Estimation of platelet-activating factor receptors in the endometrium of the pregnant rabbit: regulation of ligand availability and catabolism by bovine serum albumin.
Estimation of platelet-activating factor receptors in the endometrium of the pregnant rabbit: regulation of ligand availability and catabolism by bovine serum albumin.
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妊娠兔子宫内膜中血小板活化因子受体的估计：牛血清白蛋白对配体可用性和分解代谢的调节。
DOI：
10.1095/biolreprod43.3.368
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发表时间：
1990
影响因子：
3.6
通讯作者：
Harper,MJ
中科院分区：
文献类型：
--
作者：
Kudolo,GB;Harper,MJ
已证明多种组织（包括子宫内膜）中的强效磷脂自体、血小板激活因子（PAF C18:0；1-O-烷基-2-乙酰基-sn-甘油-3-磷酸胆碱）具有高亲和力受体。由于 PAF 相对不稳定，并且我们之前证明 PAF 的主要代谢物 lyso-PAF（1-O-烷基-2-lyso-sn-甘油-3-磷酸胆碱）从子宫内膜 PAF 受体位点取代了 [3H]PAF，因此我们检查了牛血清白蛋白 (BSA) 防止 PAF 降解的能力，并在分离的纯化兔子宫内膜中表征了 PAF 和 lyso-PAF 结合位点。怀孕第 6 天。在含有磷脂酶 A2 抑制剂、奎纳克林 (10 µM) 和二溴苯乙酮 (2 µM) 以及 0.25% BSA 的缓冲液中，添加的 [3H]PAF C18:0 在 25°C 孵育 150 分钟后，87.4 ± 3.2% 保持完整。代谢产物溶血-PAF 和 1-O-烷基-2-酰基-sn-甘油-3-磷酸胆碱（烷基酰基-GPC）仅分别达到 5.2 ± 3.2 和 3.3 ± 1.1。在相同浓度下，兔血清白蛋白（RSA）也显着保护[3H]PAF C18:0免于代谢，但牛丙种球蛋白（BGG）无效。然而，0.25% BSA 的存在并不能保护 [3H]lyso-PAF C18:0 免受广泛分解代谢：形成的主要产物是 [3H] 烷基酰基-GPC。形成微量的[3H]PAF。在相同条件（25°C，150 分钟）和 0.25% BSA 存在下，饱和度分析显示子宫内膜中存在两种类型的 PAF C18:0 受体。 1 型位点的 Kdof 为 0.42 ± 0.03 nM（平均值 ± SD；n = 3），结合能力为 0.11 ± 0.01 pmol/mg 蛋白质。 2 型受体位点的 Kdof 为 5.96 ± 0.35 nM，结合能力为 1.59 ± 0.22 pmol/mg 蛋白质。因此，在存在 BSA 的情况下，与之前不存在 BSA 时产生的值相比，两类受体的结合能力显着降低。 BSA 的存在并未显着改变 1 型位点的 Kd。 lyso-PAF C18:0 可证明一类可饱和高亲和力结合位点：Kds 范围为 0.76 ± 0.58 至 11.1 ± 0.62 nM，具体取决于使用的分析方法（Eadie-Hofstee、Scatchard-Rosenthal 或 Lundon 非线性方法）。结合能力同样变化，范围从 0.15 ± 0.08 到 15.17 ± 4.95 pmol/mg 蛋白质。这些结果证实了BSA结合PAP Cl8:0但不结合溶血-PAF C18:0的能力，其高亲和力使得BSA-PAF复合物（而非溶血-PAF-BSA复合物）对酶促降解具有抗性。由于这种高亲和力相互作用，游离的 [3H]PAF C18:0 减少，交换仅限于 PM 受体位点，PM 受体位点对 PAF 具有更大的亲和力，从而与不存在 BSA 时产生的值相比，减少了标记膜位点的数量。
High affinity receptors have been demonstrated for the potent phospholipid autacoid, platelet-activating factor (PAF C18:0; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) in a variety of tissues, including the endometrium. Because of the relative instability of PAF and our previous demonstration that lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphorylcholine), the major metabolite of PAF, displaced [3H]PAF from endometrial PAF receptor sites, we have examined the ability of bovine serum albumin (BSA) to prevent degradation of PAF and have characterized PAF and lyso-PAF binding sites in purified rabbit endometrial membranes isolated on Day 6 of pregnancy. In buffer containing the phospholipase A2inhibitors, quinacrine (10 µM) and dibromoacetophenone (2 µM), and 0.25% BSA, 87.4 ± 3.2% of added [3H]PAF C18:0 remained intact after incubation at 25°C for 150 min. The metabolic products, lyso-PAF and 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (alkylacyl-GPC), only amounted to 5.2 ± 3.2 and 3.3 ± 1.1, respectively. At the same concentration, rabbit serum albumin (RSA) also significantly protected [3H]PAF C18:0 from metabolism, but bovine gamma globulin (BGG) was ineffective. The presence of 0.25% BSA, however, did not protect [3H]lyso-PAF C18:0 from extensive catabolism: the major product formed was [3H]alkylacyl-GPC. Insignificant amounts of [3H]PAF were formed. Under the same conditions (25°C, 150 min) in the presence of 0.25% BSA, saturation analysis revealed the presence of two types of PAF C18:0 receptors in the endometrial membranes. Type 1 sites had a Kdof 0.42 ± 0.03 nM (mean ± SD; n = 3) and binding capacity of 0.11 ± 0.01 pmol/mg protein. Type 2 receptor sites had a Kdof 5.96 ± 0.35 nM and a binding capacity of 1.59 ± 0.22 pmol/mg protein. Thus, in the presence of BSA, the binding capacities of the two classes of receptors were markedly reduced compared to values generated previously in its absence. The Kdof the Type 1 sites was not significantly changed by the presence of BSA. A single class of saturable high-affinity binding sites was demonstrable for lyso-PAF C18:0: Kds ranged from 0.76 ± 0.58 to 11.1 ± 0.62 nM, depending on which method of analysis was used (Eadie-Hofstee, Scatchard-Rosenthal, or the Lundon nonlinear method). The binding capacities were equally varied, ranging from 0.15 ± 0.08 to 15.17 ± 4.95 pmol/mg protein. These results confirm the ability of BSA to bind PAP Cl8:0, but not lyso-PAF C18:0, with such high affinity that BSA-PAF complexes, but not lyso-PAF-BSA complexes, are resistant to enzymatic degradation. Because of this high affinity interaction, free [3H]PAF C18 :0 was reduced and exchange was restricted to PM receptor sites, which had even greater affinities for PAF, thus reducing the population of labeled membrane sites compared to values generated in the absence of BSA.