Determination of apo and holo retinoic acid-binding protein levels in retinoid-responsive transformed cells by high-performance size-exclusion chromatography.
Determination of apo and holo retinoic acid-binding protein levels in retinoid-responsive transformed cells by high-performance size-exclusion chromatography.
复制标题
通过高效尺寸排阻色谱法测定类维生素A反应性转化细胞中的apo和全视黄酸结合蛋白水平。
DOI：
10.1016/0003-9861(86)90585-0
复制
发表时间：
1986
影响因子：
3.9
通讯作者：
McCormick,AM
中科院分区：
文献类型：
--
作者：
Shubeita,HE;Patel,MD;McCormick,AM
A method to measure the endogenous levels of apo and holo cellular retinoic acid-binding proteins was developed using calf testis cytosol as the source of retinoic acid-binding protein. [3H]Retinoic acid-retinoic acid-binding protein complexes were assayed by high-performance size-exclusion chromatography. Preincubation of cytosol with 10 mmp-hydroxymercuribenzoate at 4 °C resulted in complete inhibition of retinoic acid binding to apo retinoic acid-binding protein. In addition, total dissociation of preformed holo retinoic acid-binding protein complexes was noted within 20 min after mercurial addition. Thus,p-hydroxymercuribenzoate converted the total pool of cellular retinoic acid-binding protein (apo plus holo) to mercurial-protein complexes unable to bind retinoic acidin vitro. Mercurial inhibition of retinoic acid-retinoic acid-binding protein complex formation was totally reversed upon the addition of 50 mmdithiothreitol. Total cytosolic retinoic acid-binding protein was determined from specific retinoic acid binding after treatment withp-hydroxymercuribenzoate and dithiothreitol. Apo cellular retinoic acid-binding protein concentration was measured by determining specific radioligand binding prior top-hydroxymercuribenzoate treatment, and correcting for exchange of endogenously bound retinoid with exogenous tritiated retinoic acid. Holo cellular retinoic acid-binding protein concentration was derived from the difference between total and apo retinoic acid-binding protein concentrations. Using this method, we have demonstrated that retinoid-responsive EJ and T24 human bladder carcinoma cell lines and AT3A and AT3B rat pancreatic acinar carcinoma cell lines lack detectable levels of either apo or holo cellular retinoic acid-binding protein. These results established that retinoid inhibition of transformed bladder and acinar cell proliferation in culture was mediated by a cellular retinoic acid-binding protein-independent mechanism.
