Studies on calciferol metabolism. 8. Evidence for a cytoplasmic receptor for 1,25-dihydroxy-vitamin D3 in the intestinal mucosa.
Studies on calciferol metabolism. 8. Evidence for a cytoplasmic receptor for 1,25-dihydroxy-vitamin D3 in the intestinal mucosa.
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骨化醇代谢研究。
DOI：
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发表时间：
1973
影响因子：
4.8
通讯作者：
A. Norman
中科院分区：
文献类型：
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作者：
H. Tsai;A. Norman
摘要先前已经从在体内条件下进行的研究中假设（Tsai，H. C.的方法，黄河，巴西-地G.，和Norman，A. W.（1972）J.Biol.Chem.247，5728-5735），在钙化醇（维生素D）代谢物1，25-二羟基胆钙化醇在肠染色质部分中的出现与归因于钙化醇的生理反应在该靶组织中的起始之间可能存在因果关系。本报告描述了通过体外技术研究肠匀浆系统的特性、特异性和要求的努力，肠匀浆系统能够影响肠染色质组分中1，25-二羟基胆钙化醇的定位。在0-4 ℃下，将肠匀浆在0.25 m蔗糖、0.02 m KCl、0.05 m MgCl 2和0.05 m Tris-Cl（pH 7.5）中与32 - 325 pmol 1，25-二羟基[3 H]胆钙化醇孵育30 min，导致随后分离的染色质部分饱和，每个鸡肠染色质中类固醇的水平为6.2 pmol。先前的实验（Tsai，H. C.的方法，黄河，巴西-地C.的方法，和Norman，A. W.（1972）J.Biol.Chem.247，5728-5735），其中将1，25-二羟基[3 H]胆钙化醇心内给予胆钙化醇缺乏的小鸡，导致5.2皮摩尔类固醇/小鸡肠染色质部分的结合。在体外相同的条件下，只有1.4皮摩尔的胆钙化醇，2.0皮摩尔的25-羟基胆钙化醇，或0.3皮摩尔的雌二醇结合到肠染色质部分。获得了1，25-二羟基[3 H]胆钙化醇与细胞质受体蛋白强制性结合的证据，然后将该类固醇与肠染色质组分结合。根据其在蔗糖梯度、凝胶过滤色谱中的迁移，判定肠胞质受体为大分子，根据其对链霉蛋白酶（而非RNA酶或DNA酶）处理的敏感性、其热不稳定性及其硫酸铵沉淀性，判定其为蛋白质。细胞质受体对1，25-二羟基胆钙化醇与肠染色质组分的结合和转移具有高度特异性;只有250倍过量的25-羟基胆钙化醇，而不是胆钙化醇、5，6-反式胆钙化醇或二氢速甾醇，才能显著（50%）减少1，25-二羟基-[3 H]胆钙化醇的染色质定位。也从细胞质受体和染色质结合的组织特异性中获得了证据。无论是肝脏，肾脏，或脾脏胞质能够有效地转移1，25-二羟基胆钙化醇肠染色质，也不是肝脏或肾脏细胞核能够接受类固醇从肠胞质受体。这些结果描述了允许1，25-二羟基胆钙化醇首先与细胞质受体然后与核受体顺序结合的体外条件，这些结果与用这种类固醇在体内获得的结果以及报道的在许多其他类固醇激素的体内和体外条件下发生的结果类似。
Abstract It has previously been postulated from studies conducted under in vivo conditions (Tsai, H. C., Wong, R. G., and Norman, A. W. (1972) J. Biol. Chem. 247, 5728–5735) that there may be a cause-and-effect relationship between the appearance of the calciferol (vitamin D) metabolite, 1,25-dihydroxycholecalciferol in the intestinal chromatin fraction, and the initiation of the physiological responses attributed to calciferol in this target tissue. The present report describes efforts to study via in vitro techniques the properties, specificity, and requirements of an intestinal homogenate system capable of effecting the localization of 1,25-dihydroxycholecalciferol in the intestinal chromatin fraction. Incubation of intestinal homogenates in 0.25 m sucrose, 0.02 m KCl, 0.05 m MgCl2, and 0.05 m Tris-Cl, pH 7.5, with 32 to 325 pmoles of 1,25-dihydroxy[3H]cholecalciferol at 0–4° for 30 min resulted in the saturation of the subsequently isolated chromatin fraction at a level of 6.2 pmoles of steroid per chick intestinal chromatin. Previous experiments (Tsai, H. C., Wong, R. C., and Norman, A. W. (1972) J. Biol. Chem. 247, 5728–5735) where 1,25-dihydroxy[3H]cholecalciferol was given intracardially to a cholecalciferol-deficient chick resulted in the binding of 5.2 pmoles of steroid per chick intestinal chromatin fraction. Under identical conditions in vitro only 1.4 pmoles of cholecalciferol, 2.0 pmoles of 25-hydroxycholecalciferol, or 0.3 pmole of estradiol were bound to the intestinal chromatin fraction. Evidence was obtained for the mandatory binding of the 1,25-dihydroxy[3H]cholecalciferol to a cytoplasmic receptor protein prior to the association of this steroid with the intestinal chromatin fraction. The intestinal cytoplasmic receptor was judged to be a macromolecule on the basis of its migration in a sucrose gradient, gel filtration chromatography, and a protein on the basis of its sensitivity to treatment with Pronase, but not RNase or DNase, its heat lability, and its precipitability by ammonium sulfate. The cytoplasmic receptor was highly specific for the binding and transfer of 1,25-dihydroxycholecalciferol to the intestinal chromatin fraction; only a 250-fold excess of 25-hydroxycholecalciferol, but not cholecalciferol, 5,6-trans-cholecalciferol, or dihydrotachysterol effected a significant (50%) reduction of the chromatin localization of 1,25-dihydroxy-[3H]cholecalciferol. Evidence was also obtained from a tissue specificity both for the cytoplasmic receptor and the chromatin binding. Neither liver, kidney, or spleen cytosol were able to transfer effectively 1,25-dihydroxycholecalciferol to intestinal chromatin, nor were liver or kidney nuclei able to accept the steroid from the intestinal cytosol receptor. These results describing the in vitro conditions which permit the sequential binding of 1,25-dihydroxycholecalciferol to first a cytoplasmic and then a nuclear receptor are analogous both to the results obtained in vivo with this steroid and to those reported to occur under in vivo and in vitro conditions for many other steroid hormones.