Abstract
A serum-free hormone-supplemented medium which was previously formulated for the growth of mouse embryonal carcinoma (EC) cells and rat mammary epithelial (REM) cells required the presence of crude bovine fetuin as a medium supplement for maintaining cell growth. This requirement could not be replaced by purified fetuin preparations. The major growth-promoting activity (embryonin) in the crude fetuin preparation has been purified to near homogeneity by carboxymethylcellulose chromatography and high performance gel filtration chromatography. Purified embryonin, 2 to 4 micrograms/ml, is able to stimulate the growth of mouse EC cells in a serum-free hormone-supplemented medium to a level that is achieved with 0.5 to 1 mg/ml of the crude fetuin preparation. The biological activity resides in a high molecular weight glycoprotein (Mr = 270,000). Three polypeptide chains are observed following reduction, a major polypeptide (Mr = 185,000) and two minor chains (Mr = 116,000 to 120,000 and 68,000). Embryonin differs from pure fetuin in molecular weight, isoelectric point, amino acid composition, and immunological reactivity. However, embryonin is similar to human alpha 2-macroglobulin (alpha 2M) in these physicochemical and immunological properties. Resemblance to alpha 2M is also indicated by the observation that alpha 2M used over the same protein concentration range as embryonin produces a comparable stimulation of EC cell growth in the absence of serum. In addition, embryonin and alpha 2M produce a 2- to 10-fold differential stimulation of collagen production in cultures of normal rat kidney and RME cells, a response which may be linked to the growth-promoting activity of these two proteins.
Highlights
A serum-free hormone-supplemented medium which method of Pedersen (9) which utilizes a 45% ammonium was previously formulated for the growth of mouse sulfate precipitate of fetal calf serum was found to be active embryonal carcinoma (EC) cells and rat mammary ep- inpromoting the growth of HeLa cells and several other ithelial (RME) cells required the presencoef crude bo- epithelial cell types in uitro in the absence of serum (1, 7, 8, vine fetuin as a medium supplement for maintaining 10, 1 1 )
Resemblance or Spiro (21, 22) are biologically inactivein promoting cell to a2M is indicated by the observation that a2M growth (1,2,12, 14,15).These resultssuggest that the growthused over thesameproteinconcentrationrangeas promoting activity may reside in one or moreof the contamembryonin produces a comparable stimulation of EC inating species or that fetuin is the acptirvineciple but during cell growth in the absence of serum
Fetuin is anacidic glycoprotein consistingof a core protein of partial degradation due to enzymes such as thrombin or ( M, = 37,300) linked tothree oligosaccharide chains,each plasmin which are activated during the preparationof serum having an apparent M, of approximately 3,400 (1)
Summary
T h e column was eluted a t a Materials-Pedersen-prepared fetuin(type 111) was purchased flow rate of 0.5 ml/min at 600 p.s.i. using a Waters HPLC chromatogfromSigma.DeutschandSpirofetuinpreparations,fetalbovine raphy pump equipped with a model 440 absorbance detector. The serum,trypsin,penicillin-streptomycin, andHepeswereobtained biologically active high molecular weight material which eluted from from Gihco. Acrylamide, hisacrylamideN, ,N,N ' ,N'-tetrameth- and stored at -20 "C in distilled water at. A concentration of 2 to 4 ylethylenediamine, SDS, ammonium persulfate, anSdDS gel electro- mg/ml. Phoretic high molecular weight standard protein mixture Ouchterlony total acrylamide monomer concentration were run at 3 mA per gel. PeviconC-870 was The gels were stained for proteins w0it.0h5%Coomassie brilliant blue from Accurate Chemical and Scientific Corp., Hicksville, NY. Buffal- in 25%.isopropanol or for glycoproteins with 0.0054 Ethyl-Stains-All yte (pH 3.0-10.0) was from Pierce
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