Mantione C R, Goldman M E, Martin B, Bolger G T, Lueddens H W, Paul S M, Skolnick P
Laboratory of Bio-organic Chemistry, NIDDK, Bethesda, MD 20892.
Biochem Pharmacol. 1988 Jan 15;37(2):339-47. doi: 10.1016/0006-2952(88)90738-1.
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 affinities 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-beta-carboline to "brain-type" benzodiazepine receptors and [3H]dihydroalprenolol binding to beta-adrenergic receptors were unaffected by this material. Subsequent column chromatography on hydroxylapatite purified this material by greater than 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 dihydropyridine Ca2+-channel antagonist binding sites.
在十八烷基硅烷柱上进行色谱分析的大鼠胃窦酸化提取物中含有一种物质，该物质分别抑制[3H]Ro 5 - 4864（4'-氯地西泮）与“外周型”苯二氮䓬受体的结合以及[3H]尼群地平与二氢吡啶Ca2+通道拮抗剂结合位点的结合。这种物质降低了两种放射性配体的表观亲和力，而对结合位点的最大数量没有显著影响。相比之下，[3H]地西泮、[3H]Ro 15 - 1788（乙基 - 8 - 氟 - 5,6 - 二氢 - 5 - 甲基 - 6 - 氧代 - 4H - 咪唑并[1,5a][1,4]苯二氮䓬 - 3 - 羧酸酯）和[3H]3 - 甲氧羰基 - β - 咔啉与“脑型”苯二氮䓬受体的结合以及[3H]二氢心得安与β - 肾上腺素能受体的结合不受该物质影响。随后在羟磷灰石上进行柱色谱分析，使该物质的纯度提高了2000倍以上。这种半纯化物质通过反相高效液相色谱分离为一个紫外吸收峰，该峰抑制[3H]Ro 5 - 4864和[3H]尼群地平的结合。这种16000道尔顿物质的活性在热处理和链霉蛋白酶作用下完全被破坏，在胰蛋白酶作用下部分降低。此外，该物质的抑制活性在0.1至10 mM的浓度范围内以浓度依赖方式被Ca2+增强。对与高效液相色谱纯化物质或正宗磷脂酶A2（眼镜蛇）一起孵育的2 - 9,10[3H]二棕榈酰磷脂酰胆碱的薄层色谱扫描比较显示，该物质具有与磷脂酶A2无法区分的酶学特性。这些发现表明，这种内源性蛋白质可能是一种磷脂酶A2同工酶，它可能修饰“外周型”苯二氮䓬受体和二氢吡啶Ca2+通道拮抗剂结合位点。
