, but pregnenolone did not diminish their affinity radioalkylation. These observations indicate that the Arg-250 and Lys-175 peptides are involved in substrate binding and suggest that His262 and Cys183 are in close proximity in the three-dimensional structure of the enzyme.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Placenta
/
Radioisótopos de Carbono
/
Marcadores de Afinidade
/
Hidroxiprogesteronas
/
3-Hidroxiesteroide Desidrogenases
Tipo de estudo:
Risk_factors_studies
Limite:
Female
/
Humans
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
1993
Tipo de documento:
Article