Purification and characterization of six fibrinolytic serine-proteases from earthworm Lumbricus rubellus.
The six lumbrokinase fractions (F1 to F6) with fibrinolytic activities were purified from earthworm Lumbricus rubellus lysates using the procedures of autolysis, ammonium sulfate fractionation, and column chromatography. The proteolytic activities on the casein substrate of the six iso-enzymes ranged from 11.3 to 167.5 unit/mg with the rank activity orders of F2 > F1 > F5 > F6 > F3 > F4. The fibrinolytic activities of the six fractions on the fibrin plates ranged from 20.8 to 207.2 unit/mg with rank orders of F6 > F2 > F5 > F3 > F1 > F4. The molecular weights of each iso-enzyme, as estimated by SDS-PAGE, were 24.6 (F1), 26.8 (F2), 28.2 (F3), 25.4 (F4), 33.1 (F5), and 33.0 kDa (F6), respectively. The plasminogen was activated into plasmin by the enzymes. The optimal temperature of the six iso-enzymes was 50 degrees C, and the optimal pH ranged from pH 4-12. The four iso-enzymes (F1-F4) were completely inhibited by PMSF. The two enzymes (F5 and F6) were completely inhibited by aprotinin, TLCK, TPCK, SBTI, LBTI, and leupeptin. The N-terminal amino acid (aa) sequences of the first 20 to 22 residues of each fraction had high homology. All six iso-enzymes had identical aa residues 2-3 and 13-15. The N-terminal 21-22 aa sequences of the F2, F3, and F4 iso-enzymes were almost the same. The N-terminal aa sequences of F5 and F6 were identical.
Department of Biological Sciences, Konkuk University, Seoul 143-701, Korea.
Il Hwan
IH
Cho
Eui Sung
ES
Choi
Hun Gil
HG
Lim
Hyung Hoan
HH
Lee
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005337:Fibrin / Q000378:metabolism; D005342:Fibrinolysis; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009835:Oligochaeta / Q000201:enzymology*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D010958:Plasminogen / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D013696:Temperature; D013997:Time Factors
D002364:Caseins; D004791:Enzyme Inhibitors; D005343:Fibrinolytic Agents; D007527:Isoenzymes; D010664:Phenylmethylsulfonyl Fluoride; D005337:Fibrin; D010958:Plasminogen; D012697:Serine Endopeptidases