PURIFICATION AND CHARACTERIZATION OF AN SDS-ACTIVATED FIBRINOLYTIC ENZYME FROM EISENIA-FETIDA

A sodium-dodecyl-sulfate activated fibrinolytic enzyme from Eisenia fe tida (the E. fetida enzyme) was purified by chromatography on DEAE Sep harose, Sephadex G-75, and Phenyl Sepharose 4. It (M-r = 45 kDa) was c omposed of two subunits (M-r = 26 kDa and M-r = 18 kDa) held together by hydrophobic interactions. The enzyme displayed four activities when we used fibrin plates to detect the proteolytic activity. These were designated as CFPg (complete fibrinolysis in the plasminogen-rich plat e), uCFPg (uncompleted fibrinolysis in the plasminogen rich Flare), CF (complete fibrinolysis in the plasminogen-free Flare), and uCF (uncom pleted fibrinolysis in the plasminogen-free plate). SDS activated CFPg and rendered the enzyme more sensitive to some inhibitors. Leupeptin, chymostatin, pepstatin, aprotinin, phenylmethylsulfonyl fluoride, and dithiothreitol had no effect on uCF. Pepstatin stimulated CFPg and uC FPg, while E-64, a thiol inhibitor, activated uCFPg and uCF. The N-ter minal sequence of the large subunit was analyzed and compared with som e known proteins. The large subunit alone had catalytic activity, whil e the small subunit did not. Using plasminogen as the substrate for de fining peptide bond specificity, the E. fetida enzyme was observed to cleave the carboxyl side of basic amino acids, small neutral amino aci ds, and Met residue. (C) 1991 Elsevier Science Inc.