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ARS Home » Research » Publications at this Location » Publication #47043


item Vallet, Jeff

Submitted to: Domestic Animal Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/16/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: The pig uterus during neonatal development and endometrium during pregnancy secrete a protein which binds vitamin A. Studies of the role of this protein during neonatal uterine development and pregnancy were hampered by the lack of a purified preparation. A method for purification of this protein from pig allantoic fluid was elucidated. The purified preparation consisted of several forms which differ in their ability to bind vitamin A. Antisera specific to the protein was also generated. Because the different forms of the protein differ in their affinity, factors which convert the tight-binding forms to loose binding forms may be important for delivery of Vitamin A to where it is required. The availability of a purified preparation of this protein will allow investigation of the molecular basis of the various forms as well as an investigation into their role during pregnancy and uterine development. Also, using the purified preparation of this protein and antiserum specific to the protein, a radioimmunoassay for the protein can be developed to study control of secretion of this protein during pregnancy and neonatal uterine development. Factors which enhance secretion of this protein during pregnancy might be expected to increase protein secretion generally, which might increase uterine capacity and litter size.

Technical Abstract: Retinol binding protein (RBP) was purified from day 60 porcine allantoic fluid. The purification procedure yielded protein which generated a single band (Mr approx. 20,000) after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and four isoelectric variants (isoelectric variants 1-4) after two dimensional PAGE (2D-PAGE). The protein 1) crossreacted with anti-human RBP antiserum and 2) bound 3H retinol, indicating that the purified protein is an RBP. Using native 2D- PAGE, six native forms of RBP were observed. Three native forms were fluorescent and three were not fluorescent. Using denaturing 2D-PAGE analysis, two of the nonfluorescent and two of the fluorescent native forms of RBP were found to correspond to isoelectric variant 1 on denaturing 2D-PAGE while the other fluorescent and nonfluorescent forms correspond to isoelectric variant 2. Removal of retinol resulted in two new nonfluorescent native forms of RBP, indicating that removal of retino does not convert all of the fluorescent forms directly to the nonfluorescent forms. Retinol treatment enhanced the amount of isoelectric variant 1, but not isoelectric variant 2, present as fluorescent RBP, indicating that the isoelectric variants differ in their affinity for retinol. G-100 Sephadex chromatography of RBP and human transthyretin yielded two peaks of RBP; 2D-PAGE and immunoblotting of proteins in each peak indicated that all four isoelectric variants were present in both peaks, however isoelectric variants 3 and 4 appeared to be greater in the late eluting peak. These data indicate that RBP can be purified from porcine ALF and suggest that the isoelectric variants differ in their affinity for retinol and transthyretin.