Effects of Yeast Proteinase and Its Inhibitor on the Inactivation of Tryptophan Synthase from Saccharomyces cerevisiae and Neurospora crassa

Abstract

This paper reports the isolation of a proteinase and a proteinase inhibitor from Saccharornyces cerevisiae S288C (a). The purified proteinase catalyzes the hydrolysis of casein a t neutral pH. It inactivates the holo-tryptophan synthase from Neurospora c r a m but does not inactivate the holo-ornithine aminotransferase from pig kidney. This proteinase can be inhibited either by phenylmethanesulfonyI fluoride (a specific inhibitor for serine proteases), the sulfhydryl reagent, p-mercuribenzoate, or the endogenous proteinase inhibitor present in yeast. The yeast proteinase inhibitor is a heat-stable material with a molecular weight of approximately 13000. This proteinase inhibitor can protect both yeast and Neurospora tryptophan synthase against proteolytic inactivation. The proteinase and its inhibitor coexist in the yeast extracts. The cellular level of the proteinase and the inhibitor activities varies with the composition of the medium in which the cells are grown. Since some yeast proteinases have been shown to form inactive complexes with their respective inhibitors, it is proposed that proteinase-inhibitor interaction is a possible mechanism for regulation of intracellular proteolytic activity.Previous work on Neurospora tryptophan synthase [l] has shown that inactivation of partially purified enzyme can be prevented by phenylmethanesulfonyl fluoride, a known inhibitor of serine protease [2,3]. From this observation, together with the fact that the highly purified enzyme is stable, i t was cancluded that the inactivation in vitro of the Neurospora synthase is due to proteolysis [l]. More recent studies by us have demonstrated that the protease which causes the inactivation of the Neurospora synthase is a serine endopeptidase [4]. Furthermore, we demonstrated that the purified endopeptidase can be strongly inhibited by a proteinaceous inhibitor present in Neurospora [4].The activity of the tryptophan synthase of Saccharomyces cerevisiae is also known to be influenced by macromolecular factors, inactivating and protecting, present in yeast [5]. Recently a partial purification of both factors was reported [6,7]; however, no information was presented regarding the mechanism of inactivation.

Three yeast proteins that specifically inhibit yeast proteases A, B, and C

“…7 is linear up to 90% inhibition of protease B and then it approaches 100% inhibition. Tsai et al (32) obtained an IB calibration curve which was linear only up to 60% inhibition. It seems probable that their soluble casein substrate competes more effectively with 'B than does our insoluble azocoll substrate.…”

“…Schott and Holzer (31) have obtained protease B in pure form and Hasilik and Holzer (9) have confirmed that protease B is capable of activating chitin synthetase. Tsai et al (32) have studied the inactivation of yeast and Neurospora tryptophan synthase by a yeast protease (presumably protease B) and the inhibition of this destruction by an inhibitor (presumably IB). Molano and Gancedo (25) have reported that a yeast protease inactivates yeast fructose 1,6-bisphosphatase and an inhibitor of this inactivation is also present in yeast.…”

On the stoichiometry reversibility of interaction between Neurospora protease I and its inhibitor

“…1) which was hardly observed under the low pH conditions [4] can be greatly stimulated by 4 M urea, by incubation with 0.1 M citrate buffer pH 4 or by extensive dialysis. Since such a phenomenon has not been observed with purified protease I, it may be attributed to a protease-inhibitor complex similar to that observed in yeast [5,10]. After heat treatment (90°C, 5 rain) the proteolytic activity of the complex was completely destroyed, while a heat stable material remained in the supernatant that was active in the inhibition of…”

“…Recently we have shown that there exists in Neurospora crassa a group of protease inhibitors which preferentially inhibit the proteolytic enzymes from the same organism [4]. The findings of the coexistence of proteases and inhibitors and their specific interactions in microorganisms led us to the postulate, that the interplay of proteases with their cognate inhibitors may play an important role in the regulation of intracellular proteolytic activity [4,5]. With an aim to understanding the mechanism of this control process, efforts were made to isolate and characterize a protease-inhibitor complex from Neurospora.…”

Formation and Location of 1,4-β-Glucanases and 1,4-β-Glucosidases from Penicillium janthinellum

“…The assay for proteolytic activity was performed according to Tsai et al (30), using casein-yellow (Calbiochem, San Diego, Calif.) as the substrate. Before assaying for protease activity, the samples were concentrated 10-fold by a Minicon-B 15 (Amicon Corp., Lexington, Mass.).…”