title: A novel metalloprotease from the wild basidiomycete mushroom Lepista nuda.
abstract: A 20.9-kDa metalloprotease was isolated from dried fruiting bodies of the wild basidiomycete mushroom Lepista nuda. The N-terminal amino acid sequence of the protease was seen to be ATFVLTAATNTLFTA, thus displaying no similarity with the sequences of previously reported metalloproteases. The protease was purified using a procedure that entailed ion-exchange chromatography on CM-Cellulose, Q-Sepharose, and Mono S, and FPLC-gel filtration on Superdex 75. The protease functioned at an optimum pH of 7.0 and an optimum temperature of 50 degrees C. It was also noted that the protease demonstrated a proteolytic activity of 1,756 U/mg toward casein. The Km of the purified protease toward casein was 6.36 mg/ml at a pH of 7.0 and with a temperature of 37 degrees C, whereas the Vmax was 9.11 microgram ml(-1) min(-1). The activity of the protease was adversely affected by EDTA-2Na, suggesting that it is a metalloprotease. PMSF, EGTA, aprotinin, and leupeptin exerted no striking inhibitory effect. The activity of the protease was enhanced by Fe2+, but was curtailed by Cd2+, Cu2+, Hg2+, Pb2+, Zn2+, and Fe3+ ions. The protease also exhibited inhibitory activity against HIV-1 reverse transcriptase with an IC50 value of 4.00 micrometer. The IC50 values toward hepatoma Hep G2 and leukemia L1210 cells in vitro were 4.99 micrometer and 3.67 micrometer, respectively.
authors.affiliation: State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, China.
authors.forename: Y Y
authors.initials: YY
authors.lastname: Wu
authors.forename: H X
authors.initials: HX
authors.lastname: Wang
authors.forename: T B
authors.initials: TB
authors.lastname: Ng
mesh_terms: D000363:Agaricales / Q000201:enzymology*; D002364:Caseins / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D048690:Fruiting Bodies, Fungal / Q000201:enzymology; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000633:toxicity; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D002364:Caseins; D002413:Cations, Divalent; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D045726:Metalloproteases