Purification and properties of two proteinases from Saccharomyces cerevisiae

Abstract

A procedure is described for the purification of two intracellular proteinases from baker's yeast ( Saccharomyces cerevisiae). These enzymes (which were not separated by gel filtration or ion-exchange chromatography) are either very similar or form a complex with one another. After the final purification step, each proteinase had a specific activity 25% greater than that of crystalline trypsin. The proteinases have isoelectric points below pH 4.8, and molecular weights of 73,000 as measured by Sephadex G-100 chromatography. Crude preparations, and a few of the purified fractions, showed good stability at 4 °. Most ion-exchange chromatography preparations gave low enzyme yields, possibly because of the loss of a stabilizing factor or the removal of a third proteinase. Metal chelators and alkaline earth metals had no effect on activity or stability, suggesting that these proteinases are not metallo-enzymes. Proteinase B is inhibited by p-chloromercuribenzoate (PCMB) and mercury ions; PCMB inhibition is partially reversed by cysteine, while mercury inhibition is fully reversed. Eighty per cent or more of each proteinase in the yeast cell is in the zymogen form and seems to be particle-bound.