Solubilization and characterization of prostaglandin E2 binding protein from porcine cerebral cortex.
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.
Yumoto
N
N
Watanabe
Y
Y
Watanabe
K
K
Watanabe
Y
Y
Hayaishi
O
O
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
D002793:Cholic Acids; D011458:Prostaglandins E; D015232:Dinoprostone; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate