Reactivation of human placental 17 beta, 20 alpha-hydroxysteroid dehydrogenase: affirmation of affinity labeling principles.

Abstract:

:Human placental 17 beta, 20 alpha-hydroxysteroid dehydrogenase was completely inactivated by the affinity alkylator, 3-bromoacetoxy-1,3,5(10)-estratrien-17-one (estrone 3-bromoacetate). The inactivated enzyme was then reactivated to 100% of the enzyme activity by base-catalyzed hydrolysis of the steroidalester-enzyme conjugate. After the reactivated enzyme was repurified by dialysis, re-inactivation studies were performed on it. The reactivated enzyme could not be re-inactivated by the original alkylator, estrone 3-bromoacetate. However, 16 alpha-bromoacetoxyestradiol-17 beta 3-methyl ether caused a loss of reactivated enzyme activity at a rate comparable to that for the native enzyme. These observations demonstrate that a specific amino acid modification within the enzyme active site was produced by estrone 3-bromoacetate alkylation and suggest that the conformation of the active center was essentially unaltered. Thus, these successful reactivation studies of 17 beta, 20 alpha-hydroxysteroid dehydrogenase affirm the specificity of affinity labeling. This methodology also offers a new tool to investigate the steroid binding regions of macromolecular proteins.

journal_name

Steroids

journal_title

Steroids

authors

LaRochelle MC,Thomas JL,Strickler RC

doi

10.1016/0039-128x(84)90039-4

subject

Has Abstract

pub_date

1984-02-01 00:00:00

pages

209-17

issue

2

eissn

0039-128X

issn

1878-5867

pii

0039-128X(84)90039-4

journal_volume

43

pub_type

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