Studies on the Apparent Instability of Neurospora Tryptophan Synthase

Neurospora tryptophan synthase prepared by a standard procedure was found to be contaminated with endogenous protease(s). Such a protease is responsible for the apparent instability of the tryptophan synthase. The rate of inactivation of tryptophan synthase was found to be directly proportional to the level of proteolytic activity. The protease is synthesized during all phases of cell growth. However, the proteolytic activity is most pronounced in the aged cultures. The protease which has been purified by Sephadex G‐200 column chromatography and poly‐acrylamide‐gel electrophoresis exhibits the properties of an endopeptidase. It is relatively insensitive to p‐mercuribenzoate and is insensitive to EDTA. It is very sensitive, however, to the proteolytic inhibitor phenylmethanesulfonyl fluoride. During the course of this study, a potent protease inhibitor has also been isolated from Neurospora. It is concentrated in a fraction precipitated with ammonium sulfate at 30 to 45% saturation. The isolated inhibitor is heat‐resistant, non‐dialyzable and is excluded by Sephadex G‐25. It strongly inhibits the Neurospora protease but has no effect on trypsin.