A new photoactivatable reagent capable of transferring a radiolabel to target proteins. Application to the human growth hormone-rat liver prolactin receptor interaction.
A new photoactivatable cross-linking reagent, 1-(2'-dithiopyridyl)-2-(5'-azidosalicylamido)ethane (ASDPE), was synthesized. This probe can be easily labeled with 125I in the azidosalicylamido ring and contains an activated disulfide bridge. After reaction of [125I]ASDPE with proteins, the radiolabeled moiety of the probe becomes attached to cysteine residues. Upon partial reduction of human growth hormone (hGH) with dithiothreitol, its C-terminal disulfide bond between residues 182 and 189 was cleaved and the nascent thiol groups were modified with [125I]ASDPE to yield [125I]ASET-hGH [1-(thio-hGH)-2-(3'-[125I]iodo-5'-azidosalicylamido)ethane]. After binding of this hormone derivative to rat liver microsomes, followed by photolysis and subsequent reduction of disulfide bridges, the specific transfer of the radiolabeled moiety to prolactin receptor (PRL-R) was achieved. Partial purification of the radiolabeled receptor by size exclusion chromatography was performed. We anticipate that [125I]ASDPE will be generally useful in pursuing structural and functional studies of target proteins which interact specifically with protein ligands.
Instituto de Química y Fisicoquímica Biológicas, Universidad de Buenos Aires, CONICET, Buenos Aires, Argentina.
N T
NT
Masckauchán
J M
JM
Delfino
H N
HN
Fernández
D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry; D019382:Human Growth Hormone / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods*; D008862:Microsomes, Liver / Q000378:metabolism / Q000648:ultrastructure*; D011725:Pyridines / Q000138:chemical synthesis / Q000737:chemistry; D051381:Rats; D011981:Receptors, Prolactin / Q000737:chemistry* / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
C113824:1-(2'-dithiopyridyl)-2-(5'-azidosalicylamido)ethane; D001386:Azides; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D011725:Pyridines; D011981:Receptors, Prolactin; D019382:Human Growth Hormone