Vallet J L
USDA, ARS, Roman L. Hruska U.S. Meat Animal Research Center, Clay Center, Nebraska 68933, USA.
Domest Anim Endocrinol. 1996 Mar;13(2):127-38. doi: 10.1016/0739-7240(95)00063-1.
Retinol-binding protein (RBP) was purified from Day 60 porcine allantoic fluid by a combination of diethylaminoethyl cellulose, G-100 Sephadex, G-50 Sephadex, Phenyl-Sepharose, and Reactive Green 19-dye-agarose chromatography. the yield was 1 to 2 mg of RBP, which generated a single M(r) approximately 20,000 band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and up to four isoelectric variants (isoelectric variants 1 to 4) after two-dimensional PAGE (2D-PAGE). The protein cross-reacted with antiserum raised against human RBP. When incubated with [3H]retinol and subjected to G-100 Sephadex chromatography, [3H]retinol coeluted with the protein. These results indicate that the purified protein is an RBP. When purified RBP was subjected to native 2D-PAGE, six forms of RBP were observed. Three native forms were fluorescent, and three were not fluorescent, suggesting that these forms were RBP with and without retinol, respectively. Denaturing 2D-PAGE analysis of each native form of RBP suggested that two of the nonfluorescent and two of the fluorescent native forms of RBP corresponded to isoelectric variant 1 on denaturing 2D-PAGE, whereas the other fluorescent and nonfluorescent forms corresponded to isoelectric variant 2. The incubation of RBP with 50 microM retinol enhanced the amount of both isoelectric variants present as fluorescent RBP, but uptake by isoelectric variant 1 was greater than that by isoelectric variant 2. These data indicate that RBP can be purified from porcine allantoic fluid and suggest that the isoelectric variants may differ in their affinity for retinol.
通过二乙氨基乙基纤维素、G - 100葡聚糖凝胶、G - 50葡聚糖凝胶、苯基琼脂糖凝胶和活性绿19 - 染料琼脂糖凝胶色谱法相结合，从第60天的猪尿囊液中纯化视黄醇结合蛋白（RBP）。产量为1至2毫克RBP，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）后产生一条单一的分子量约为20,000的条带，在二维聚丙烯酰胺凝胶电泳（2D - PAGE）后产生多达四种等电变体（等电变体1至4）。该蛋白质与针对人RBP产生的抗血清发生交叉反应。当与[3H]视黄醇一起孵育并进行G - 100葡聚糖凝胶色谱分析时，[3H]视黄醇与该蛋白质共洗脱。这些结果表明纯化的蛋白质是一种RBP。当纯化的RBP进行天然2D - PAGE时，观察到六种形式的RBP。三种天然形式有荧光，三种没有荧光，这表明这些形式分别是结合和未结合视黄醇的RBP。对RBP的每种天然形式进行变性2D - PAGE分析表明，两种无荧光的天然形式和两种有荧光的天然形式的RBP在变性2D - PAGE上对应于等电变体1，而其他有荧光和无荧光的形式对应于等电变体2。RBP与50微摩尔视黄醇孵育增加了作为有荧光RBP存在的两种等电变体的量，但等电变体1的摄取量大于等电变体2。这些数据表明RBP可以从猪尿囊液中纯化出来，并表明等电变体对视黄醇的亲和力可能不同。
