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LA Landon1 *, A McLain1 , RM Roberts1 *, JA Green1
Dept of Animal Science, Univ of Missouri, Columbia, Missouri 1
Different pregnancy-associated glycoproteins (PAG) are expressed by the fetal chorionic membranes prior to and after implantation in cattle, sheep and pigs. PAG are structurally related to aspartic proteinases (AP), such as pepsin, and retain the AP substrate-binding cleft. We hypothesize that PAG form a spectrum of ligand-binding proteins with overlapping peptide-binding specificities and affinities. Affinity chromatography with immobilized pepstatin, an AP inhibitor, was used to rapidly produce enriched native PAG protein fractions. Bovine and ovine placental lysates (Day 80-100) were passed over pepstatin-agarose columns equilibrated to neutral (pH 7.0) or to acidic pH (pH 5.0) in the presence of 150 mM NaCl. Unbound PAG passed through the column and were not studied further. To elute bound proteins, the salt concentration was raised to 1 M NaCl and the buffer pH increased in steps to pH 10.0. Protein bands in the pH 5.0 column eluent, migrating with apparent Mr of 40,000-80,000 during SDS-PAGE, were distinct from those observed in the pH 7.0 column eluents. In western blots, different polyclonal anti-PAG antisera differentially recognized the eluted PAG fractions. N-terminal sequencing indicated that the bands represented distinct PAG. These results indicate that PAG can be enriched and fractionated based upon pH-dependent binding and elution of PAG from a single affinity column, that PAG have ligand-binding function and that the variations in PAG affinity for pepstatin may reflect the broad specificity range of PAG peptide-binding. USDA grant #96-01842.
This abstract is being presented on Tuesday, August 3 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.