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United States Department of Agriculture

Agricultural Research Service

Title: Purification and Characterization of Porcine Folate Binding Proteins from Uterine Flushings and Allantoic Fluid

Author
item Vallet, Jeffrey

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 25, 1997
Publication Date: N/A

Technical Abstract: The objectives of these experiments were to (1) determine if a folate binding protein (FBP) is present in uterine flushings or allantoic fluid, (2) purify and characterize the FBP, (3) generate FBP antisera and, (4) determine whether FBP in uterine flushings changes from day 10 to 15 of the cycle or pregnancy. Samples of both D60 pseudopregnant uterine flushings and D60 allantoic fluid were incubated with 10 uCi 3H-folic acid and then subjected to G-100 Sephadex chromatography. For each sample, a peak (Mr 30,000) of 3H-folic acid was found indicating the presence of FBP. Folate-Sepharose chromatography followed by G-100 chromatography was used to purify FBP from each source. For uterine flushings, the major component had an Mr of 20,000. N-terminal sequencing of this band gave the sequence FNWDHXGKMEPAXKRHFXXXTXLY which was 72% identical homology to bovine milk FBP beginning at amino acid 64 and suggests that this band represents truncated FBP. For allantoic fluid, the preparation had an Mr of 30,000. N-terminal sequencing of this band gave the sequence ARAKTDMLNVXMDAKHHKPKPS XED which was 68% identical to bovine milk FBP beginning at amino acid 4. Allantoic fluid FBP was used to immunize rabbits and the resulting antiserum was used for immunoblotting. Immunoblots of uterine flushings collected on days 10, 11, 12, 13 and 15 of the cycle or pregnancy indicated that FBP was undetectable on D 10 and became strongly detectable by D 15. Furthermore, multiple bands (Mr 30,000, 27,000) were present on D15 of the cycle. These results indicate that an FBP is present in uterine flushings during the cycle and early pregnancy and increases between days 10 and 15. These data suggest that FBP may be involved in folate delivery to the developing conceptus.

Last Modified: 11/28/2014