Purification and characterization of fibrinolytic enzyme from cultured mycelia of Armillaria mellea.
A fibrinolytic enzyme was purified from the cultured mycelia of Armillaria mellea by ion-exchange chromatography followed by gel filtration, and was designated A. mellea metalloprotease (AMMP). The purification protocol resulted in a 627-fold purification of the enzyme, with a final yield of 6.05%. The apparent molecular mass of the purified enzyme was estimated to be 21kDa by SDS-PAGE, fibrin-zymography and gel filtration chromatography, which revealed a monomeric form of the enzyme. The optimal reaction pH value and temperature were, pH 6.0, and 33 degrees C, respectively. This protease effectively hydrolyzed fibrinogen, preferentially digesting the Aalpha-chain over the Bbeta- and r-chains. Enzyme activity was inhibited by Cu(2+) and Co(2+), but enhanced by the addition of Ca(2+) and Mg(2+) ions. Furthermore, AMMP activity was potently inhibited by EDTA, and was found to exhibit a higher specificity for the substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 24 amino acid residues of the N-terminal sequence were MFSLSSRFFLYTLCL SAVAVSAAP, which is extremely similar to the 24 amino acid residues of the N-terminal sequence of the fruiting body of A. mellea. These data suggest that the fibrinolytic enzyme AMMP, obtained from the A. mellea exhibits a profound fibrinolytic activity. The mycelia of A. mellea may thus represent a potential source of new therapeutic agents to treat thrombosis.
Lee
Sook-Young
SY
Department of Biology Research Center for Industrial Accelerators, Dongshin University, 252 Daeho-dong, Naju 520-714, Republic of Korea.
Kim
Jae-Sung
JS
Kim
Ji-Eun
JE
Sapkota
Kumar
K
Shen
Ming-Hua
MH
Kim
Seung
S
Chun
Hong-Sung
HS
Yoo
Jin-Cheol
JC
Choi
Han-Suk
HS
Kim
Myung-Kon
MK
Kim
Sung-Jun
SJ
D000363:Agaricales / Q000201:enzymology*; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D025282:Mycelium / Q000201:enzymology*; D013696:Temperature
D005343:Fibrinolytic Agents; C505389:AMMP protein, Armillaria mellea; D045726:Metalloproteases