The Occurrence of the Growth Factor Strepogenin in Purified Proteins¹

Abstract

The bacterial growth factor, tentatively called strepogenin, which stimulates the growth of a variety of bacteria, has been found in partial hydrolysates of vitamin-free casein.* It has therefore seemed advisable to examine several purified proteins for their ability to yield the factor on partial hydrolysis. Since Wright and Skeggsb have reported strepogenin-like activity for enzymic digests of casein, we have tested such preparations and found them to be more active than the partial hydrolysates prepared with dilute acid as described from this L a b~r a t o r y .~ Therefore, proteins to be examined were treated with trypsin rather than with acid.Tryptic digests of many proteins were found to be much richer sources of the growth factor than was the liver extract previously used as a starting point for purification. Most of these proteins were highly purified, and had been recrystallized repeatedly, All proteins, however, did not yield strepogenin activity when trypsinized. For example, dialyzed egg white, a m d e proteinaceous material, proved to be almost inactive, while crystalline proteins such as insulin and trypsinogen gave rise to activity far greater than that of liver extracts.The rate of liberation of strepogenin by the action of trypsin varied with different proteins. Maximal activity was obtained with digestion periods of twenty hours or less. In the case of casein, more strepogenin was released by trypsin than by pepsin.Since tryptic digests of certain proteins were richer in the growth factor than sources previously examined, these digests have been used as starting points for the purification of strepogenin. Several schemes for concentration of the sub? stance have been tested, and a few will be outlined in the experimental section. Some properties of the active agent which shed light on its chemical nature also will be included. ExperimentalMethods.-The strepogenin assays were performed with the aid of Lactobacillus casei according to the method described earlier.* It is necessary to emphasize, as indicated previously, that a small inoculum was used in this test, since with larger seedIngs the entire effect may be less well marked. In the preparation of the basal medium, it was found advisable to use the casein hydrolysates made with sulfuric acid as recommended rather than the hydrochloric acid hydrolysates commercially available. In the case of the sulfuric acid procedure, traces of strepogenin which survived the acid hydrolysis were removed by the barium (1) An abstract of this paper was published: Woolley and Sprince.(2) Fellow of The Xutrition Foundation, Inc. (3) With the technical assistance of B. Bailey and M. L. Collyer. (4) Sprince and Woolley, J . E x p . M e d . , 80, 213 (1944). ( 5 ) Wright and Skegns, J, Bncl., 48, 117 (1944). Fcdnation Proceedings, 4, 164 (10433.sulfate,' which was used to rid the solution of sulfate ion. When commercial hydrochloric acid hydrolysates were employed, the growth in the blank tubes was appreciable, but acceptable assays could nevertheless be obtained....

Effect of 'Insulinase-Inhibitor' on Destruction of Insulin by Intact Mouse.

“…The inhibitor utilized in the present study was prepared from fresh beef liver and after ashing it was ineffective in vitro. The identity of insulinase-inhibitor remains unknown other than it is a peptide (or peptides) of low molecular weight which is resistant to hyd'rolysis by acid, trypsin and pepsin and which resembles strepogenin (9) in a number of characteristics.…”

On the Nutrition of Desulphovibrio desulphuricans

“…Sprince & Woolley (1945) have shown that a substance of this nature, strepogenin, is required for the growth of certain lactic bacteria. No preparation of strepogenin was available for testing, but the partial tryptic casein digests were prepared so as to conserve this material as much as possible (Dr Woolley, personal communication).…”

An Unidentified Growth Factor in Distillers’ Dried Solubles Essential for the Chick

“…It is unlikely that this factor is strepogenin, because Sprince and Woolley (1945) showed that strepogenin is present in commercial casein. It probably is not any of the factors which recently has been shown to be related to or identical with folic acid (Peterson, 1946).…”