Purification and characterisation of two hemorrhagic metalloproteinases from the venom of the long-nosed viper, Vipera ammodytes ammodytes.
Two hemorrhagic proteins, VaH1 and VaH2, have been purified from Vipera ammodytes ammodytes venom. They are monomeric glycoproteins of an apparent molecular mass of 70kDa and multiple isoelectric points around pH 5.5. Both molecules are proteolytically active against azocasein as substrate. VaH1, which was characterised in detail, showed maximum activity at pH 7.5. Ethylenediaminetetraacetic acid eliminated the proteolytic as well as the hemorrhagic activity of VaH1 while iodoacetamide, phenylmethylsulfonyl fluoride and pepstatin A, inhibitors of cysteine, serine and aspartic proteinases respectively, had no effect. VaH1 is therefore a metalloproteinase whose hemorrhagic activity is very likely the result of its proteolytic activity. VaH1 is a fibrinogenase, hydrolysing exclusively the Aalpha-chain of fibrinogen. In the B-chain of insulin it cleaved with a high preference the bond between Ala(14) and Leu(15). Based on its molecular mass, VaH1 (as well as VaH2) is a Class P-III metalloproteinase. Partial amino acid sequences of its CNBr fragments demonstrated a high level of identity with the reprolysin subfamily of zinc-metalloproteinases.
Leonardi
Adrijana
A
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, 1000, Ljubljana, Slovenia.
Gubensek
Franc
F
Krizaj
Igor
I
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000032:analysis / Q000302:isolation & purification*; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism; D008666:Metalloendopeptidases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014757:Viper Venoms / Q000201:enzymology*; D017819:Viperidae
D002364:Caseins; D004791:Enzyme Inhibitors; D005343:Fibrinolytic Agents; D007328:Insulin; D014757:Viper Venoms; C026018:azocasein; D005340:Fibrinogen; D004492:Edetic Acid; D008666:Metalloendopeptidases