Affinity radiolabeling identifies peptides associated with the isomerase activity of human type I (placental) 3beta-hydroxysteroid dehydrogenase/isomerase.
Biochemistry
; 36(29): 9029-34, 1997 Jul 22.
Article
em En
| MEDLINE
| ID: mdl-9220991
3beta-Hydroxysteroid dehydrogenase and steroid Delta5-->4-isomerase (3beta-HSD/isomerase) were purified as a single protein from human term placenta. The affinity alkylator, 5,10-secoestr-4-yne-3,10, 17-trione (secosteroid), was incubated with the purified enzyme (30/1 secosteroid/enzyme molar ratio) to produce an 80% loss of initial isomerase activity over 90 min in a time-dependent, irreversible manner. The secosteroid inactivated 3beta-HSD by only 20% during the same 90 min. Incubations containing the isomerase substrate steroid, 5-androstene-3,17-dione, completely protected the isomerase activity from inactivation by the secosteroid and did not slow the inactivation of 3beta-HSD. The enzyme containing covalently bound steroid was separated from unreacted secosteroid by reversed phase HPLC. Ketones on the protein-bound secosteroid were radiolabeled by reduction with sodium boro[3H]hydride (specific radioactivity 50 microCi/micromol for the transferred tritium). After removal of the unreacted sodium boro[3H]hydride, the affinity-radiolabeled enzyme was digested with trypsin-TPCK, and the peptides were isolated by reversed phase HPLC. The radiolabeled peptide fractions were sequenced. The secosteroid alkylated three tryptic peptides: 251GQFYYISDDTPHQSYDNLNYTLSK274, tritiated His262; 176NGGTLYTCALR186, tritiated Cys183; and 353TVEWVGSLVDR363, tritiated Trp356. Coincubation with the isomerase substrate blocked the labeling of these three peptides and shifted the alkylation by secosteroid to a single tryptic peptide (135EIIQNGHEEEPLENTWPAPYPHSK159, tritiated His142). Using substrate protection to validate specificity, the affinity labeling secosteroid has identified peptides in the enzyme that are associated with isomerase activity.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Placenta
/
Progesterona Redutase
/
Esteroide Isomerases
/
Marcadores de Afinidade
/
Estrenos
/
Complexos Multienzimáticos
Tipo de estudo:
Risk_factors_studies
Limite:
Humans
Idioma:
En
Revista:
Biochemistry
Ano de publicação:
1997
Tipo de documento:
Article
País de afiliação:
Estados Unidos
País de publicação:
Estados Unidos