Uterine Milk Protein, a Novel Activin-Binding Protein, Is Present in Ovine Allantoic Fluid

• Published in: Endocrinology (Philadelphia) 1999-10, Vol.140 (10), p.4745-4752
• Main Authors: McFarlane, James R, Foulds, Lynda M, Anne E O’Connor, Phillips, David J, Jenkin, Graham, Hearn, Milton T W, de Kretser, David M
• Format: Article
• Language: English
• Subjects: Activin; Affinity; Allantoic fluid; Bioassays; Biological activity; Fetuses; Follistatin; Fractionation; Growth factors; Ligands; Liquid chromatography; Plasmacytoma; Pluripotency; Protease inhibitors; Protein purification; Proteinase inhibitors; Proteins; Sequences; Serine proteinase; Sheep's milk; Surface plasmon resonance; Uterus
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/endo.140.10.7072

Activins are pluripotent growth factors that have recently been shown to be present in placental and fetal membrane preparations. Our previous studies have identified and purified activin A from ovine amniotic and allantoic fluids. In this study, ligand blots of side fractions from the isolation of activin A from allantoic fluid suggested the presence of activin-binding proteins other than follistatin. Further purification of one of these fractions involved two sequential reverse phase HPLC steps and a Superose 12HR fractionation. SDS-PAGE revealed a single protein band of 55 kDa, which was identified by NH2-terminal sequencing as ovine uterine milk protein (UTMP), a member of the serine protease inhibitor (serpin) superfamily of proteins. Further binding studies, using ligand blot techniques and Superose 12HR fractionation in the presence of[ 125I]activin, demonstrated UTMP to be an activin-binding protein with a lower affinity for activin than that of follistatin. A study of the specific binding behavior of UTMP to activin, using surface plasmon resonance, revealed an apparent equilibrium dissociation constant (Kd) of 49 ± 25 nm, compared with the follistatin-activin Kd of 379 ± 51 pm. Similar to another activin-binding protein,α 2-macroglobulin, UTMP was unable to neutralize the bioactivity of activin in a bioassay based on the capacity of activin to inhibit the proliferation of an MPC-11 plasmacytoma cell line. The high concentrations of this protein in uterine fluid during pregnancy and its ability to bind activin suggest that UTMP may act as a low affinity, high capacity binding protein to sequester activin in the local uterine environment.