Bailey J S, Siu C H
Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, Canada.
J Biol Chem. 1988 Jul 5;263(19):9326-32.
This report describes the purification and partial characterization of a novel retinoic acid-binding protein (CRABP-II) from neonatal rat pups. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-cellulose, and high performance liquid chromatography (HPLC) on a DEAE 5PW column. Two retinoic acid-binding peaks were resolved at the DEAE-cellulose step, with CRABP-I in the major peak and CRABP-II in the minor peak. Apparent homogeneity was achieved for both binding proteins after the HPLC step. CRABP-II consists of a single polypeptide, migrating with an apparent Mr of 15,000 in sodium dodecyl sulfate-polyacrylamide gel. It has an isoelectric point of 5.0. The dissociation constant for CRABP-II of retinoic acid was estimated to be 65 nM by fluorescence titration. Amino-terminal sequence analysis showed that CRABP-II has a distinct sequence, while the CRABP-I sequence is exactly identical to that of the rat testis CRABP. Despite the extensive sequence homology between CRABP-I and CRABP-II, antibodies directed against CRABP-I did not cross-react with CRABP-II.
本报告描述了从新生大鼠幼崽中纯化一种新型视黄酸结合蛋白（CRABP-II）并对其进行部分特性鉴定的过程。分离步骤包括在Sephadex G-75上进行凝胶过滤、在DEAE-纤维素上进行离子交换色谱以及在DEAE 5PW柱上进行高效液相色谱（HPLC）。在DEAE-纤维素步骤中分离出两个视黄酸结合峰，主要峰中为CRABP-I，次要峰中为CRABP-II。经过HPLC步骤后，两种结合蛋白均达到了表观均一性。CRABP-II由一条单一多肽组成，在十二烷基硫酸钠-聚丙烯酰胺凝胶中迁移时表观分子量为15,000。其等电点为5.0。通过荧光滴定法估计CRABP-II与视黄酸的解离常数为65 nM。氨基末端序列分析表明CRABP-II具有独特的序列，而CRABP-I的序列与大鼠睾丸CRABP的序列完全相同。尽管CRABP-I和CRABP-II之间存在广泛的序列同源性，但针对CRABP-I的抗体与CRABP-II没有交叉反应。
