ABSTRACT
In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography on CM Sepharose. The enzyme called leucurolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH2-terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity=21.6 units/mg and 17.5 units/microg, respectively; crude venom=8.0 units/mg and 9.5 units/microg). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a cleaved the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 microg) subcutaneously into mice.
Subject(s)
Bothrops , Crotalid Venoms/enzymology , Endopeptidases , Amino Acid Sequence , Animals , Blood Platelets/metabolism , Crotalid Venoms/genetics , Endopeptidases/chemistry , Endopeptidases/isolation & purification , Endopeptidases/metabolism , Enzyme Stability , Fibrin/metabolism , Fibrinogen/metabolism , Fibronectins/metabolism , Humans , Laminin/metabolism , Membrane Glycoproteins/metabolism , Mice , Molecular Sequence Data , Plasminogen/metabolism , Sequence Alignment , Substrate SpecificityABSTRACT
The bacterial physiology of streptonigrin toxicity was further investigated. An optimal oxygen concentration for toxicity was inferred from data showing that streptonigrin at 5 micrograms/mL was rapidly lethal to aerobic cultures of Escherichia coli K12JF361, but was without effect on anaerobic cultures and was bacteriostatic to cultures inhibited in 5 atm of oxygen plus 1 atm of air (5 atm O2 plus air) (1 atm = 101.325 kPa). Escherichia coli were protected from a potentially lethal concentration of streptonigrin during anaerobic incubation, whether previously grown anaerobically, aerobically, or in 5 atm O2 plus air. Superoxide dismutase activity increased with increasing oxygen tension in the medium, but was not significantly changed by a lethal concentration of streptonigrin. Although the superoxide dismutase activity was four times greater in E. coli grown in 5 atm O2 plus air than those grown in air alone, the aerobic survival in 5 micrograms/mL streptonigrin was identical, which suggested that superoxide dismutase was not rate limiting for toxicity. Escherichia coli K12 strains deficient in glutathione (KMBL54-129, AB1157-821, and AB1157-830) were protected from streptonigrin poisoning. Dithiothreotol (5.0 mM), diamide (1 mM), methyl viologen (1 mM), and cyanide (10 mM) protected aerobic E. coli from 5 micrograms/mL streptonigrin. These data are also consistent with a model of in vivo streptonigrin toxicity that requires a favorable intracellular oxidation--reduction state and an optimal concentration of molecular oxygen.
