Purification and characterization of intrauterine folate-binding proteins from swine.
Folate-binding proteins (FBP) from Day 60 pseudopregnant uterine flushings and Day 60 allantoic fluid were purified by affinity chromatography on folate-Sepharose followed by G-100 Sephadex chromatography. FBP from uterine flushings had a molecular weight of 20000; the N-terminal sequence was FNWDHXGKMEPAXKRHFXXXTXLYX, which is 72% identical to bovine milk FBP beginning at amino acid 64. Allantoic fluid FBP had a molecular weight of 30000; and the N-terminal sequence ARAKTDMLNVXMDAKHHKPKPSXED, which is 68% identical to bovine milk FBP starting at amino acid 4. Scatchard analysis of purified allantoic fluid FBP using [3H]folic acid as ligand indicated a dissociation constant of 0.54 nM, and the protein was saturated at 20 nM. Antiserum to the purified allantoic fluid FBP was generated in rabbits and used for immunoblotting. Uterine flushings were collected from pregnant and nonpregnant gilts on Days 10, 11, 12, 13, and 15. Immunoblotting indicated that FBP concentrations increased in uterine flushings from both pregnant and nonpregnant gilts between Days 10 and 15. Total uterine flush specific binding of [3H]folic acid increased from 0.015 nmol on Day 10 to 2.14 nmol on Day 15. These results indicate that an FBP similar to other known FBPs is present in uterine flushings and allantoic fluid and that its level increases at about the time of blastocyst elongation and initiation of conceptus hematopoiesis. These results suggest a role for FBP in the delivery of folate to the developing conceptus.
Vallet
J L
JL
USDA, ARS, Roman L. Hruska U.S. Meat Animal Research Center, Clay Center, Nebraska 68933, USA. vallet@email.marc.usda.gov
Christenson
R K
RK
Klemcke
H G
HG
D000482:Allantois / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001826:Body Fluids / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011247:Pregnancy; D011555:Pseudopregnancy; D011956:Receptors, Cell Surface; D013552:Swine; D007507:Therapeutic Irrigation; D014599:Uterus / Q000737:chemistry*
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D010446:Peptide Fragments; D011956:Receptors, Cell Surface