Metalloproteinase with factor X activating and fibrinogenolytic activities from Vipera berus berus venom.
We have previously shown that Vipera berus berus venom contains several factor X activating enzymes. In the present study we have investigated one of them. The enzyme was separated from venom by gel filtration on Sephadex G-100 superfine and chromatography on agarose HPS-7 and phenyl-agarose. The enzyme is a glycosylated metalloproteinase containing hexoses, hexosamines and neuraminic acid. The purified factor X activating enzyme consists of two equal chains (59 kDa). The specificity studies have shown that enzyme is nonspecific factor X activating proteinase hydrolysing also proteins such as azocasein, gelatin and fibrinogen. The enzyme hydrolyses oxidized insulin B-chain at the positions Ala(14)-Leu(15) and Tyr(16)-Leu(17) but it is inactive on fibrin, plasminogen and prothrombin. We used 8-10 amino acid residues containing peptides, which reproduce the sequence around the cleavage sites in factor X, factor IX and fibrinogen, as potential substrates for enzyme. Cleavage products of peptide hydrolysis were determined by MALDI-TOF MS. The peptide Asn-Asn-Leu-Thr-Arg-Ile-Val-Gly-Gly-factor X fragment was cleaved by enzyme at positions Leu(3)-Thr(4) and Arg(5)-Ile(6). The fibrinogen peptide fragment Glu-Tyr-His-Thr-Glu-Lys-Leu-Val-Thr-Ser was hydrolysed at position Lys(6)-Leu(7).
Samel
Mari
M
National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia.
Vija
Heiki
H
Subbi
Juhan
J
Siigur
Jüri
J
D000818:Animals; D005170:Factor X / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D045726:Metalloproteases / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014757:Viper Venoms / Q000201:enzymology*; D017819:Viperidae / Q000378:metabolism*
D010455:Peptides; D014757:Viper Venoms; D005170:Factor X; D005340:Fibrinogen; D045726:Metalloproteases