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Solubilization and Characterization of Prostaglandin E 2 Binding Protein from Porcine Cerebral Cortex
Author(s) -
Yumoto Noboru,
Watanabe Yasuyoshi,
Watanabe Kikuko,
Watanabe Yumiko,
Hayaishi Osamu
Publication year - 1986
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1986.tb12934.x
Subject(s) - chemistry , chaps , incubation , chromatography , size exclusion chromatography , trypsin , binding site , prostaglandin , biochemistry , binding protein , membrane , enzyme , gene
The specific binding protein for prostaglandin (PG) E 2 was solubilized in an active form from the crude mitochondrial (P 2 ) fraction of porcine cerebral cortex. After incubation with 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propane sulfonate (CHAPS) at 4°C for 30 min, the PGE 2 binding to the supernatant fraction (103,000 g , 60 min) was determined by the polyethylene glycol method. The maximum yield (approximately 30% of the binding activity to the P 2 fraction) was obtained with 10 m M CHAPS. The specific [ 3 H]PGE 2 binding to the solubilized fraction was time‐dependent and the equilibrium was reached at around 60 min at 37°C. By dilution of the reaction mixture, the binding site‐[ 3 H]PGE 2 complex formed after 5‐min incubation slowly dissociated, whereas that formed after 60‐min incubation did not dissociate to a significant extent. The binding was highly specific for PGE 2 and inhibited by unlabeled PGs in the following order: PGE 2 > PGE, × PGE 2 α > PGE, methyl ester > PGA 2 > 13,14‐dihydro‐15‐keto‐PGE 2 > PGD 2 . Scatchard analyses of the solubilized fraction suggested the presence of high‐ and low‐affinity sites. Heat treatment and preincubation with trypsin or proteinase K markedly reduced the binding. The binding activity was eluted in a single peak both from gel filtration and from ion‐exchange columns using HPLC. These results suggest that a specific protein solubilized may be responsible for the binding site.