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Coagulant thrombin-like enzymes from the venoms of Brazilian and Peruvian bushmaster (Lachesis muta muta) snakes.
- Source :
-
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology [Comp Biochem Physiol B Biochem Mol Biol] 2003 Oct; Vol. 136 (2), pp. 255-66. - Publication Year :
- 2003
-
Abstract
- Two isoforms of a thrombin-like enzyme designated TLE-B and TLE-P were purified from the venoms of Lachesis muta muta (bushmaster) snakes captured in two different geographical localities, Manaus (Brazil) and Pucallpa (Perú). TLE-B and TLE-P showed Mr values of 44000 and 43000 under reducing conditions on SDS-PAGE, which decreased to 27000 after deglycosylation with N-glycosidase F (PNGase F). The purified proteinases split off fibrinopeptide A rapidly from human fibrinogen and fibrinopeptide B more slowly. In addition, both enzymes released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bbeta-chain. Their specific clotting activities were equivalent to 1000 and 900 NIH thrombin units/mg on human fibrinogen and 526 and 606 NIH thrombin units/mg on bovine fibrinogen for TLE-B and TLE-P, respectively. Kinetic properties of these enzymes were determined using representative chromogenic substrates. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Purified rabbit IgG against TLE-B reacted with both enzymes forming a continuous precipitin line on immunodiffusion. Furthermore, Western blot and indirect ELISA were used to compare the antigenic cross-reactivity for both enzymes as well as the venoms of L. muta muta and Bothrops snakes. Incubation of human alpha2-macroglobulin (alpha2-M) with each enzyme at molar ratios of 1:1, 1:2 and 1:4 enzyme:inhibitor resulted in retarding their clotting activities by approximately 12 times, whereas their amidolytic activities were not affected. However, the Mr 180000 subunits of alpha2-M were not cleaved by these enzymes, suggesting that alpha2-M inhibits TLEs by steric hindrance. Similarly, inhibitions of their clotting activities were obtained using high concentrations of rabbit IgG (40 microg, corresponding to molar ratio enzyme:inhibitor of 1:2) against TLE-B.
- Subjects :
- Animals
Brazil
Cattle
Chromatography, Affinity
Chromatography, Gel
Cross Reactions
Crotalid Venoms immunology
Enzyme Inhibitors pharmacology
Fibrinogen chemistry
Fibrinogen metabolism
Humans
Immunohistochemistry
Isoenzymes chemistry
Isoenzymes metabolism
Kinetics
Metalloendopeptidases pharmacology
Peptide Fragments chemistry
Peptide Fragments metabolism
Peptide Mapping
Peru
Rabbits
Thrombin antagonists & inhibitors
Thrombin chemistry
Trypsin metabolism
alpha-Macroglobulins pharmacology
Crotalid Venoms enzymology
Thrombin isolation & purification
Thrombin metabolism
Viperidae
Subjects
Details
- Language :
- English
- ISSN :
- 1096-4959
- Volume :
- 136
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology
- Publication Type :
- Academic Journal
- Accession number :
- 14529751
- Full Text :
- https://doi.org/10.1016/s1096-4959(03)00202-1