Inagami S, Ong D E
Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN 37232.
J Nutr. 1992 Mar;122(3):450-6. doi: 10.1093/jn/122.3.450.
Two forms of human cellular retinol-binding protein, type II [hCRBP(II)A and hCRBP(II)B], were purified from the small intestine in a three-step purification procedure. The N-terminal sequence of CRBP(II)A was determined to 36 residues and found to have only a single residue difference when compared with the N-terminal sequence in rat; the amino acid change maintained charge. hCRBP(II)B was blocked at the N-terminus, presumably because of N-acetylation of the initial threonine residue as previously established for CRBP(II)B from rat. This blockage did not affect the binding properties of the protein. The ability of pure hCRBP(II) to bind all-trans-retinol, retinal and retinoic acid was examined by competitive binding assay and compared with the binding specificity of pure human cellular retinol-binding protein (hCRBP). Retinoic acid did not compete with retinol for binding to either protein. Retinal competed with retinol for binding to hCRBP(II) but not to hCRBP, consistent with what was observed for the homologous proteins of rats. Polyclonal antiserum against hCRBP(II)B was produced that recognized both forms of hCRBP(II) in Western blotting and RIA but did not react with hCRBP. Distribution of hCRBP(II) was determined in the jejunum. Higher concentrations of hCRBP(II) were observed in the proximal portion compared with the distal portion. Although concentrations varied in individual intestinal samples, concentrations up to 1% of total mucosal protein were observed in some samples. These results provide tools for further investigation of the role of hCRBP(II) and suggest that previous results from rats, in this important aspect, are relevant to human metabolism of vitamin A.
通过三步纯化程序从小肠中纯化出了两种形式的人细胞视黄醇结合蛋白II [hCRBP(II)A和hCRBP(II)B]。确定了CRBP(II)A的N端序列至36个残基，发现与大鼠的N端序列相比只有一个残基差异；氨基酸变化保持了电荷。hCRBP(II)B在N端被封闭，推测是由于初始苏氨酸残基的N-乙酰化，这与先前确定的大鼠CRBP(II)B情况相同。这种封闭并不影响该蛋白的结合特性。通过竞争性结合试验检测了纯hCRBP(II)结合全反式视黄醇、视黄醛和视黄酸的能力，并与纯人细胞视黄醇结合蛋白(hCRBP)的结合特异性进行了比较。视黄酸不与视黄醇竞争结合这两种蛋白中的任何一种。视黄醛与视黄醇竞争结合hCRBP(II)，但不与hCRBP竞争结合，这与在大鼠同源蛋白中观察到的情况一致。制备了针对hCRBP(II)B的多克隆抗血清，该抗血清在蛋白质印迹和放射免疫分析中能识别两种形式的hCRBP(II)，但不与hCRBP反应。测定了空肠中hCRBP(II)的分布。与远端部分相比，近端部分观察到更高浓度的hCRBP(II)。尽管在各个肠道样本中浓度有所不同，但在一些样本中观察到浓度高达总黏膜蛋白的1%。这些结果为进一步研究hCRBP(II)的作用提供了工具，并表明在这一重要方面，先前来自大鼠的结果与人类维生素A代谢相关。
