Fogh K, Voorhees J J, Aström A
Department of Dermatology, University of Michigan, Ann Arbor 48109.
Arch Biochem Biophys. 1993 Feb 1;300(2):751-5. doi: 10.1006/abbi.1993.1104.
Human cellular retinoic acid-binding protein (CRABP) type I and type II were expressed in Escherichia coli from cloned cDNAs. Expressed proteins were purified by gel filtration and ion-exchange chromatography, resulting in highly pure proteins. The yield after gel filtration was approximately 50 mg/liter bacterial culture. In binding studies the equilibrium dissociation constant, Kd, of retinoic acid (RA) for E. coli-derived CRABP-I and CRABP-II was 6.8 and 39 nM, respectively. The Kd of the synthetic retinoid analog CD 367 was 2.2 nM for CRABP-I and 3.0 nM for CRABP-II. RA competed with the binding of CD 367 to CRABP-I and CRABP-II with IC50 values of 20.0 and 90.0 nM, respectively. Retinoid analogs competed with the binding of CD 367 to CRABP-I and CRABP-II in the following order: (p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphtyl)-1-propenyl]-benzoic acid (TTNPB) > 4-oxo-RA > 4-OH-RA > 13-cis-RA = 9-cis-RA. m-carboxy-TTNPB and CD 271 were found not to compete with the binding of CD 367 to CRABP-I or CRABP-II even at 500-fold molar excess. These data demonstrate that E. coli-derived CRABP-I has a higher affinity for RA than CRABP-II and that retinoic acid metabolites have a lower affinity for these proteins. The observed difference in affinity for RA supports the idea that CRABP-I, which is constitutively expressed, and CRABP-II, which is induced by RA, have different functions in the cell. In addition, 9-cis-RA, a natural ligand for the retinoid X receptors, is not a physiological ligand for either CRABP-I or CRABP-II.
人细胞视黄酸结合蛋白（CRABP）I型和II型通过克隆的cDNA在大肠杆菌中表达。表达的蛋白通过凝胶过滤和离子交换色谱法纯化，得到高纯度的蛋白。凝胶过滤后的产量约为每升细菌培养物50毫克。在结合研究中，视黄酸（RA）对大肠杆菌来源的CRABP-I和CRABP-II的平衡解离常数Kd分别为6.8和39 nM。合成类视黄醇类似物CD 367对CRABP-I的Kd为2.2 nM，对CRABP-II的Kd为3.0 nM。RA与CD 367与CRABP-I和CRABP-II的结合竞争，IC50值分别为20.0和90.0 nM。类视黄醇类似物与CD 367与CRABP-I和CRABP-II的结合竞争顺序如下：（对-[(E)-2-(5,6,7,8-四氢-5,5,8,8-四甲基-2-萘基)-1-丙烯基]-苯甲酸（TTNPB）> 4-氧代-RA > 4-羟基-RA > 13-顺式-RA = 9-顺式-RA。发现间羧基-TTNPB和CD 271即使在500倍摩尔过量时也不与CD 367与CRABP-I或CRABP-II的结合竞争。这些数据表明，大肠杆菌来源的CRABP-I对RA的亲和力高于CRABP-II，并且视黄酸代谢产物对这些蛋白的亲和力较低。观察到的对RA亲和力的差异支持了以下观点：组成性表达的CRABP-I和由RA诱导的CRABP-II在细胞中具有不同的功能。此外，9-顺式-RA是类视黄醇X受体的天然配体，既不是CRABP-I也不是CRABP-II的生理配体。
