IDENTIFICATION OF THE STEROL-BINDING AND ACTIN-BINDING DOMAINS OF PLASMA VITAMIN-D BINDING-PROTEIN (GC-GLOBULIN)
IDENTIFICATION OF THE STEROL-BINDING AND ACTIN-BINDING DOMAINS OF PLASMA VITAMIN-D BINDING-PROTEIN (GC-GLOBULIN)
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DOI：
10.1021/bi00146a021
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发表时间：
1992-08-11
期刊：
影响因子：
2.9
通讯作者：
COOKE, NE
中科院分区：
文献类型：
--
作者：
HADDAD, JG;HU, YZ;COOKE, NE
哺乳动物血浆维生素 D 结合蛋白 (DBP) 或 Gc 球蛋白被认为具有至少两个功能特性：甾醇结合和 G 肌动蛋白隔离。用放射性亲电子配体 3-β-(溴乙酰氧基)-25-羟基胆钙化醇对甾醇结合位点进行亲和标记，然后进行有限的蛋白水解，从而可以分离和鉴定分子氨基末端的三个重叠肽。 当 G-肌动蛋白亲和层析应用于其他蛋白水解片段时，分离并鉴定了来自分子羧基末端的两个片段。另一个大的胰蛋白酶片段显示出甾醇和肌动蛋白结合特性。甾醇结合结构域的氨基末端分配通过编码在其羧基末端截短的蛋白质的突变大鼠DBP cDNA的体外转录和翻译产物证明甾醇特异性结合来证实。甾醇结合结构域位于第一个氨基末端二硫键之间的区域，肌动蛋白结合结构域位于残基 350 和 403 之间。在人类、大鼠和小鼠 DBP 中发现这些区域具有高度的序列保守性。这些功能域分配证实了这两种结合活性的明显独立性，并有助于解释观察到的 DBP-肌动蛋白-DNase I 三蛋白复合物以及 DBP 和 profilin 之间对 G-肌动蛋白结合的竞争。我们的发现应该有助于通过定点诱变实验更精确地描述结合域。
The mammalian plasma vitamin D binding protein (DBP), or Gc-globulin, is recognized to have at least two functional properties: sterol binding and G-actin sequestration. Affinity labeling of thc sterol binding site with the radioactive electrophilic ligand, 3-beta-(bromoacetoxy)-25-hydroxycholecalciferol, followed by limited proteolysis, permitted the isolation and identification of three overlapping peptides in the amino terminus of the molecule. When G-actin affinity chromatography was applied to other proteolytic fragments, two fragments from the carboxy terminus of the molecule were isolated and identified. Another, large, tryptic fragment displayed both sterol- and actin-binding properties. The amino-terminal assignment of the sterol-binding domain was confirmed by demonstrating sterol-specific binding by an in vitro transcribed and translated product of a mutated rat DBP cDNA encoding a protein truncated in its carboxy terminus. The sterol-binding domain was localized to the region between the first-amino-terminal disulfide bond, and the actin-binding domain was found between residues 350 and 403. A high degree of sequence conservation in these regions was found among human, rat, and mouse DBP's. These functional domain assignments confirm the apparent independence of these two binding activities and help to explain the observed triprotein complex of DBP-actin-DNase I and the competition between DBP and profilin for G-actin binding. Our findings should facilitate more precise delineation of the binding domains by site-directed mutagenesis experiments.