Pleureryn, a novel protease from fresh fruiting bodies of the edible mushroom Pleurotus eryngii.
A protease designated pleureryn, with an N-terminal sequence dissimilar from previously reported mushroom metalloendopeptidases and showing only limited resemblance to aspartic proteinases, albeit considerable homology to DNA replication licensing factor, was isolated from fresh fruiting bodies of the edible mushroom Pleurotus eryngii. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel Blue gel, ion exchange chromatography on CM-Sepharose, and FPLC-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel Blue gel and CM-Sepharose. It demonstrated a single band with a molecular weight of 11.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pleureryn demonstrated a protease activity of 9364 U/mg toward casein. It exhibited a pH optimum of 5.0 and a temperature optimum of 45 degrees C, with substantial activity remaining at high temperatures and pH 4 and 12. The activity of the protease was adversely affected by pepstatin A, indicating that it is an aspartic protease. PMSF, trypsin inhibitor, and EDTA exerted no striking effect, suggesting that it is neither a serine protease nor a metalloprotease. It inhibited translation in a rabbit reticulocyte lysate system with an IC(50) of 20 nM. Pleureryn also exhibited some inhibitory activity against HIV-1 reverse transcriptase, reminiscent of a suppressive action of HIV-1 protease on its homologous reverse transcriptase but was devoid of ribonuclease, deoxyribonuclease, and antifungal activities.
Wang
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Department of Microbiology, China Agricultural University, Beijing, China.
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D000595:Amino Acid Sequence; D000818:Animals; D019380:Anti-HIV Agents / Q000494:pharmacology; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D010436:Pepstatins / Q000494:pharmacology; D020076:Pleurotus / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011817:Rabbits; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D019380:Anti-HIV Agents; D010436:Pepstatins; D011480:Protease Inhibitors; D018894:Reverse Transcriptase Inhibitors; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin