Partial characterization of the cellular proteolytic system of Schizophyllum commune

Abstract

Under nitrogen deprivation conditions, cells of the advancing hyphal margin of Schizophyllum commune colonies use amino acids released by proteolysis occurring in the older portions of the mycelium. Assays of activity of cell free extracts against the nonnative protein substrate azocasein revealed the presence of all four mechanistic classes of proteases. Based on inhibitor studies, metalloproteases appeared to be the most active proteases in the extracts, and the activity of this class of enzymes increased during nitrogen starvation. Serine proteases also constituted a large proportion of the proteolytic activity at pH 6. This is consistent with the observation that two protease bands detected in native gelatin-containing Polyacrylamide gels (ScPrB and ScPrH) that appear during nitrogen deprivation are metalloproteases; another (ScPrA) is a serine protease. Chromogenic peptide substrates used in activity assays and in native Polyacrylamide gels revealed the presence of two major aminopeptidase activities. One of these is specific for aromatic and aliphatic amino acid residues; the other, a Zn²⁺-dependent enzyme, has specificity for positively charged amino acids. Multiple forms of both enzymes are produced and their expression is altered by nitrogen deprivation. Three dipeptidyl aminodipeptidases, one specific for gly-phe-X (having three electrophoretically distinguishable forms), one specific for gly-pro-X and one specific for lys-ala-X were detected. Schizophyllum commune produces at least three chymotrypsin-like activities specific for N-succinyl-ala-ala-pro-phe-X. The protease/esterase substrate N-acetyl-phe-β-naphthylester (APNE) produced the most complex banding pattern in native gels with two of the bands corresponding to the chymostrypsin-like activities. Correlating bands produced by staining with chromogenic substrates with those in gelatin gels indicated that ScPrA is a chymotrypsin-like enzyme, that ScPrC and ScPrD are likely to be dipeptidyl aminodipeptidases, and that ScPrH is a Zn²⁺-dependent carboxypeptidase.

Key Words:

• aminopeptidase
• dipeptidase
• endopeptidase
• fungi
• metalloprotease
• protease
• protein degradation
• Schizophyllum commune