Solubilization, Characterization and Partial Purification of [3H]Mepyramine-Binding Protein, a Possible Histamine Hi Receptor, from Rat Liver Membrane

Abstract

[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 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, (^-chlorpheniramine and tri -prolidine were all 2.9±0.8/βM, 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 H1receptor 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.