Continuing studies will be made of the biochemical mechanisms by which snake venom proteinases retain or regain activity in the presence of molar excesses of plasma proteinase inhibitors (PPI), the major host defense against exogenous proteolytic activity. The active modified C1 esterase inhibitor (C1INH) formed by Crotalus atrox Alpha protease and by C. adamanteus Proteinase H, the hemorrhagic proteinase from this venon will be studied and reaction products isolated and characterized to determine the regions of the C1INH molecule which are susceptible to limited proteolysis. The modified inhibitor will be isolated in homogenous condition, and the stoichiometry of complex formation between modified C1INH and plasmin, kallikrein and C1 esterase determined and compared to that of intact C1INH. Stability of the complexes will be studied by electrophoresis and assays for reappearance of proteolytic activity. Susceptibility of modified C1INH to leucocyte elastase will be determined and compared with intact C1INH to establish whether modification by exogenous venom proteinases enhances C1INH inactivation by endogenous proteinases. Alpha1 antichymotrypsin (Alpha1X) was the only PPI studied which was inactivated by Elapid venoms, and the most active venom, Dendroaspis angusticeps, will be fractionated and the proteinase which inactivates Alpha1 X purified and characterized. Possible effects on other PPI will be examined to determine whether this proteinase is indeed specific for Alpha1X. Studies of the inactivation reaction, including isolation of products, will be made and compared to Alpha1X inactivation by C. atrox Alpha protease. High molecular weight venom proteinases (C. adamanteus Proteinases H and B, and C. atrox Gamma protease) are now available in nearly homogeneous form. The pure enzymes will be incubated with Alpha2-macroglobulin (Alpha2M) at varying ratios, residual proteinase and inhibitor activity measured, and the reaction monitored electrophoretically to determine the extent of interaction with Alpha2M and the stability of any complexes formed, and to show how venom proteinases retain activity in the presence of Alpha2M. These studies should increase understanding of the role of proteolysis in snakebite pathogenesis by demonstrating that enzymatc inactivation of PPI is a mechanism by which trace amounts of venom proteinases can effectively disrupt the PPI system. Enzymatic inactivation of PPI by specific venom proteinases could also prove useful in animal model systems for determining the physiological roles of PPI and for studying diseases, e.g., emphysema, involving deficiencies of a specific PPI.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL022996-09
Application #
3337102
Study Section
Toxicology Study Section (TOX)
Project Start
1978-04-01
Project End
1987-08-31
Budget Start
1986-04-01
Budget End
1987-08-31
Support Year
9
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Roswell Park Cancer Institute Corp
Department
Type
DUNS #
City
Buffalo
State
NY
Country
United States
Zip Code
14263
Gomis-Ruth, F X; Meyer, E F; Kress, L F et al. (1998) Structures of adamalysin II with peptidic inhibitors. Implications for the design of tumor necrosis factor alpha convertase inhibitors. Protein Sci 7:283-92
Bode, W; Kress, L F; Meyer, E F et al. (1994) The crystal structure of adamalysin II, a zinc-endopeptidase from the snake venom of the eastern diamondback rattlesnake Crotalus adamanteus. Braz J Med Biol Res 27:2049-68
Catanese, J J; Kress, L F (1993) Opossum serum alpha 1-proteinase inhibitor: purification, linear sequence, and resistance to inactivation by rattlesnake venom metalloproteinases. Biochemistry 32:509-15
Gomis-Ruth, F X; Kress, L F; Bode, W (1993) First structure of a snake venom metalloproteinase: a prototype for matrix metalloproteinases/collagenases. EMBO J 12:4151-7
Grams, F; Huber, R; Kress, L F et al. (1993) Activation of snake venom metalloproteinases by a cysteine switch-like mechanism. FEBS Lett 335:76-80
Catanese, J J; Kress, L F (1992) Isolation from opossum serum of a metalloproteinase inhibitor homologous to human alpha 1B-glycoprotein. Biochemistry 31:410-8
Kress, L F (1986) Inactivation of human plasma serine proteinase inhibitors (serpins) by limited proteolysis of the reactive site loop with snake venom and bacterial metalloproteinases. J Cell Biochem 32:51-8
Kurecki, T; Kress, L F (1985) Purification and partial characterization of the hemorrhagic factor from the venom of Crotalus adamanteus (eastern diamondback rattlesnake). Toxicon 23:657-68
Kruzel, M; Kress, L F (1985) Separation of Crotalus atrox (western diamondback rattlesnake) alpha-proteinase from serine proteinase and hemorrhagic factor activities. Anal Biochem 151:471-8
Catanese, J J; Kress, L F (1985) Enzymatic digestion of human plasma inter-alpha-trypsin inhibitor by snake venom metalloproteinases. Comp Biochem Physiol B 80:507-12

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