The above experiments indicate that the growth-stimulating substance found in embryonic tissue extract, which has been responsible for the continuous growth of fibroblasts in vitro for 14 years, is either protein in nature or closely associated with the protein of the extract and adsorbed by it. If any specific hormone responsible for cell division is present, it is united to the protein or carried along with it in its first precipitation. It seems probable that the tissues utilize this protein for the nitrogen which they build into protoplasm. Whether it is first hydrolyzed before adsorption by the tissues has not been ascertained as yet. It has been shown in other experiments reported in the following paper that the amino acids of the tissue juice do not suffice for the growth of fibroblasts and that hydrolyzed tissue juice is toxic in the same way that a too concentrated mixture of amino acids is toxic.
The results of the foregoing experiments may be summarized as follows:
1. Fractionation of embryo tissue juice has shown that it is the protein fraction that contains the activating substance.
2. Tissues continue to grow for a long time in the protein of the extract precipitated by CO2 and at a rate approximately equal to that in the original extract diluted to the same nitrogen concentration.
3. The non-protein nitrogen gives slight stimulation to growth.
4. Purification of the protein by repeated precipitation destroys its growth-promoting properties, but whether this is due to a denaturing of the protein,—which occurs very readily,—or to loss of some substance possibly an enzyme attached to it, has not been ascertained.
5. Preparations of purified proteins from embryonic tissue and egg white have shown no marked nutritive or stimulating action. A number of other pure substances have been tried without effect.