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 Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Strickler, R. C. Articles by Warren, J. C. Search for Related Content PubMed PubMed Citation Articles by Strickler, R. C. Articles by Warren, J. C. Social Bookmarking                      What's this?

JBC, Vol. 250, Issue 19, 7656-7662, Oct, 1975

Affinity labeling of steroid binding sites. Study of the active site of 20beta-hydroxysteroid dehydrogenase with 2alpha-bromoacetoxyprogesterone and 11alpha-bromacetoxyprogesterone

R. C. Strickler, F. Sweet and J. C. Warren

To further characterize the active site of 20beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) from Streptomyced hydrogenans we synthesized 2alpha-bromoacetoxyprogesterone, a substrate for the enzyme in 0.05 M phosphate buffer at 25 degrees, pH 7.0, with Km and Vmax values of 1.90 X 10(-5) M and 6.09 nmol/min/mg of enzyme, respectively. This affinity labeling steroid inactivates 20beta-hydroxysteroid dehydrogenase in an irreversible and time-dependent manner which follows pseudo-first order kinetics with a t1/2 value of 4.6 hours. 2alpha-[2-3H]Bromoacetoxyprogesterone was synthesized and used to radiolabel the enzyme active site. Amino acid analysis of the acid hydrolysate of the radiolabeled enzyme supports a mechanism whereby the steroid moiety delivers the alkylating group to the steroid binding site of the enzyme where it reacts with a methionyl residue. Both 2alpha- and 11alpha-bromoacetoxyprogesterone alkylate a methionyl residue at the active site of 20beta-hydroxysteroid dehydrogenase. The enzyme was inactivated with a mixture containing both 2alpha-[2-3H]Bromoacetoxyprogesterone and 11alpha-2[2-14C]bromoacetoxyprogesterone. Following degradation of separate aliquots of the radiolabeled enzyme by cyanogen bromide or trypsin, the protein fragments were separated by gel filtration and ion exchange chromatography. Resolution of peptides carrying the 3H label from those possessing the 14C label demonstrates that 2alpha-bromoacetoxyprogesterone and 11alpha-bromoacetoxyprogesterone each label a different methionine at the steroid binding site of 20beta-hydroxysteroid dehydrogenase.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				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]