Open AccessIsolation of Substance P Binding Protein from Rat Brain.
Author(s) -
Yoshihiro Nakata,
Hiroyuki Takamatsu,
Naoyoshi Kuroyanagi,
Hiroaki Nishio,
Tomio Segawa,
Toshifumi Akizawa,
Yuko Hirai,
Mitoshi Akiyama
Publication year - 1992
Publication title -
japanese journal of pharmacology/japanese journal of pharmacology
Language(s) - English
Resource type - Journals
eISSN - 1347-3506
pISSN - 0021-5198
DOI - 10.1254/jjp.59.313
Subject(s) - polyclonal antibodies , antiserum , digitonin , affinity chromatography , concanavalin a , microbiology and biotechnology , blot , biochemistry , sodium dodecyl sulfate , chemistry , gel electrophoresis , polyacrylamide gel electrophoresis , lectin , antibody , chaps , biology , solubilization , membrane , enzyme , in vitro , gene , immunology
Substance P (SP) binding protein of rat brain was solubilized by digitonin. The solubilized proteins were then purified by sequential gel filtration, concanavalin A lectin Sepharose, and SP-affinity chromatography. The calculated molecular weight of this purified SP binding protein was 76-74 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rabbits were immunized with the purified protein and resulting polyclonal anti-sera were tested. The immune serum significantly inhibited [3H]SP binding to the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate solubilized membrane fractions from rat brain, whereas pre-bleed antiserum failed to inhibit the binding. This polyclonal antibody also inhibited the activity of 45Ca influx into astroglioma cells stimulated by SP, but does not inhibit that stimulated by histamine. Furthermore, this polyclonal antibody recognized the 76-74 kDa band as assessed by Western blotting. These data strongly suggest that this polyclonal antibody could recognize a part of the natural SP receptor site.