Design of an affinity matrix for purification of the histamine H1 receptor from guinea pig cerebellum.
H1 receptors from guinea pig cerebellum were solubilized using digitonin, and [125I]iodobolpyramine was used as a probe. [125I]Iodobolpyramine binding to this solubilized preparation occurred with a KD of 0.1 nM and a Bmax of 220 fmol/mg of protein and was inhibited by various H1 ligands with the expected potencies. Using a gel filtration procedure, a very sensitive radioassay was set up for detecting H1 activity in the solubilized preparation: 0.1 nM [125I]iodobolpyramine specific binding represented greater than 90% of total binding. Moreover, the synthesis is described of potent H1 antagonists that are mepyramine derivatives with an amino alkyl acylamido alkyl spacer arm. One of them, UCL 1057 (Ki = 0.5 nM), has been coupled to a Sepharose epoxy-activated resin. The resulting affinity matrix adsorbed selectively [125I]iodobolpyramine binding sites from the guinea pig cerebellum soluble preparation. In contrast, a Sepharose-glycine matrix was not able to adsorb these sites.
Ruat
M
M
Unité de Neurobiologie et Pharmacologie (U. 109) de l'INSERM, Centre Paul Broca, Paris, France.
Traiffort
E
E
Garbarg
M
M
Schwartz
J C
JC
Demonchaux
P
P
Ife
R J
RJ
Tertiuk
W
W
Ganellin
C R
CR
D000327:Adsorption; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002531:Cerebellum / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D006168:Guinea Pigs; D008024:Ligands; D008297:Male; D011869:Radioligand Assay; D011969:Receptors, Histamine H1 / Q000302:isolation & purification* / Q000378:metabolism; D012116:Resins, Plant; D012995:Solubility
D008024:Ligands; D011969:Receptors, Histamine H1; D012116:Resins, Plant