HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Penning, T. M. Articles by Pawlowski, J. E. Search for Related Content PubMed PubMed Citation Articles by Penning, T. M. Articles by Pawlowski, J. E. Social Bookmarking                      What's this?

J. Biol. Chem., Vol. 266, Issue 14, 8826-8834, May, 1991

Affinity labeling of 3 alpha-hydroxysteroid dehydrogenase with 3 alpha- bromoacetoxyandrosterone and 11 alpha-bromoacetoxyprogesterone. Isolation and sequence of active site peptides containing reactive cysteines; sequence confirmation using nucleotide sequence from a cDNA clone

TM Penning, WR Abrams and JE Pawlowski
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.

Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) of rat liver cytosol is potently inhibited at its active site by nonsteroidal anti-inflammatory drugs (NSAIDs). Using 3 alpha- bromoacetoxy-5 alpha-androstan-17-one (BrAnd, a substrate analog) and 11 alpha-bromoacetoxyprogesterone (Br11P, a glucocorticoid analog) as affinity-labeling agents, kinetic evidence was obtained that these agents alkylate this site. Inactivation of 3 alpha-HSD with either [14C]BrAnd or [14C] Br11P led to the incorporation of 1 mol of affinity- labeling agent per enzyme monomer. Complete acid hydrolysis of 3 alpha- HSD radiolabeled with either agent followed by amino acid analysis led to the identification of [14C]carboxymethylcysteine indicating that [14C]BrAnd and [14C]Br11P covalently tag discrete reactive cysteine(s) at the enzyme active site. Trypsin digestion of [14C]BrAnd-inactivated 3 alpha-HSD followed by peptide mapping led to the purification of a single radiolabeled peptide (3A1) which gave the following sequence: H2N-Ser-Ile-Gly-Val-Ser-Asn-Phe-Asn-X-Arg-CO2H. Identical experiments on [14C] Br11P-inactivated 3 alpha-HSD led to the purification of three radiolabeled peptides (11P1-11P3). The major radiolabeled peptide (11P1) had an identical sequence to 3A1 which was tagged with [14C]BrAnd. The minor radiolabeled peptides had the following sequences: H2N-Ser-Lys-Asp-Ile-Ile-Leu-Val-Ser-Tyr-X-Thr-Leu-Gly-Ser- Ser-Arg-CO2H (11P2) and H2N-Ser-Pro-Val-Leu-Leu-Asp-Asp-Pro-Val-Leu-X- Ala-Ile-Ala-Lys-CO2H (11P3). In each peptide group X was identified as carboxymethylcysteine. Alignment of the peptide sequences with the primary structure of 3 alpha-HSD, deduced from its cDNA clone, assigned peptide 11P1 to residues 162-171, peptide 11P2 to residues 208-223, and peptide 11P3 to residues 232-246 of the amino acid sequence. The reactive cysteines correspond to Cys170, Cys217, and Cys242. We propose that Cys170 labeled by BrAnd may lie within the catalytic pocket of the enzyme. By contrast the 11 alpha-bromoacetoxy group in Br11P labeled several reactive cysteines which may be involved in the binding of glucocorticoids and NSAIDs.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. M. Jez, B. P. Schlegel, and T. M. Penning Characterization of the Substrate Binding Site in Rat Liver 3alpha -Hydroxysteroid/Dihydrodiol Dehydrogenase. THE ROLES OF TRYPTOPHANS IN LIGAND BINDING AND PROTEIN FLUORESCENCE J. Biol. Chem., November 22, 1996; 271(47): 30190 - 30198. [Abstract] [Full Text] [PDF]

				J. L. Thomas, W. E. Nash, M. W. Crankshaw, and R. C. Strickler Affinity Labeling in the Presence of the Reduced Diphosphopyridine Nucleotide NADH Identifies Peptides Associated With the Activities of Human Placental 3{beta}-Hydroxy-{Delta}5- Steroid Dehydrogenase/Isomerase Reproductive Sciences, April 1, 1994; 1(2): 155 - 163. [Abstract] [PDF]