Filter assay for 1alpha, 25-dihydroxyvitamin D3. Utilization of the hormone's target tissue chromatin receptor.
Filter assay for 1alpha, 25-dihydroxyvitamin D3. Utilization of the hormone's target tissue chromatin receptor.
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1α, 25-二羟基维生素 D3 的过滤测定。
DOI：
10.1021/bi00717a005
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发表时间：
1974
期刊：
影响因子：
2.9
通讯作者：
M. Haussler
中科院分区：
文献类型：
--
作者：
P. Brumbaugh;D. Haussler;K. Bursac;M. Haussler
已经通过利用与来自鸡肠粘膜sa的受体系统的竞争性结合开发了1α，25-二羟基维生素D3(1a，25-(OH)2-D3)的放射性受体测定法。该靶组织结合系统由高亲和力胞质受体蛋白和核染色质上的受体位点组成。将重构的胞质-染色质与放射性1a，25-(OH)2-D3一起孵育，并通过将激素-受体-染色质复合物捕获在玻璃纤维过滤器上来分离结合的和游离的甾醇。使用 1% Triton X-100 洗去游离或非特异性结合的甾醇。特异性结合的甾醇的量与孵育中的胞质染色质的量线性相关，并且受体系统在5nM 1a，25-(OH) 2-[3H]D3时饱和。用增加量的真实非放射性1a，25-(OH) 2-D3 获得的同位素稀释标准曲线表明，通过该测定可以检测到17 pg的激素。从正常鸡和人血浆中提取并广泛纯化可疑的1a，25-(OH)2-D3级分后，循环-^^。显然，1α，25-二羟基维生素D3(1a，25-(OH)2-D3)'是维生素D3的代谢产物，其介导导致肠道钙吸收增加(Haussler等人，1971；Holick等人，1971)和骨钙吸收(Holick等人，1972；Wong等人，1972)增加的生化事件。在实验动物中。这种代谢物是由 25-羟基维生素 D3 (25-OH-D3) 在肾脏中羟基化产生的，被认为是维生素的激素形式（Fraser 和 Kodicek，1970；Lawson 等人，1971；Gray 等人，1971）。为了进一步研究维生素 D3 向 1a, 25-(OH) 2-D3 的代谢，并研究该激素在人体钙代谢中的作用，我们利用鸡肠道中该激素的染色质受体开发了一种灵敏的、竞争性的 1a, 25-(OH) 2-D3 蛋白结合测定法。已有报道利用类固醇激素各自的细胞质受体进行测定（Korenman，1968；Ballard 和 Baxter，1974）。这些细胞内蛋白由于其巨大的特异性和亲和力而非常适合测量激素结合活性（Jensen 等，1971）。然而，观察到的鸡肠细胞质受体1a，25-(OH) 2-D3 的热稳定性阻碍了我们开发单独使用该蛋白质的激素测定法。最近的实验（Brumbaugh 和 Haussler，1973，1974a，b）还表明，1a,25-(OH)2-D3 的染色质受体对该激素具有与胞质受体相似的亲和力和特异性。另外，一旦la，25-(OH) 2-
A radioreceptor assay for 1 a, 25-dihydroxyvitamin D3 (la, 25-(OH) 2-D3) has been developed by utilizing competitive binding to the receptor system from chick intestinal muco-sa. This target tissue binding system consists of a high affinity cytosol receptor protein and acceptor sites on nuclear chromatin. Reconstituted cytosol-chromatin is incubated with ra-dioactive la, 25-(OH) 2-D3, and the bound and free sterol are separated by trapping the hormone-receptor-chromatin com-plex on glass fiber filters. Washing away of free or nonspecifi-cally bound sterol is carried out with 1% Triton X-100. The amount of specifically bound sterol is linearly related to the quantity of cytosol-chromatin in the incubation and the receptor system is saturated at 5 nM la, 25-(OH) 2-[3H] D3. An iso-tope-dilution standard curve obtained with increasing amounts of authentic nonradioactive la, 25-(OH) 2-D3 indicates that 17 pg of hormone can be detected by this assay. After extraction and extensive purification of the suspected la, 25-(OH) 2-D3 fraction from normal chickand human plasma, the circulating-^^. pparently, 1 a, 25-dihydroxyvitamin D3(la, 25-(OH) 2-D3)'is the metabolite of vitamin D3 that mediates the biochemical events that result in increased calcium absorption from the intestine (Haussler et al., 1971; Holick et al., 1971) and bone calcium resorption (Holick et al., 1972; Wong et al., 1972) in experimental animals. This metabo-lite is produced by hydroxylation of 25-hydroxyvitamin D3 (25-OH-D3) in the kidney and is considered to be the hormonal formof the vitamin (Fraser and Kodicek, 1970; Lawson et al., 1971; Gray et al., 1971). In order to study further the metabolism of vitamin D3 to la, 25-(OH) 2-D3 as well as investigate the role of this hormone in calcium me-tabolism in humans, we have developed a sensitive, competitive protein binding assay for la, 25-(OH) 2-D3 utilizing the hormone’s chromatin receptor in the chick intestine. Assays of steroid hormones employing their respective cytoplasmic receptors have been reported (Korenman, 1968; Ballard and Baxter, 1974). These intracellular proteins are ideally suited for measurement of hormone binding activity due to their great specificity and affinity (Jensen et al., 1971). How-ever, the observed thermolability of the chick intestinal cytoplasmic receptor for la, 25-(OH) 2-D3 precluded our develop-ment of an assay for the hormone using this protein alone. Re-cent experiments (Brumbaugh and Haussler, 1973, 1974a, b) also indicate that the chromatin receptor for la, 25-(OH) 2-D3 has an affinity and specificity for this hormone similar to that of the cytosol receptor. In addition, once the la, 25-(OH) 2-