A specific binding protein for cardiac glycosides exists in bovine serum
A specific binding protein for cardiac glycosides exists in bovine serum
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DOI：
10.1074/jbc.273.26.16259
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发表时间：
1998-06-26
影响因子：
4.8
通讯作者：
Schoner, W
中科院分区：
文献类型：
--
作者：
Antolovic, R;Kost, H;Schoner, W
为了寻找血液中可能参与强心苷特异性转运至钠泵受体位点的结合蛋白，我们通过亲和层析，在哇巴因-琼脂糖 4B 柱上，纯化因子为 5000，从牛血清的球蛋白部分中分离出 26 kDa 的强心苷结合蛋白 (CGBG)。强心苷结合球蛋白被蛋白质反应性蛋白特异性共价标记。地高辛衍生物 HDMA（N-羟基琥珀酰亚胺地高辛-3-O-甲基羰基-ε-氨基己酸酯）。即使非常高浓度的其他类固醇，如雌激素、睾酮、黄体酮和可的松，也不能阻止 CGBG 的 HDMA 标记（5 和 100 nM），但卡烯内酯哇巴因和地高辛或蟾蜍二烯内酯 proscillaridin A 却可以。 CGBG是两个26kDa亚基形成二硫键的同型二聚体，由于在SDS变性过程中不存在β-巯基乙醇时，在SDS-聚丙烯酰胺凝胶电泳中观察到53kDa蛋白质的HDMA标记，其N端氨基酸序列K-D-V-Y-R-A-P-D-G-T-Q-S-A与基因和蛋白质序列数据库中记录的蛋白质没有序列相似性。牛肾中存在 90 kDa 胞质 CGBG，并与 CGBG 抗体发生反应。通过内在色氨酸荧光的猝灭来监测哇巴因与强心苷结合球蛋白的结合。此类研究使用 Koshland-Nemethy-Filmer 模型揭示了两个负协同哇巴因结合位点，K-d' 为 1.52 nM 和 K-d" = 75 nM，相互作用因子为 50。血浆中强心苷结合球蛋白的证明与最近在哺乳动物中发现的内源性强心苷一致。
Searching for a binding protein in blood, which may be involved in the specific transport of cardiac glycosides to their receptor sites on the sodium pump, we isolated a cardiac glycoside-binding protein (CGBG) of 26 kDa from the globulin fraction of bovine serum by affinity chromatography and on a ouabain-Sepharose 4B column by a purification factor of 5000. The cardiac glycoside-binding globulin was labeled specifically and covalently by the protein-reactive digoxigenin derivative HDMA (N-hydroxysuccimidyldigoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate). Even very high concentrations of other steroids, such as estrogen, testosterone, progesterone, and cortisone, did not prevent HDMA-labeling (at 5 and 100 nM) of CGBG, but the cardenolides ouabain and digoxin or the bufadienolide proscillaridin A did so. CGBG is a homodimer of two 26-kDa subunits forming disulfide bonds, since HDMA labeling of a protein of 53 kDa was observed in SDS-polyacrylamide gel electrophoresis when beta-mercaptoethanol was absent during SDS denaturation, The N-terminal amino acid sequence K-D-V-Y-R-A-P-D-G-T-Q-S-A showed no sequence similarity with proteins recorded in gene and protein sequence data banks. A 90-kDa cytosolic CGBG exists in bovine kidneys and reacts with antibodies against CGBG. Binding of ouabain to the cardiac glycoside-binding globulin was monitored by quenching of intrinsic tryptophan fluorescence. Such studies reveal two negatively cooperative ouabain binding sites with K-d' of 1.52 nM and K-d" = 75 nM and with an interaction factor of 50 using a Koshland-Nemethy-Filmer model. The demonstration of a cardiac glycoside-binding globulin in plasma is consistent with the recent finding of endogenous cardiac glycosides in mammals.