Purification and properties of porcine allantoic fluid retinol-binding protein.
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.
Vallet
J L
JL
USDA, ARS, Roman L. Hruska U.S. Meat Animal Research Center, Clay Center, Nebraska 68933, USA.
D000482:Allantois / Q000378:metabolism*; D000818:Animals; D001826:Body Fluids / Q000737:chemistry*; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing; D011247:Pregnancy; D012177:Retinol-Binding Proteins / Q000302:isolation & purification*; D013552:Swine; D013997:Time Factors
D012177:Retinol-Binding Proteins