title: Solubilization, characterization and partial purification of [3H]mepyramine-binding protein, a possible histamine H1 receptor, from rat liver membrane.
abstract: [3H]Mepyramine binding protein, a possible subtype of histamine H1 receptors, was solubilized from rat liver membrane with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and Tween 60 as detergents and glycerol as an enhancer of solubilization. The optimal concentration of CHAPS was 10 mM and that of glycerol was 20% or more (v/v). The molecular weight of the [3H]mepyramine binding protein-detergent complex was determined to be 670K by Sepharose CL-4B gel filtration and 800K by sucrose density gradient sedimentation. By target size analysis, the molecular weights of both the membrane-bound and solubilized [3H]mepyramine binding protein were determined to be 162K. These values are similar to those of other well-characterized H1-receptor proteins, though slightly different. Simultaneous computerized analysis of the data obtained by [3H]mepyramine binding to the solubilized [3H]mepyramine binding protein indicated the presence of a single binding site with a KD value of 19.0 +/- 5.6 nM and a binding capacity (Bmax) of 6.6 +/- 2.1 pmole/mg protein. The Ki value of cold mepyramine for [3H]mepyramine binding to the solubilized receptor was 20 +/- 4 nM, whereas those of diphenhydramine, d-chlorpheniramine and triprolidine were all 2.9 +/- 0.8 microM, or about 150 times that of mepyramine. These data on the molecular and binding characteristics of the solubilized protein reported here suggest that there is a subtype of histamine H1 receptor in rat liver membrane. The solubilized preparation retained 90% and 75% of its [3H]mepyramine binding activity after storage at -80 degrees C and 4 degrees C, respectively, for 20 days. The solubilized [3H]mepyramine binding protein was purified 30-fold by Sepharose CL-4B gel filtration, Bio Gel HTP hydroxylapatite, Octyl Sepharose 4B and hydroxylapatite HPLC column chromatographies.
authors: Department of Pharmacology II, Osaka University School of Medicine, Japan.
authors: H
authors: H
authors: Fukui
authors: N P
authors: NP
authors: Wang
authors: T
authors: T
authors: Watanabe
authors: H
authors: H
authors: Wada
mesh_terms: D000631:Aminopyridines / Q000378:metabolism*; D000818:Animals; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005720:Gamma Rays; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D011485:Protein Binding; D011738:Pyrilamine / Q000378:metabolism*; D051381:Rats; D011968:Receptors, Histamine / Q000378:metabolism*; D011969:Receptors, Histamine H1 / Q000302:isolation & purification / Q000378:metabolism*; D013395:Sucrose
chemical_list: D000631:Aminopyridines; D011968:Receptors, Histamine; D011969:Receptors, Histamine H1; D013395:Sucrose; D011738:Pyrilamine