Solubilization and characterization of the pyrilamine-binding protein from cultured smooth muscle cells.
The cultured smooth muscle cell line DDT1MF-2 expresses a large number (9.7 x 10(6) receptors/cell) of functional histamine H1-type receptors [J. Cell. Physiol. 134:367-375 1988]. Two different binding assays, gel filtration and polyethylene glycol precipitation, indicated that the [3H]pyrilamine binding activity was solubilized by 1% digitonin with binding characteristics similar to those of intact cells. The solubilized proteins were then purified by sequential gel filtration, chromatofocusing, and reverse phase high pressure liquid chromatography. The calculated molecular weight of this purified pyrilamine-binding protein was 38-40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]Pyrilamine binding to these 38-40-kDa proteins indicated a single class of binding site with a Kd of 288 nM, which is equivalent to that of intact cells and digitonin-solubilized proteins. The computer analysis Scatfit also indicated that one molecule of [3H]pyrilamine bound to one molecule of purified protein. Furthermore, a polyclonal antibody raised against the purified protein recognized the 38-40-kDa band by Western blotting techniques, specifically bound to the cell surface of DDT1MF-2 cells, and inhibited [3H]pyrilamine binding to these cells in a dose-dependent manner. These data strongly suggest that the purified 38-40-kDa protein is part of an antagonist binding domain on the histamine H1 receptor on DDT1MF-2 cells.
Mitsuhashi
M
M
Howard Hughes Medical Institute Laboratories, University of California, Medical Center, San Francisco 94143.
Payan
D G
DG
D000631:Aminopyridines / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism*; D011092:Polyethylene Glycols; D011738:Pyrilamine / Q000378:metabolism*; D011969:Receptors, Histamine H1 / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity
D000631:Aminopyridines; D011969:Receptors, Histamine H1; D011092:Polyethylene Glycols; D011738:Pyrilamine