Solubilization and Characterization of Prostaglandin E2 Binding Protein from Porcine Cerebral Cortex

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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°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°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 > PGE, × PGE2α > PGE, methyl ester > PGA2 > 13,14‐dihydro‐15‐keto‐PGE2 > 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.