Chemical modification and structural analysis of the progesterone membrane binding protein from porcine liver membranes
Chemical modification and structural analysis of the progesterone membrane binding protein from porcine liver membranes
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DOI：
10.1023/a:1007269507856
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发表时间：
2001-02-01
影响因子：
4.3
通讯作者：
Wehling, M
中科院分区：
文献类型：
--
作者：
Falkenstein, E;Eisen, C;Wehling, M
除了对转录和蛋白质合成的调节的经典基因组类固醇作用之外，已经描述了各种类固醇的快速非基因组作用。这些对细胞信号和功能的影响被认为是通过与经典细胞内受体无关的膜结合位点传递的。最近，一个高亲和力的孕酮膜结合蛋白（mPR）的特点是在猪肝膜。在本研究中，孕酮结合到猪肝微粒体mPR所必需的氨基酸残基已被确定通过使用蛋白质修饰试剂。在所有测试的试剂中，对羧基、甲硫氨酸和色氨酸具有特异性的试剂，如N，N '-二环己基碳二亚胺、氯胺T和N-溴代琥珀酰亚胺诱导[H-3]孕酮结合减少。为了评价是否存在必需的二硫键，将猪肝微粒体与二硫键还原剂二硫苏糖醇（DTT）一起孵育，并测量[H-3]孕酮结合。该处理还导致结合活性降低，DTT的IC 50为20 mM。在存在或不存在还原剂的情况下进行的蛋白质印迹分析表明，mPR -在其结合状态下-由至少两个相同的亚基组成，其表观分子量为28 kDa，并通过二硫键连接。总之，在本研究中的证据参与羧基，色氨酸和蛋氨酸残基[H-3]孕酮结合到猪肝微粒体。此外，它表明，mPR可以形成二硫键连接的同源二聚体。
In addition to the classical genomic steroid actions on modulation of transcription and protein synthesis, rapid, nongenomic effects have been described for various steroids. These effects on cellular signaling and function are supposed to be transmitted by membrane binding sites unrelated to the classical intracellular receptors. Recently, a high affinity progesterone membrane binding protein (mPR) has been characterized in porcine liver membranes. In the present study, amino acid residues that are essential for progesterone binding to porcine liver microsomal mPR have been identified by the use of protein modifying reagents. Among all reagents tested, agents with specificity for carboxyl groups, methionine and tryptophan such as N,N'-dicyclohexylcarbodiimide, chloramine T and N-bromosuccinimide induced a reduction in [H-3]progesterone binding. To evaluate the presence of essential disulfide bridges, porcine liver microsomes were incubated with the disulfide reducing agent dithiothreitol (DTT) and [H-3]progesterone binding was measured. This treatment also resulted in a reduction of binding activity with an IC50 of 20 mM for DTT. Western-blotting analysis in the presence or absence of the reducing agent suggested that mPR - in its binding state - consists of at least two identical subunits with an apparent molecular mass of 28 kDa which are linked by a disulfide bridge. In conclusion, in the present study evidence for an involvement of carboxyl-, tryptophan- and methionine residues in [H-3]progesterone binding to porcine liver microsomes is given. In addition, it is shown that mPR can form disulfide-linked homodimers.