The intracellular proteases of Ṉe̲u̲ṟo̲s̲p̲o̲ṟa̲ c̲ṟa̲s̲s̲a̲

Lyopilized mycelia were fractionated three times with ammonium sulfate and chromatographed on diethylaminoethyl cellulose. The chromatographic fractions were then assayed for the presence of acidic, neutral, and alkaline proteolytic activities. Multiple proteolytic activities were demonstrated, based on the criteria of chromatographic location, pH optimum, inhibition by commercial and/or endogenous inhibitors, location in a particular ammonium sulfate preparation, growth stage of organism, and crypticity. Cryptic behavior was expressed by incubating column fractions at 4°C for several days and periodically assaying for the presence of proteolytic activities.

Substrate-specific peptidases were revealed by electrophoresing column fractions on 7% polyacrylamide slab gels and then incubating the gels in the presence of a peptide/dye solution, which displayed peptide digestion as sharp blue bands. Eighteen distinct peptidases were shown with this assay. It is concluded that N. Crassa likely contains many more endogenous proteases than heretofore described for this, or any other, organism. The finding warrents further investigation to determine the number, location, and function of the endogenous multiple proteolytic activities.