Photoaffinity labeling of the rat plasma vitamin D binding protein with [26,27-3H]-25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate].
Photoaffinity labeling of the rat plasma vitamin D binding protein with [26,27-3H]-25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate].
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用 [26,27-3H]-25-羟基维生素 D3 3 beta-[N-(4-叠氮基-2-硝基苯基)甘氨酸] 光亲和标记大鼠血浆维生素 D 结合蛋白。
DOI：
10.1021/bi00365a001
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Holick,MF
中科院分区：
文献类型：
--
作者：
Ray,R;Holick,SA;Hanafin,N;Holick,MF
摘要：维生素D结合蛋白（DBP）在维生素ind、25-羟基维生素D （25-OH-D）及其代谢产物的转运中起着重要作用。为了更好地了解这种普遍存在的蛋白质的分子结合特性，我们设计并合成了25-OH-D3及其放射性标记对偶物的光亲和类似物。这种类似物25-羟基维生素D3 3/3-[N-（4-叠氮-2-硝基苯基）甘氨酸]（25-OH-D3- ang）被大鼠DBP识别，其结合活性比25-OH-D3低约10倍。[3H] 25-OH-D3或[3H] 25-OH-D3- ang与大鼠DBP孵生发现，这两种化合物都能特异性结合一种沉淀系数为4.1 s的蛋白质，每一种化合物都被超过500倍的25-OH-D3取代。当[3H] 25-OH-D3- ang在大鼠DBP存在的情况下暴露于紫外线辐射，然后添加500倍的25-OH-D3时，没有从4中置换氚。是峰。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳-射线自显像分析[3H] 25-OH-D3- ang在大鼠DBP存在下暴露于紫外线辐射，然后添加500倍过量的25-OH-D3，发现一个分子量为52000的主要波段。这些数据为[3H] 25-OH-D3-ANG与大鼠DBP共价连接提供了强有力的证据。这种光亲和探针将为分析这种转运蛋白的结合位点提供有价值的工具。在世界各地超过300个不同的人群中，血液中存在一种特定群体成分（Gc）。其规律性的出现频率和良好的电泳多态性模式确定了它是一种遗传标记。然而，直到1975年才发现它具有特定的生物学作用，当时人们偶然发现它以一种非常特殊的方式识别并结合维生素D及其代谢物（Daiger等人，1975;Daiger, 1979； Thomas等人，1959；Haddad和Walgate， 1976a, b； Van Baelen等人，1978）。现在已经确定Gc是负责维生素D及其代谢物从循环到目标组织的运输的蛋白质。DBP由分子量为51000- 58000的单肽链组成(Haddad &
Revised Manuscript Received June 18, 1986 abstract: It is well recognized that the vitamin D binding protein (DBP) is important for the transport of vitaminD, 25-hydroxyvitamin D (25-OH-D), and its metabolites. In an attempt to better understand the molecular-binding properties of this ubiquitous protein, we designed and synthesized a photoaffinity analogue of 25-OH-D3 and its radiolabeled counterpart. This analogue, 25-hydroxyvitamin D3 3/3-[N-(4-azido-2-nitrophenyl) glycinate](25-OH-D3-ANG), was recognized by the rat DBP and was about 10 times less active than 25-OH-D3 in terms of binding. Incubation of [3H] 25-OH-D3 or [3H] 25-OH-D3-ANG with rat DBP revealed that both compounds were specifically boundto a protein with a sedimentation coefficient of 4.1 S. Each was displaced with a 500-fold excess of 25-OH-D3. When [3H] 25-OH-D3-ANG was exposed to UV radiationin the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3, there was no displacement of tritium from the 4. IS peak. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisautoradiographic analysis of [3H] 25-OH-D3-ANG exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3 revealed one major band with a molecular weight of 52000. These data provide strong evidence that [3H] 25-OH-D3-ANG was covalently linked to the rat DBP. This photoaffinity probe should provide a valuable tool for the analysis of the binding site on this transport protein. e group-specific component (Gc) in blood occurs in more than 300 distinct populations throughout the world. Its regular frequency of occurrence and well-characterized polymorphic pattern in electrophoresis identify it as a genetic marker. However, no specific biologic role was assigned to it until 1975, when the discovery was made accidentallythat Gcrecognizes and binds to vitamin D and its metabolites in a very specific manner(Daiger et al., 1975; Daiger, 1979; Thomas et al., 1959; Haddad & Walgate, 1976a, b; Van Baelen et al., 1978). It is now firmly established that Gc is the protein responsible for the transport of vitamin D and its metabolites from the circulation to target tissues. DBP consists of a single poly-peptide chain of molecular weight 51000-58 000 (Haddad &