Photoaffinity labeling of homologous Met-133 and Met-139 amino acids of rabbit and sheep sex hormone-binding globulins with the unsubstituted Delta 6-testosterone photoreagent.
Purified rabbit and sheep sex hormone-binding globulins (SHBGs) were photolabeled by Delta 6-testosterone. The maximal levels of specific incorporation were respectively 0.33 and 0.30 mol of label/mol of homodimer. Tryptic cleavage of photolabeled SHBGs gave a single radioactive peptide for rabbit SHBG and two major radioactive peptides S1 and S2 for sheep SHBG. Edman sequencing of the photolabeled peptide of rabbit SHBG revealed a single sequence corresponding to peptidic fragment Leu-118-Lys-134. Subcleavage of this peptide with elastase led to a single radioactive peptidic fragment corresponding to dipeptide Met-133-Lys-134, identified by mass spectrometry, while deletion of the C-terminal residue with carboxypeptidase B showed that all the radioactivity remained on peptide Leu-118-Met-133, thus demonstrating that photolabeling occurred exclusively on Met-133, the only residue common to the two radioactive subcleaved peptides. Edman sequencing of peptides S1 and S2 of sheep SHBG showed a same single sequence corresponding to residues Gln-126-Arg-140 which contained no identifiable phenylthiohydantoin derivative at cycle 14, thus indicating that in both cases the corresponding Met-139 residue is the main site of photolabeling, as confirmed for peptide S1 by the presence at this cycle of a major peak of radioactivity while in peptide S2 the photoattachment of Delta 6-testosterone was found labile in the conditions of sequencing. The photolabeled peptide S1 was characterized by mass spectrometry which showed the covalent fixation of one mole of Delta 6-testosterone and the presence of a biantennary oligosaccharide attached at Asn-133, which suggests that the steroid-binding site is probably not deeply buried in the SHBG homodimer.
Institut National de la Santé et de la Recherche Médicale, Unité INSERM U 329, Pathologie Hormonale Moléculaire, Hôpital Debrousse, Lyon, France.
D
D
Kassab
S
S
Pichat
C
C
Chambon
T
T
Blachère
M
M
Rolland de Ravel
E
E
Mappus
C
C
Grenot
C Y
CY
Cuilleron
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005720:Gamma Rays; D006868:Hydrolysis; D007700:Kinetics; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D012756:Sheep; D013739:Testosterone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D019920:Photoaffinity Labels; D012738:Sex Hormone-Binding Globulin; C023630:6-dehydrotestosterone; D013739:Testosterone; D008715:Methionine; D014357:Trypsin