title: Solubilization and characterization of prostaglandin E2 binding protein from porcine cerebral cortex.
abstract: The specific binding protein for prostaglandin (PG) E2 was solubilized in an active form from the crude mitochondrial (P2) fraction of porcine cerebral cortex. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 4 degree C for 30 min, the PGE2 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 P2 fraction) was obtained with 10 mM CHAPS. The specific [3H]PGE2 binding to the solubilized fraction was time-dependent and the equilibrium was reached at around 60 min at 37 degrees C. By dilution of the reaction mixture, the binding site-[3H]PGE2 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 PGE2 and inhibited by unlabeled PGs in the following order: PGE2 greater than PGE1 much greater than PGF2 alpha greater than PGE2 methyl ester greater than PGA2 greater than 13,14-dihydro-15-keto-PGE2 greater than PGD2. 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.
authors.forename: N
authors.initials: N
authors.lastname: Yumoto
authors.forename: Y
authors.initials: Y
authors.lastname: Watanabe
authors.forename: K
authors.initials: K
authors.lastname: Watanabe
authors.forename: Y
authors.initials: Y
authors.lastname: Watanabe
authors.forename: O
authors.initials: O
authors.lastname: Hayaishi
mesh_terms: D000818:Animals; D001001:Anura; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D002793:Cholic Acids; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D015232:Dinoprostone; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D011458:Prostaglandins E / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine
chemical_list: D002793:Cholic Acids; D011458:Prostaglandins E; D015232:Dinoprostone; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate