Solubilization, characterization and partial purification of (3H)mepyramine-binding protein, a possible histamine H1 receptor, from rat liver membrane.
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- FUKUI Hiroyuki
- Department of Pharmacology II, Osaka University School of Medicine
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- WANG Nai Ping
- Department of Pharmacology II, Osaka University School of Medicine
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- WATANABE Takehiko
- Department of Pharmacology I, Tohoku University School of Medicine
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- WADA Hiroshi
- Department of Pharmacology II, Osaka University School of Medicine
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説明
[3H] Mepyramine binding protein, a possible subtype of histamine H, 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 membranebound and solubilized [3H]mepyramine binding protein were determined to be 162K. These values are similar to those of other well-characterized H, -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 μM, 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°C and 4°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.
収録刊行物
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- Jpn.J.Pharmacol.
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Jpn.J.Pharmacol. 46 (2), 127-139, 1988
公益社団法人 日本薬理学会
