C. Richard Savage

Interaction of epidermal growth factor with adult rat liver parenchymal cells in primary culture

Abstract Adult rat liver parenchymal cells in primary culture exhibit specific saturable binding of 125 I-labeled murine epidermal growth factor (EGF). The Scatchard plot of the binding data obtained at 36 °C was curvilinear yielding two apparent dissociation constants of 1.5 × 10 −10 m and 1.2 × 10 −9 m with 27,000 and 57,000 sites per cell, respectively. The binding data obtained at 2 °C yielded a linear Scatchard plot with an apparent dissociation constant of 4.4 × 10 −9 m and 78,000 sites per cell. Exposure of the hepatocytes to EGF at 36 °C resulted in a loss of EGF binding capacity due to down regulation of receptors. The cells recovered the capacity to bind EGF upon incubation in medium which did not contain EGF; this recovery was inhibited by cycloheximide. The cultures appeared to internalize and degrade bound EGF at 36 °C but not at 2 °C. The degradation of EGF was inhibited by chloroquine, an inhibitor of lysosomal enzymes. These data indicate that liver specifically binds and further processes EGF, and therefore, may be a physiological target tissue for this growth factor.

Extended expression of differentiated function in primary cultures of adult liver parenchymal cells maintained on nitrocellulose filters. I. Induction of phosphoenolpyruvate carboxykinase and tyrosine aminotransferase.

Abstract A new support system has been developed which provides long-term maintenance of non-dividing adult rat liver parenchymal cells in monolayer cultures. The hepatocytes, attached to Millipore (MP) filters, are maintained as free-floating cultures which express differentiated liver cell functions for up to 13 days. After 8 days of culture on MP filters, the hepatocytes are still capable of inducing tyrosine aminotransferase 3- to 4-fold and phosphoenolpyruvate carboxykinase 10-to 15-fold. The advantage of using floating MP filters to support the hepatocytes over the more conventional culture supports such as glass or plastic dishes are: (1) the functional lifespan of cultured hepatocytes is doubled, permitting experiments requiring 4–8 days to complete; (2) it permits rapid and easy transfer of cells from one set of culture conditions to another; (3) sections can be cut from one filter permitting multiple samples from a single culture; (4) the filters containing the cells can be processed without losing the orientation of cell surfaces, an important consideration when employing techniques such as autoradiography and/or electron microscopy; and (5) this culture technique can readily be adapted for co-cultivation experiments in order to directly examine biological and biochemical effects of secreted products of one cell type on another.

Human epidermal growth factor/urogastrone: rapid purification procedure and partial characterization.

Abstract A new procedure for the isolation of two biologically active forms of human epidermal growth factor (h-EGF-1) and (h-EGF-2) has been devised. Starting with 20 liters of raw human urine, the method employs a six-step fractionation procedure which yields 100–150 μg of h-EGF-1 and 50–100 μg of h-EGF-2. Initial studies suggest that h-EGF-2 may have been derived from h-EGF-1 by removal of the carboxy-terminal arginine or leucine-arginine residue(s). Based on immunological data and electrophoretic mobility at pH 9.5, h-EGF-2 appears to be identical to authentic h-EGF isolated by Cohen and Carpenter ( Proc. Natl. Acad. Sci. , 1975, 72 , 1317). Using the antibody to authentic h-EGF, single precipitin lines of identity are observed between h-EGF-1, h-EGF-2, and authentic h-EGF. Both forms of h-EGF have comparable biological activity in stimulating the growth of adult human skin fibroblasts in culture.

Isolation of rat epidermal growth factor (r-EGF): chemical, biological and immunological comparisons with mouse and human EGF

Rat epidermal growth factor, (r-EGF), was isolated from adult male rat submandibular glands, with final yields of 4-6 mg r-EGF from 20 to 25 g wet weight of tissue. Amino acid analysis of r-EGF indicated a high degree of homology with murine EGF (m-EGF) and human EGF, (h-EGF). However, r-EGF contains 49 amino acid residues, versus 53 for human and murine EGFs, and lacks two characteristic tryptophan residues present in the other two species. The lack of tryptophan residues did not affect cellular binding or mitogenic activity or r-EGF. Polyclonal antisera to each of the three separate species demonstrated crossreactivity with the other species of EGF. A sensitive radioimmunoassay was developed for r-EGF which can detect 25 pg of hormone.

Improved method for the purification of biologically active transcobalamin II.

Transcobalamin II (TC II) was purified about 300,000-fold from Cohn fraction III using a modification of the procedure described by Allen and Majerus (J. Biol. Chem. 247, 7709-7717 (1972)). The simplified method incorporated isoelectric precipitation of the TC II into the purification scheme which permitted the elimination of two column chromatographic steps originally reported by the above workers. The final preparation had 26.7 mug of vitamin B12 (B12) bound per mg of protein and an A280/A361 ratio of 2.05, both of which are in good agreement with the reported values. The purified TC II was biologically active with respect to its ability to facilitate penetration of B12 into HeLa cells in tissue culture.

[1] Purification of human epidermal growth factor by monoclonal antibody affinity chromatography

Publisher Summary Epidermal growth factor (EGF) is a single chain polypeptide containing amino acid residues that exhibits potent mitogenic activity for a variety of cell types both in vivo and in vitro . Many reviews are available concerning the mechanism of action and biological effects of EGF. This molecule was first described by Cohen, who isolated EGF from the submaxillary glands of adult male mice (mEGF). Since then, EGF has been isolated from rat, guinea pig prostate, and human urine (hEGF). This chapter discusses a part of the standard published procedure and presents the use of monoclonal antibody affinity chromatography in the light of other published reports using this technology.