Characterization of histamine H1-receptor binding peptides in guinea pig brain using [125I]iodoazidophenpyramine, an irreversible specific photoaffinity probe.
Aminophenpyramine--i.e., N-(5-[2-(4-aminophenyl)ethanamidopentyl])-N'-(4-methoxybenzyl)-N-m ethyl-N'- (2-pyridinyl)-1,2-ethanediamine, a derivative of mepyramine (pyrilamine), a typical antagonist of histamine at its H1 receptor--was synthesized and converted into [125I)iodoazidophenpyramine, a potential photoaffinity probe for the H1 receptor. In the dark, reversible binding of this probe to cerebellar membranes occurred with a Kd of 1.2 x 10(-11) M and a Bmax of 240 fmol/mg of protein and was inhibited by various H1-receptor antagonists with the expected potencies. These features establish the compound as one of the most potent H1-receptor antagonists known so far. Upon UV irradiation, 5% of the bound radioactivity was covalently incorporated into cerebellar membrane polypeptides as shown by standard NaDodSO4/PAGE. Two bands of 47 and 56 kDa were consistently labeled, labeling being prevented by various H1-receptor antagonists with the expected potencies and stereoselectivity. In the presence of protease inhibitors, labeling of the 56-kDa peptide increased at the expense of the 47-kDa peptide, suggesting that the latter was produced by hydrolysis of the former under the action of membrane proteases. In the absence of 2-mercaptoethanol, a band of 350-400 kDa appeared, apparently at the expense of the lighter bands, suggesting that the latter might be linked by one or more disulfide bridges to a higher molecular mass complex. We propose that at least part of the ligand binding domain of the histamine H1 receptor resides within a subunit of apparent molecular mass 56,000.
Ruat
M
M
Unité de Neurobiologie de l'Institut National de la Santé et de la Recherche Médicale (Unité 109), Centre Paul Broca, Paris, France.
Körner
M
M
Garbarg
M
M
Gros
C
C
Schwartz
J C
JC
Tertiuk
W
W
Ganellin
C R
CR
D000345:Affinity Labels; D000631:Aminopyridines / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008623:Mercaptoethanol / Q000494:pharmacology; D010777:Photochemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D011738:Pyrilamine / Q000031:analogs & derivatives / Q000378:metabolism*; D011968:Receptors, Histamine / Q000378:metabolism*; D011969:Receptors, Histamine H1 / Q000378:metabolism*
D000345:Affinity Labels; D000631:Aminopyridines; D011480:Protease Inhibitors; D011968:Receptors, Histamine; D011969:Receptors, Histamine H1; C055549:3-iodo-4-azidophenpyramine; D008623:Mercaptoethanol; D011738:Pyrilamine