Purification and characterization of an endogenous protein modulator of radioligand binding to "peripheral-type" benzodiazepine receptors and dihydropyridine ca²⁺-channel antagonist binding sites

Acidified extracts of rat antral stomach chromatographed on octadecylsilane cartridges contained material that inhibited the binding of [³H]Ro 5-4864 (4'-chlorodiazepam) and [³H]nitrenidipine to "peripheral-type" benzodiazepine receptors and dihydropyridine Ca²⁺-channel antagonist binding sites respectively. This material reduced the apparent affinites of both radioligands without significantly affecting the maximum number of binding sites. In contrast, the binding of [³H]diazepam, [³H]Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a][1,4]benzodiazepine-3-carboxylate), and [³H]3-carbomethoxy-β-carboline to "brain-type" benzodiazepine receptors and [³H]dihydroalprenolol binding to β-adrenergic receptors were unaffected by this material. Subsequent column chromatography on hydroxylapatite purified this material by > 2000-fold. This semi-purified substance was resolved by reverse phase HPLC as one u.v. adsorbing peak that inhibited both [³H]Ro 5-4864 and [³H]nitrendipine binding. The activity of this 16,000 dalton substance was destroyed completely by both heat treatment and pronase and partially reduced by trypsin. Furthermore, the inhibitory activity of this substance was enhanced by Ca²⁺ in a concentration-dependent fashion (0.1 to 10 mM). Comparison of TLC scans of 2-9,10[³H]dipalmitoyl-phosphatidylcholine incubated with either the HPLC purified material or authentic phospholipase A₂(PLA₂) (Naja naja) revealed that this substance has enzymatic properties indistinguishable from PLA₂. These findings suggest that this endogenous protein may be a PLA₂ isoenzyme which may modify both "peripheral-type" benzodiazepine receptors and dihydro-pyridine Ca²⁺-channel antagonist binding sites.