Identification and partial characterization of multiple forms of biologically active EGF in rat milk

Milk from lactating Sprague-Dawley rats was assayed for epidermal growth factor (EGF)-like activities. A homologous radioimmunoassay (RIA) indicated the presence of immunoreactive material that competed in a nonparallel fashion with submandibular gland rat EGF (sm-r-EGF). The activity in the milk was extracted using antibodies to sm-r-EGF covalently linked to acrylamide beads. This activity was characterized by RIA, nondenaturing polyacrylamide gel electrophoresis, enzymatic digestion, radioreceptor assay, and ability to stimulate incorporation of [3H]thymidine into cultured fibroblasts. The presence of three distinct immunoreactive forms of EGF in rat milk were detected that competed with 125I-labeled r-EGF for binding to the EGF receptor and stimulated DNA synthesis in growth-arrested fibroblasts. Two of the forms are converted to the sm-r-EGF species by tryptic digestion as determined by RIA and migration rates in a nondenaturing polyacrylamide gel. The biological activities are stable to heating in 0.1 M acetic acid and also are stable at pH 9.0. Levels of r-EGF equivalents in milk were low at time of birth (6.3 +/- 1.7 ng/ml) and rose to 35.4 +/- 14.6 by days 4 to 6.