Rao M N, Ghosh P, Lakshman M R
Department of Medicine, George Washington University, Washington, D. C. 20037, USA.
J Biol Chem. 1997 Sep 26;272(39):24455-60. doi: 10.1074/jbc.272.39.24455.
A cellular carotenoid-binding protein was purified to homogeneity from beta-carotene-fed ferret liver utilizing the following steps: ammonium sulfate precipitation, ion exchange, gel filtration, and affinity chromatography. The final purification was 607-fold. [14C]beta-Carotene co-purified with the binding protein throughout the purification procedures. SDS-PAGE of the purified protein showed a single band with an apparent molecular mass of 67 kDa. Scatchard analysis of the specific binding of the purified protein to beta-carotene showed two classes of binding sites, a high affinity site with an apparent Kd of 56 x 10(-9) M and a low affinity site with a Kd of 32 x 10(-6) M. The Bmax for beta-carotene binding to the high affinity site was 1 mol/mol, while that for the low affinity site was 145 mol/mol. The absorption spectrum of the complex showed a 32-nm bathochromic shift in lambdamax with minor peaks at 460 and 516 nm. Except for alpha-carotene and cryptoxanthin, none of the model carotenoids or retinol competed with beta-carotene binding to the protein. Thus, a specific carotenoid-binding protein of 67 kDa has been characterized in mammalian liver with a high degree of specificity for binding only carotenoids with at least one unsubstituted beta-ionone ring.
利用以下步骤从喂食β-胡萝卜素的雪貂肝脏中纯化出一种细胞类胡萝卜素结合蛋白，使其达到同质：硫酸铵沉淀、离子交换、凝胶过滤和亲和色谱。最终纯化倍数为607倍。在整个纯化过程中，[14C]β-胡萝卜素与结合蛋白共同纯化。纯化蛋白的SDS-PAGE显示一条单一带，表观分子量为67 kDa。对纯化蛋白与β-胡萝卜素的特异性结合进行Scatchard分析，结果显示有两类结合位点，一个高亲和力位点，表观Kd为56×10(-9) M，一个低亲和力位点，Kd为32×10(-6) M。β-胡萝卜素与高亲和力位点结合的Bmax为1 mol/mol，而与低亲和力位点结合的Bmax为145 mol/mol。复合物的吸收光谱显示λmax处有32 nm的红移，在460和516 nm处有小峰。除α-胡萝卜素和隐黄质外，模型类胡萝卜素或视黄醇均不与β-胡萝卜素竞争与该蛋白的结合。因此，已在哺乳动物肝脏中鉴定出一种67 kDa的特异性类胡萝卜素结合蛋白，其对仅结合具有至少一个未取代β-紫罗兰酮环的类胡萝卜素具有高度特异性。
