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Chemical structure and biochemical action of staphylococcal α-toxin
4th International Symposium on Animal, Plant and Microbial Toxins
129
Endotoxin and biogenic amines cause similar disturbances in the capillary bed. The changes that are observed in the content of the vessels, the vessel wall, and the perivascular region are the following: slowing down of the blood stream, degranulation of perivascular mast cells, granulocytosis, wall adhering granulocytes, plasma skimming, rouleaux-formation of erythrocytes, reduction in plasticity of many erythrocytes, acanthocytes, acanthocytosis, appearance of spherocytes and microcytes, and formation of massive aggregates of platelets and of microthrombi . Also, occasionally, cell aggregates dissolve and microemboli form new thrombi. Swelling of pericytes, endothelial, and periendothelial cells is observed, and dissocation and deformation of the endothelium cells occur. By means of contact of the vessel contents with collagen, there is an additional activation of the coagulation system by factor XIL. The changes of the epithelial lining and the wall adhering cells enhance the narrowing of the vessel lumen. Prestasis and occasionally stasis occur . One observes increased swelling of the endothelial and periendothelial cells, increased permability of the vessel wall, passage of plasma and occasionally blood cells, especially erythrocytes, through the endothelium and massive microbleedings. While stasis is observed in the nitritive capillaries, in regions where arteriolar-venular shunts exist the flow continues. The systemic blood pressure may therefore remain unchanged during this phase, although the severe disturbances described occurin the capillary bed. Three-dimensional scanning electron microscopy was used to study the changing plasticity of the blood cells and to determine the time in which microthrombi with fibrin are formed . After pretreatment with detoxified endotoxin no changes in the capillary bed occur when endotoxin is administered, and the animals survive lethal doses of endotoxins . Also, the severe disturbances of the microcirculation after experimental bum trauma are mitigated after pretreatment with endotoxid or fail to appear. In addition, following endotoxid administration, the mortality of whole body X-irradiation (WBI) in mice is reduced from LD 70/30 to ID 5. Haematological and histological studies in these animals at various time intervals showed increased cellularity of the bone marrow, increased numbersof granulocyte precursors, and increased numbers of circulating mature granulocytes as compared to animals receiving WBI alone. Bacteremia was also observed in endotoxoid-treated mice ; however, the usual consequences of bacteremia did not occur in these animals. Species differences and comparisons of the effect of endotoxins to the effect of suspensions of Gram-negative bacteria are discussed. B. and UxsASCtEK, R., Institut fit- Hygiene und Medizinische Mikrobiologie der Universität Heidelberg, Klinikum, 68 Mannheim, Germany. ÜRBAscfK,
Film: EFFECT OF ENDOTOXINS AND BIOGENIC AMINES ON THE CAPILLARY BED M. and KATo, L, The Institute of Medical Science, The University of Tokyo, P.O . Takanawa, Minato-ku, Tokyo 108, Japan.
WATANAm,
CIiEMICAL STRUCTURE AND BIOCHEMICAL ACTION OF STAPHYLOCOCCAL a-TOXIN Although highly purified staphylococcal a-toxin has been obtained, which by a number of criteria behaved as a single protein, none of the methods has resulted thus farin crystallization. Purified a-toxin was obtained from the culture supernatant in growing Staphylococcus aureus (strain Wood 46) using the followprocedures : precipitation with zinc chloride, gel-flltration on Sephadex G-25, zone electrophoresis on starch and pevikon, and column chromatography on CM-Sephadex C-50. Crystallization of a-toxin was achieved by dialysis a purified preparation after zone electrophoresis on pevikon against a saturated ammonium sulfate solution, pH 7-0 at 4°C for 3 days . A white precipitate formed in a Visking cellulose tube was collected by gentle centrifugation and dissolved with a small volume of 0-05 M phosphate buffer, pH 7-0. Recrystallization of the toxic protein was carried out by 2 x dialysis against a large volume of saturated ammonium sulfate solution of pH 7-0. Examination by microscope revealed the crystalline nature of the precipitate. The crystals tended to assume plate or rhomboidal forms. Crystallized a-toxin showed a single band both by polyacrylamide gel electrophoresis in the presence of 0-1 per cent sodium dodecyl sulfate (SDS) and by agar gel immunodiffusion test against rabbit antiserum from the purified a-toxin immunization . The preparation thus obtained revealed a 435-fold increase in specific hemolyticactivity (HU) and approximately 24 per cent of thecrude toxin was recovered in pure form . The isoelectric point (pI) of the crystalline toxin was determined by electrofocusing analysis . A single peak fraction, accounting for 95 per cent of the recovered hemolytic activity, had a pI of 7-98 ± 0-05. The molecular weight of the crystalline a-toxin has been calculated to be approximately 36,000 f 2000 by SDSpolyacrylamide gel electrophoresis. The noteworthy aspects of the amino acid composition of the crystalline a-toxin protein consisting of 267 amino acid residues are the complete absence of half-cystine and the high content of aspartic acid, lysine and threonine. Theresults of determination forthe presence of carbohydrate by the anthrone reaction andphenol-H,SO, reaction show that the toxin contains less than 1 per cent carbohydrate as glucose. And the crystalline a-toxin does not appear to have proteolytic enzyme activity. rOXICON 1975 Vol. 13
130
4th International Symposium on Animal, Plant and Microbial Toxins
Themost convenient and sensitive method of assaying a-toxin is a measurement of its hemolyticactivity. Hemolytic activity against 2 per cent suspension of rabbit erythrocytes was determined by measurement of 50 per cent hemolysis at 541 nm in a Hitachi spectrophotometer. One mg of the crystalline toxin is calculated to have 1 x 105 hemolytic units (HU) . Although the specific activity of any given preparation is not always correlated to the degree of purity, published results for the hemolytic unit in terms of micrograms of protein vary between 0-008 gg (LomiNsxt et al., 1963) and 0-05 Rg (BERNHLRAIIt et al., 1963 ; ARBuTHNOTT et al., 1967 ; Sic et al., 1973). Our value for this crystalline a-toxin was 0-01 pg per HU. Crystalline a-toxin possesses dermonecrotic and lethal activities besides hemolytic activity . The dermonecrotic dose forrabbits was approximately 0-03 gg and the Li sa for mice was approximately 1-Oltg . Effect of crystalline a-toxin on the hemolysis of rabbit reticulocytes was investigated. HU of reticulocytes induced by subcutaneous injections of pheny1hydrazine decreased approximately half in comparison with the HU of normal erythrocytes. The mode of action of crystallinea-toxin on hemolysis of rabbit erythrocytes will be discussed. WATANABE, M., YASOHARA, T. and NAKAnmA, T., Institute of Pharmacological Science, Hiroshima University, Hiroshima, Japan. NEW BRADYKIN IN ANALOGUES IN WASP (POLISTES ROTHNEYI IWATAI) VENOM We have demonstrated in the Japanese wasp (Polistes rothneyi twatai) the presence of three major and at least two minor active principles which contract the rat uterus, guinea pig ileum and lowered blood pressure. The three major active principles (P-II-1, P-II-2, P-III) were further purified by SE Sephadex column chromatography and/or gel permeation chromatography . P-II-1 and P-III were susceptible to digestion with chymotrypsin but not by trypsin, while P-II-2 did not lose its activity by treatment with these enzymes, and was identified as serotonin by u.v. spectrum and chromatographic behavior of its dansyl derivative . Amino acid analysis of P-II-1 showed the following composition ; Arg,, Thrl, Pro,, Glyl, Plie s, which was similar to that of bradykinin, except 1 mole of serine was replaced by threonine. The peptide contained the same N-terminal amino acid as bradykinin. Dansylated P-1I-1 was cleared by chymotrypsin treatment, and produced the same DNS-fragment as bradykinin . These results indicate the following structure for P-II-1 : Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg . The more basic peptide, P-III, which contained arginine and alanine in addition to the amino acid composition of P-II-1, was converted to P-II-1 and Ala-Arg by digestion with trypsin. The N-terminal amino acid was alanine and sequencing study by Edman degradation showed Ala-Arg-Arg-Pro- at the N-terminus. These findings indicate the following structure for P-III: Ala-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg. These results show that active peptides in wasp venom may be different in each species and occurrence of Thr'-bradykinin analogues is widespread in the animal kingdom. WEBER, M., Institut Pasteur, Paris, France . BINDING OF A TRITIATED a TOXIN FROM NAJA NIGRICOLLIS TO THE CHOLUVERGIC RECEPTORS FROM ELECTROPHORUS AND TORPEDO ELECTRIC ORGANS al Isotoxin from NiVa nigricollis venom was tritiated specifically on Histidine 4 by iodination and subsequent catalytic deshalogenation in the presence of tritium gas (MENEz et al., 1971). The binding of this ('H) a toxin on membrane fragments purified from the electric tissues of Electrophorus electricus and Torpedo marntorata was studied by an ultrafiltration method. On both preparations of membrane fragments, and in the domain of concentrations explored (0-2-2 nM), ('H) a toxin binds to one class of sites with a very high affinity : Ka= 5 x 101° M-1. The kinetics of binding is compatible with a simple bimolecular mechanism, with a second order rate constant kl = 3 x 10 6 mol per sec. Half reversion in the presence of an excess of unlabelled toxin occurs in 55-60 hr (WEBER and CI3AN(JEUx,1974). Both cholinergic agonists and antagonists decrease the initial rate of ('H) a toxin binding on the membrane fragments, and complete protection occurs at high concentrations of cholinergic effectors . Comparison of the protecting effect of cholinergic effectors against (3H) a toxin binding with their specific binding on . the cholinergic receptor site measured directly leads to the conclusion that in first approximation both a toxin andcholinergic effectors bind to the same site (or at least to overlapping sites) in a mutually exclusive manner. The a toxin from N. nigricollis thus binds with a high specificity to the cholinergic (nicotinic) receptor site . By themethod of protection against (I'M a toxin binding, we determined the affinities for the cholinerigc receptor site of a large number of agonists and antagonists. The pharmacology of the receptors from ElectroTOXICON 1975 Vol. 13
129
Endotoxin and biogenic amines cause similar disturbances in the capillary bed. The changes that are observed in the content of the vessels, the vessel wall, and the perivascular region are the following: slowing down of the blood stream, degranulation of perivascular mast cells, granulocytosis, wall adhering granulocytes, plasma skimming, rouleaux-formation of erythrocytes, reduction in plasticity of many erythrocytes, acanthocytes, acanthocytosis, appearance of spherocytes and microcytes, and formation of massive aggregates of platelets and of microthrombi . Also, occasionally, cell aggregates dissolve and microemboli form new thrombi. Swelling of pericytes, endothelial, and periendothelial cells is observed, and dissocation and deformation of the endothelium cells occur. By means of contact of the vessel contents with collagen, there is an additional activation of the coagulation system by factor XIL. The changes of the epithelial lining and the wall adhering cells enhance the narrowing of the vessel lumen. Prestasis and occasionally stasis occur . One observes increased swelling of the endothelial and periendothelial cells, increased permability of the vessel wall, passage of plasma and occasionally blood cells, especially erythrocytes, through the endothelium and massive microbleedings. While stasis is observed in the nitritive capillaries, in regions where arteriolar-venular shunts exist the flow continues. The systemic blood pressure may therefore remain unchanged during this phase, although the severe disturbances described occurin the capillary bed. Three-dimensional scanning electron microscopy was used to study the changing plasticity of the blood cells and to determine the time in which microthrombi with fibrin are formed . After pretreatment with detoxified endotoxin no changes in the capillary bed occur when endotoxin is administered, and the animals survive lethal doses of endotoxins . Also, the severe disturbances of the microcirculation after experimental bum trauma are mitigated after pretreatment with endotoxid or fail to appear. In addition, following endotoxid administration, the mortality of whole body X-irradiation (WBI) in mice is reduced from LD 70/30 to ID 5. Haematological and histological studies in these animals at various time intervals showed increased cellularity of the bone marrow, increased numbersof granulocyte precursors, and increased numbers of circulating mature granulocytes as compared to animals receiving WBI alone. Bacteremia was also observed in endotoxoid-treated mice ; however, the usual consequences of bacteremia did not occur in these animals. Species differences and comparisons of the effect of endotoxins to the effect of suspensions of Gram-negative bacteria are discussed. B. and UxsASCtEK, R., Institut fit- Hygiene und Medizinische Mikrobiologie der Universität Heidelberg, Klinikum, 68 Mannheim, Germany. ÜRBAscfK,
Film: EFFECT OF ENDOTOXINS AND BIOGENIC AMINES ON THE CAPILLARY BED M. and KATo, L, The Institute of Medical Science, The University of Tokyo, P.O . Takanawa, Minato-ku, Tokyo 108, Japan.
WATANAm,
CIiEMICAL STRUCTURE AND BIOCHEMICAL ACTION OF STAPHYLOCOCCAL a-TOXIN Although highly purified staphylococcal a-toxin has been obtained, which by a number of criteria behaved as a single protein, none of the methods has resulted thus farin crystallization. Purified a-toxin was obtained from the culture supernatant in growing Staphylococcus aureus (strain Wood 46) using the followprocedures : precipitation with zinc chloride, gel-flltration on Sephadex G-25, zone electrophoresis on starch and pevikon, and column chromatography on CM-Sephadex C-50. Crystallization of a-toxin was achieved by dialysis a purified preparation after zone electrophoresis on pevikon against a saturated ammonium sulfate solution, pH 7-0 at 4°C for 3 days . A white precipitate formed in a Visking cellulose tube was collected by gentle centrifugation and dissolved with a small volume of 0-05 M phosphate buffer, pH 7-0. Recrystallization of the toxic protein was carried out by 2 x dialysis against a large volume of saturated ammonium sulfate solution of pH 7-0. Examination by microscope revealed the crystalline nature of the precipitate. The crystals tended to assume plate or rhomboidal forms. Crystallized a-toxin showed a single band both by polyacrylamide gel electrophoresis in the presence of 0-1 per cent sodium dodecyl sulfate (SDS) and by agar gel immunodiffusion test against rabbit antiserum from the purified a-toxin immunization . The preparation thus obtained revealed a 435-fold increase in specific hemolyticactivity (HU) and approximately 24 per cent of thecrude toxin was recovered in pure form . The isoelectric point (pI) of the crystalline toxin was determined by electrofocusing analysis . A single peak fraction, accounting for 95 per cent of the recovered hemolytic activity, had a pI of 7-98 ± 0-05. The molecular weight of the crystalline a-toxin has been calculated to be approximately 36,000 f 2000 by SDSpolyacrylamide gel electrophoresis. The noteworthy aspects of the amino acid composition of the crystalline a-toxin protein consisting of 267 amino acid residues are the complete absence of half-cystine and the high content of aspartic acid, lysine and threonine. Theresults of determination forthe presence of carbohydrate by the anthrone reaction andphenol-H,SO, reaction show that the toxin contains less than 1 per cent carbohydrate as glucose. And the crystalline a-toxin does not appear to have proteolytic enzyme activity. rOXICON 1975 Vol. 13
130
4th International Symposium on Animal, Plant and Microbial Toxins
Themost convenient and sensitive method of assaying a-toxin is a measurement of its hemolyticactivity. Hemolytic activity against 2 per cent suspension of rabbit erythrocytes was determined by measurement of 50 per cent hemolysis at 541 nm in a Hitachi spectrophotometer. One mg of the crystalline toxin is calculated to have 1 x 105 hemolytic units (HU) . Although the specific activity of any given preparation is not always correlated to the degree of purity, published results for the hemolytic unit in terms of micrograms of protein vary between 0-008 gg (LomiNsxt et al., 1963) and 0-05 Rg (BERNHLRAIIt et al., 1963 ; ARBuTHNOTT et al., 1967 ; Sic et al., 1973). Our value for this crystalline a-toxin was 0-01 pg per HU. Crystalline a-toxin possesses dermonecrotic and lethal activities besides hemolytic activity . The dermonecrotic dose forrabbits was approximately 0-03 gg and the Li sa for mice was approximately 1-Oltg . Effect of crystalline a-toxin on the hemolysis of rabbit reticulocytes was investigated. HU of reticulocytes induced by subcutaneous injections of pheny1hydrazine decreased approximately half in comparison with the HU of normal erythrocytes. The mode of action of crystallinea-toxin on hemolysis of rabbit erythrocytes will be discussed. WATANABE, M., YASOHARA, T. and NAKAnmA, T., Institute of Pharmacological Science, Hiroshima University, Hiroshima, Japan. NEW BRADYKIN IN ANALOGUES IN WASP (POLISTES ROTHNEYI IWATAI) VENOM We have demonstrated in the Japanese wasp (Polistes rothneyi twatai) the presence of three major and at least two minor active principles which contract the rat uterus, guinea pig ileum and lowered blood pressure. The three major active principles (P-II-1, P-II-2, P-III) were further purified by SE Sephadex column chromatography and/or gel permeation chromatography . P-II-1 and P-III were susceptible to digestion with chymotrypsin but not by trypsin, while P-II-2 did not lose its activity by treatment with these enzymes, and was identified as serotonin by u.v. spectrum and chromatographic behavior of its dansyl derivative . Amino acid analysis of P-II-1 showed the following composition ; Arg,, Thrl, Pro,, Glyl, Plie s, which was similar to that of bradykinin, except 1 mole of serine was replaced by threonine. The peptide contained the same N-terminal amino acid as bradykinin. Dansylated P-1I-1 was cleared by chymotrypsin treatment, and produced the same DNS-fragment as bradykinin . These results indicate the following structure for P-II-1 : Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg . The more basic peptide, P-III, which contained arginine and alanine in addition to the amino acid composition of P-II-1, was converted to P-II-1 and Ala-Arg by digestion with trypsin. The N-terminal amino acid was alanine and sequencing study by Edman degradation showed Ala-Arg-Arg-Pro- at the N-terminus. These findings indicate the following structure for P-III: Ala-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg. These results show that active peptides in wasp venom may be different in each species and occurrence of Thr'-bradykinin analogues is widespread in the animal kingdom. WEBER, M., Institut Pasteur, Paris, France . BINDING OF A TRITIATED a TOXIN FROM NAJA NIGRICOLLIS TO THE CHOLUVERGIC RECEPTORS FROM ELECTROPHORUS AND TORPEDO ELECTRIC ORGANS al Isotoxin from NiVa nigricollis venom was tritiated specifically on Histidine 4 by iodination and subsequent catalytic deshalogenation in the presence of tritium gas (MENEz et al., 1971). The binding of this ('H) a toxin on membrane fragments purified from the electric tissues of Electrophorus electricus and Torpedo marntorata was studied by an ultrafiltration method. On both preparations of membrane fragments, and in the domain of concentrations explored (0-2-2 nM), ('H) a toxin binds to one class of sites with a very high affinity : Ka= 5 x 101° M-1. The kinetics of binding is compatible with a simple bimolecular mechanism, with a second order rate constant kl = 3 x 10 6 mol per sec. Half reversion in the presence of an excess of unlabelled toxin occurs in 55-60 hr (WEBER and CI3AN(JEUx,1974). Both cholinergic agonists and antagonists decrease the initial rate of ('H) a toxin binding on the membrane fragments, and complete protection occurs at high concentrations of cholinergic effectors . Comparison of the protecting effect of cholinergic effectors against (3H) a toxin binding with their specific binding on . the cholinergic receptor site measured directly leads to the conclusion that in first approximation both a toxin andcholinergic effectors bind to the same site (or at least to overlapping sites) in a mutually exclusive manner. The a toxin from N. nigricollis thus binds with a high specificity to the cholinergic (nicotinic) receptor site . By themethod of protection against (I'M a toxin binding, we determined the affinities for the cholinerigc receptor site of a large number of agonists and antagonists. The pharmacology of the receptors from ElectroTOXICON 1975 Vol. 13