1887

Abstract

Summary: Proteolytic enzyme activities of the wood-decaying basidiomycetes and , have been characterized using azocasein as substrate and by electrophoretic analysis with gelatin-containing polyacrylamide gels (gelatin-SDS-PAGE). In , intracellular and extracellular azocaseinase activity was optimal at pH 5-6 and was inhibited by pepstatin A. Gelatin-SDS-PAGE revealed two highly active proteinases, S1 and S4 (apparent 65000 and 30000, respectively) and two less active enzymes, S2 and S3 (apparent 47000 and 43000, respectively). S1, the predominant intracellular proteinase, was present at all ages of the mycelium (tested up to 3 months). It is active over a broad pH range, with highest activity around neutral pH. As S1 was partially inhibited by 1,10-phenanthroline, the enzyme was considered to be a metalloproteinase although EDTA and phosphoramidon had no effect. A proteinase apparently identical to S1 was also detected in the medium of older cultures. S4 is a pepstatin-sensitive aspartic proteinase; its activity was highly pH-dependent and it was inactive in gelatin gels at pH 5-0 and above. S2 and S3 were identified as intracellular metalloproteinases, present in relatively young and growing cultures. They were distinct from S1 as they were inhibited by EDTA and phosphoramidon. During starvation-induced autolysis of , proteinase S1 was the only enzyme present throughout (and the intracellular azocaseinase activity increased), which suggested a likely role of S1 in intra-hyphal protein mobilization. S4 is more likely to play a part in extracellular digestion of protein. The azocaseinase activities of cultures of were optimal at pH 7-0 (intracellular) and pH 5-6 (extracellular). Mycelial extracts gave one major band of proteinase activity in gelatin gels, C1 (apparent 62-64000). Since the activity was sensitive to inhibitors of both serine and metalloproteinases, there may have been overlapping bands due to enzymes of both types. Extracellular samples gave a more complex pattern, (five bands, C2-C6, 50000-100000). C2 and C4 are PMSF-sensitive proteinases, C5 and C6 are probably metalloproteinases, while C3, which was most active at pH 4-0, was unaffected by any of the inhibitors tested, including pepstatin A. No aspartic proteinase equivalent to S4 appeared to be produced by . From the information gained about the intracellular or extracellular location of these enzymes, and the conditions under which they are active, an role may be tentatively ascribed to some of them.

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1995-07-01
2021-10-23
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