Separation of the estrogen-activated growth-regulatory forms of alpha-fetoprotein in mouse amniotic fluid

We have previously reported that murine fetal alpha-fetoprotein (AFP) incubated for 1.0 h at room temperature in the presence of high concentrations of estradiol (E2) generates a growth-regulatory product designated AFP/E2. Subsequently we developed a bioassay in the immature mouse uterus to measure both the growth-inhibitory and growth-enhancing properties of AFP. In the present study, we have employed this bioassay to monitor each of the amniotic fluid-derived AFP isolates fractionated by various chromatographic and electrophoretic techniques. The objective of this investigation was to partition and isolate the various molecular forms of AFP contained in amniotic fluid and determine whether the growth-regulatory activities resided with one or more of the fractions. AFP was fractionated by three different chromatographic/electrophoretic methods: E2 affinity chromatography, preparative polyacrylamide-gel electrophoresis (PAGE), and high-performance liquid chromatography (HPLC); and one immunoaffinity method: gel-entrapped antibody filtration (GAF). Whereas E2 affinity chromatography separated the biological activity of AFP into inhibitory and possibly enhancing activities, PAGE purification yielded three fractions: an inhibitor, an enhancer, and a fraction without growth-regulatory activity. Immunoaffinity separation yielded an AFP product with only inhibitory activities. In comparison, fractionation by HPLC produced seven AFP fractions in which only three displayed growth-regulatory activities: two inhibitory and one enhancing. After subsequent HPLC rechromatography of these fractions, none displayed any biological activity. Thus, murine AFP derived from amniotic fluid is composed of potential heterofunctional forms that, depending on their relative abundance in the preparation, constitute a mixture capable of either (a) growth inhibition, (b) no effect, or (c) growth enhancement.