J. Biol. Chem., Vol. 265, Issue 15, 8431-8435, 05, 1990
Histidine 235 of human sex hormone-binding globulin is the covalent site of attachment of the nucleophilic steroid derivative, 17 beta- bromoacetoxydihydrotestosterone
MS Khan and W Rosner
Department of Medicine, St. Luke's/Roosevelt Hospital Center, New York, New York 10019.
This article deals with the elucidation of the steroid-binding site of
human sex hormone-binding globulin (SHBG). 17 beta-
Bromoacetoxydihydrotesterone (BA-DHT) reacted with highly purified SHBG in
a time-dependent and irreversible fashion. The interaction could be totally
inhibited by the simultaneous addition of an excess of dihydrotesterone. At
the completion of the reaction, the molar ratio of BA-DHT to SHBG was
approximately unity. SHBG was affinity labeled with [14C]BA-DHT and
submitted to acid hydrolysis. The released amino acids were evaluated on
high performance liquid chromatography, and virtually all of the 14C was
identified as 3-[14C]carboxymethylhistidine. Furthermore,
[14C]BA-DHT-labeled SHBG was digested with trypsin, followed by isolation
of the released tryptic peptides by reverse-phase high performance liquid
chromatography. The 14C was localized to a single tryptic peptide. It
contained 2' histidyl residues, corresponding to residues 235 and 251 in
the known amino acid sequence of SHBG. Although most of the
3-[14C]carboxymethylhistidine, or its phenylthiohydantoin derivative, was
trapped on the filter of the amino acid sequenator, sufficient
radioactivity emerged to identify histidyl residue 235 as the labeled amino
acid.
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