EFFECTS OF SULFHYDRYL-REAGENTS, RETINOIDS, AND SOLUBILIZATION ON THE ACTIVITY OF MICROSOMAL RETINOL DEHYDROGENASE
EFFECTS OF SULFHYDRYL-REAGENTS, RETINOIDS, AND SOLUBILIZATION ON THE ACTIVITY OF MICROSOMAL RETINOL DEHYDROGENASE
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DOI：
10.1006/abbi.1995.1415
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发表时间：
1995-08-20
影响因子：
3.9
通讯作者：
NAPOLI, JL
中科院分区：
文献类型：
--
作者：
BOERMAN, MHEM;NAPOLI, JL
识别孔细胞视黄醇结合蛋白（CRBP）为底物的微粒体视黄醇脱氢酶（RoDH）在10 mM半胱氨酸存在下被氧化苯丙氨酸（IC50 = 3 μ M）抑制，二硫苏糖醇的抑制是可逆的，表明两个接近的半胱氨酸残基对RoDH活性至关重要。溴苯larsin氧化物是一种不可逆抑制剂（IC50 = 0.2 μ M），表明亲核试剂位于两个半胱氨酸残基附近。n -乙基马来酰亚胺抑制了由holo-CRBP支持的反应，但没有抑制游离视黄醇的反应，这表明它在不影响催化位点的情况下阻碍了holo-CRBP接近RoDH。在维生素a充足或维生素a缺乏的大鼠微粒体中，RoDH活性相似，并且不受相对高浓度（5 μ M）的全反式维甲酸、全细胞维甲酸结合蛋白、猿细胞维甲酸结合蛋白或g-顺式维甲酸的抑制。Triton X-100在洗剂与蛋白质的比例为0.25比1 （w/w）时刺激了8倍的RoDH活性。Tween 80、Brij 92和Triton X-100（2:1:2）的组合在洗涤剂与蛋白质的比例为2.5比1 （w/w）时刺激了8倍的RoDH活性，洗涤剂溶解的RoDH通过PAO-Sepharose树脂部分纯化，首选NADP(H)作为辅助因子，从holo-CRBP合成视网膜的K-m为0.6 μ M （V-max = 115 pmol/min/mg蛋白），视网膜还原到CRBP的K-m为0.6 μ M （V-max = 613 pmol/min/mg蛋白）。这项工作为微粒体RoDH提供了进一步的见解，并加强了RoDH和holo-CRBP之间相互作用的证据。(C) 1995学术出版社，Inc。
A microsomal retinol dehydrogenase (RoDH) that recognizes hole-cellular retinol binding protein (CRBP) as substrate is inhibited by phenylarsine oxide (IC50 = 3 mu M) in the presence of 10 mM cysteine, Inhibition was reversible with dithiothreitol, indicating that two cysteine residues in close proximity are essential for RoDH activity. Bromophenylarsine oxide was an irreversible inhibitor (IC50 = 0.2 mu M)) suggesting that a nucleophile lies close to the two cysteine residues. N-Ethylmaleimide inhibited reactions supported by holo-CRBP, but not from free retinol, suggesting that it obstructed holo-CRBP access to RoDH without affecting the catalytic site. RoDH activity was similar in microsomes from vitamin A-sufficient or vitamin A-deficient rats and was not inhibited by relatively high concentrations (5 mu M) of all-trans-retinoic acid, holo-cellular retinoic acid binding protein, ape-cellular retinoic acid binding protein, or g-cis-retinoic acid, Triton X-100 stimulated RoDH activity eightfold at a detergent to protein ratio of 0.25 to 1 (w/w). A combination of Tween 80, Brij 92, and Triton X-100 (2:1:2) stimulated RoDH activity eightfold at a detergent to protein ratio of 2.5 to 1 (w/w), Detergent-solubilized RoDH, partially purified through a PAO-Sepharose resin, preferred NADP(H) as cofactor, had a K-m for retinal synthesis from holo-CRBP of 0.6 mu M (V-max = 115 pmol/min/mg protein) and a K-m for reduction of retinal bound to CRBP of 0.6 mu M (V-max = 613 pmol/min/mg pro tein). This work provides further insight into microsomal RoDH and strengthens the evidence of an interaction between RoDH and holo-CRBP. (C) 1995 Academic Press, Inc.