Glycosylated trypsin-like proteases from earthworm Eisenia fetida.
Although groups of earthworm proteases have been found by several laboratories, it is still unclear how many of the isolated trypsin-like fibrinolytic enzymes are in glycosylated form. Here, eight glycosylated fibrinolytic proteases (EfP-0-1, EfP-0-2, EfP-I-1, EfP-I-2, EfP-II-1, EfP-II-2, EfP-III-1 and EfP-III-2) were isolated from an earthworm species (Eisenia fetida) through a stepwise-purification procedure: ammonium sulfate precipitation, affinity chromatography on a Sepharose-4B column coupled with soybean trypsin inhibitor (SBTI), and ionic chromatography with a DEAE-Cellulose-52 column. Among the eight purified trypsin-like glyco-proteases, EfP-0-2 and EfP-II-2 were newly isolated isozymes. Glycoprotein staining of the proteases on native-PAGE with a Schiff's reagent (sodium meta-periodate) revealed that the eight proteases were glycoproteins. Measurements of the glycan content with sodium meta-periodate and glycoprotein-test reagent showed that these proteases had different carbohydrate contents. Dot-blotting assay with ConA suggested the oligosaccharides were composed of mannose residues.
Wu
Jin Xia
JX
College of Life Science, Hebei University, 180 Wusi East Road, Baoding City 071002, Hebei, China.
Zhao
Xiao Yu
XY
Pan
Rong
R
He
Rong Qiao
RQ
D000595:Amino Acid Sequence; D000818:Animals; D050260:Carbohydrate Metabolism; D004789:Enzyme Activation; D005340:Fibrinogen / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000201:enzymology*; D010434:Pepsin A / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D006023:Glycoproteins; D007527:Isoenzymes; D005340:Fibrinogen; D014357:Trypsin; D010434:Pepsin A