Affinity labeling of rat GST A1-1 by 3β-(iodoacetoxy)dehydroisoandrosterone

R. F. Colman, J. J. Barycki

Research output: Contribution to journalArticle

Abstract

3β-(Iodoacetoxy)dehydroisoandrosterone (3β-IDA), a reactive steroid analogue, functions as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent inactivation is observed upon incubation of enzyme with 3β-IDA at pH 7.5 and 37°C. The rate of enzyme inactivation exhibits a nonlinear dependence on 3β-IDA concentration, yielding an apparent K(i) of 21 μM. Protection against inactivation is provided by the non-substrate steroid ligand 17β-estradiol-3,17-disulfate but not by Δ5-androstene 3,17-dione or any other electrophilic substrate tested. These results suggest that reaction occurs within a non-substrate steroid site, which is distinguishable from the electrophilic substrate binding site. Two cysteines, Cys17 and Cys111, are modified in equal amounts, although the average reagent incorporation is 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer upon complete inactivation. Molecular modeling suggests that Cys17 and Cys111 are located in the non-substrate steroid binding site, within the cleft between the two subunits of the dimeric enzyme.

Original languageEnglish (US)
Pages (from-to)255-259
Number of pages5
JournalClinical Chemistry and Enzymology Communications
Volume8
Issue number4-6
StatePublished - Dec 1 1999

Fingerprint

Labeling
Rats
Steroids
Enzymes
Binding Sites
Affinity Labels
Molecular modeling
Substrates
Glutathione Transferase
Dimers
Liver
Isoenzymes
Cysteine
3-(iodoacetoxy)dehydroisoandrosterone
Ligands

Keywords

  • Affinity labeling
  • Glutathione S-transferase
  • Steroid sites

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Affinity labeling of rat GST A1-1 by 3β-(iodoacetoxy)dehydroisoandrosterone. / Colman, R. F.; Barycki, J. J.

In: Clinical Chemistry and Enzymology Communications, Vol. 8, No. 4-6, 01.12.1999, p. 255-259.

Research output: Contribution to journalArticle

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AB - 3β-(Iodoacetoxy)dehydroisoandrosterone (3β-IDA), a reactive steroid analogue, functions as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent inactivation is observed upon incubation of enzyme with 3β-IDA at pH 7.5 and 37°C. The rate of enzyme inactivation exhibits a nonlinear dependence on 3β-IDA concentration, yielding an apparent K(i) of 21 μM. Protection against inactivation is provided by the non-substrate steroid ligand 17β-estradiol-3,17-disulfate but not by Δ5-androstene 3,17-dione or any other electrophilic substrate tested. These results suggest that reaction occurs within a non-substrate steroid site, which is distinguishable from the electrophilic substrate binding site. Two cysteines, Cys17 and Cys111, are modified in equal amounts, although the average reagent incorporation is 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer upon complete inactivation. Molecular modeling suggests that Cys17 and Cys111 are located in the non-substrate steroid binding site, within the cleft between the two subunits of the dimeric enzyme.

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