1887

Abstract

SUMMARY

type-A -toxin (phospholipase C) was purified on a preparative scale by a simple, rapid, two-stage procedure involving precipitation of culture-supernatant fluids with ammonium sulphate at 35 to 50% saturation, followed by isoelectric focusing in a H 4.6 gradient. Milligram yields of highly purified -toxin with 10 to 15% recovery of activity were obtained in single fractions.

Two forms of -toxin were identified by electrofocusing. The major peak of activity possessed a I of 5.49±0.06( ) and the minor, a I of 5.25( ). The former was free from detectable collagenase (I 4.54), hyaluronidase (I 4.73), θ-toxin (I 6.56) and neuraminidase. It gave a single precipitin arc on immunoelectrophoresis, but analysis by SDS polyacrylamide disk-gel electrophoresis at high protein-loading revealed a major protein component of molecular weight 53,800 and two minor protein bands. Atomic emission spectro-scopy did not detect the presence of zinc in such preparations. The latter showed a single line of identity with in Ouchterlony gel diffusion tests and contained a protein component with the same molecular weight as that of .

Fractions and both possessed hot-cold haemolytic, phospholipase-C, and lethal activities. Both hydrolysed lecithin and sphingomyelin. Electro-focusing of -toxin in the presence of 6m urea resulted in the detection of only one component, , with a I identical to . It also possessed all three biological activities of -toxin. Removal of the urea and refocusing was accompanied by the reappearance of . The occurrence and formation of could not be interpreted in terms of artefactual causes of multiple forms of proteins identified by isoelectric focusing. These studies provided evidence favouring the suggestion that and could be related as conformers, although aggregation or polymerisation could not be entirely excluded as possible alternative explanations.

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1974-02-01
2024-03-29
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References

  1. Adams M. H., Hendee E. D., Pappenheimer A. M. Jr 1947; Factors affecting the production of Clostridium welchii alpha toxin. J. exp. Med. 85:701
    [Google Scholar]
  2. Allen R. J. L. 1940; The estimation of phosphorus. Biochem. J. 34:858
    [Google Scholar]
  3. Bangham A. D., Dawson R. M. C. 1962; Electrokinetic requirements for the reaction between Cl. perfringens α-toxin (phospholipase C) and phospholipid substrates. Biochim. biophys. Acta 59:103
    [Google Scholar]
  4. Bernheimer A. W., Grushoff P. 1967; Cereolysin: production, purification and partial characterisation. J. gen. Microbiol 46:143
    [Google Scholar]
  5. Bernheimer A. W., Grushoff P., Avigad L. S. 1968; Isoelectric analysis of cytolytic bacterial proteins. J. Bact 95:2439
    [Google Scholar]
  6. Berson S. A., Yalow R. S. 1966; Deamidation of insulin during storage in the frozen state. Diabetes 15:875
    [Google Scholar]
  7. Bull H. B., Breese K., Ferguson G. L., Swenson C. A. 1964; The pYL of urea solutions. Archs Biochem. Biophys 104:297
    [Google Scholar]
  8. Burk N. F., Greenberg D. M. 1930; The physical chemistry of the proteins in nonaqueous and mixed solvents. I. The state of aggregation of certain proteins in urea-water solutions. J. biol. Chem. 87:197
    [Google Scholar]
  9. Carlstrom A. 1966; Induced heterogeneity in lactoperoxidase. Acta chem. scand. 20:1426
    [Google Scholar]
  10. Casu A., Pala V., Monacelli R., Nanni G. 1971; Phospholipase C from Clostridium perfringens: purification by electrophoresis on acrylamide-agarose gel. Ital. J. Biochem. 20:166
    [Google Scholar]
  11. Chilson O. P., Costello L. A., Kaplan N. O. 1965; Effects of freezing on enzymes. Fed. Proc. 24 (Suppl. 15) S55:
    [Google Scholar]
  12. Dawson R. M. C. 1968; In Biological membranes—physical fact and function. edited by Chapman D. London and New York: p 59
    [Google Scholar]
  13. Dawson R. M. C., Elliot D. C., Elliot W. H., Jones K. M. 1969; Preparation and composition of biochemical reagents. In Data for biochemical research. 2nd ed Oxford: Section 25, p 615
    [Google Scholar]
  14. Delaunay M., Guillaumie M., Delaunay A. 1949; fitudes sur le collagene. I. A propos des collagenases bacteriennes. Annls Inst. Pasteur 76:16
    [Google Scholar]
  15. Diner B. A. 1970; Purification and properties of Clostridium welchii phospholipase C. Biochim. biophys. Acta 198:514
    [Google Scholar]
  16. Dolby D. E., Macfarlane M. G. 1956; Variation in the toxicity of lecithinases (α-toxin) from different strains of Clostridium welchii. Br. J. exp. Path. 37:324
    [Google Scholar]
  17. Dorfmann A. 1955; Mucopolysaccharidases. In Methods in enzymology. edited by Colowick S. P., Kaplan N.O. New York and London: vol. 1, p 166
    [Google Scholar]
  18. Flatmark T., Vesterberg O. 1966; On the heterogeneity of beef heart cytochrome C. IV. Isoelectric fractionation by electrolysis in a natural pH. gradient. Acta chem., scand. 20:1497
    [Google Scholar]
  19. Florkin M., Stotz E. H. 1964; Report of the Commission on Enzymes of the International Union of Biochemistry. In Comprehensive biochemistry. Amsterdam: vol. 13: appendix B
    [Google Scholar]
  20. Gerding J. J. T., Koppers A., Hagel P., Bloemendal H. 1971; Cyanate formation in solutions of urea. II. Effect of urea on the eye lens protein a-crystallin. Biochim. biophys. Acta 243:374
    [Google Scholar]
  21. Glushkova A. I., Nenashev V. P., Golshmid V. K. 1969; The study of nutrient medium and the toxin of Cl. perfringens type A by means of gel filtration through sephadex gel. In Toxins, toxoids, and antitoxins. Proceedings of the Inter-Institute Conference, Stavropol, 1966 Moscow: p 97 (in Russian)
    [Google Scholar]
  22. Goodenough D. A., Revel J. -P. 1971; The permeability of isolated and in situ mouse hepatic gap junctions studied with enzymatic tracers. J. Cell Biol 50:81
    [Google Scholar]
  23. Habermann E. 1958; Ober α-Toxin/Lecithinase, θ-Toxin, Hyaluronidase und gelatinolytisches Prinzip des Giftes von Clostridium welchii Typ A. Naturwissenschaften 45:366
    [Google Scholar]
  24. Habermann E. 1959; Ober die Giftkomponenten des Gasbranderregers Clostridium welchii Typ A. Naunyn Schmiedebergs Arch. exp. Path. Pharmakol 235:513
    [Google Scholar]
  25. Haglund H. 1971; Isoelectric focusing in pH gradients—a technique for fractionation and characterisation of ampholytes. In Methods of biochemical analysis. edited by Glick D. New York, London, and Sydney: vol. 19, p 1
    [Google Scholar]
  26. Hanahan D. J., Turner M. B., Jayko M. E. 1951; The isolation of egg phosphatidyl choline by an adsorption column technique. J. biol. Chem. 192:623
    [Google Scholar]
  27. Heyningen W. E. van. 1941a; The biochemistry of gas gangrene toxins. 1. Estimation of the α-toxin of Cl. welchii type A. Biochem. J 35:1246
    [Google Scholar]
  28. Heyningen W. E. van. 1941b; The biochemistry of gas gangrene toxins. 2. Partial purification of the toxins of Cl. welchii type A. Separation of a and 6 toxins. Biochem. J. 35:1257
    [Google Scholar]
  29. Heyningen W. E. van, Bidwell E. 1948; The biochemistry of gas gangrene toxins. 4. The reaction between the α-toxin (lecithinase) of Clostridium welchii and its antitoxin. Biochem. J. 42:130
    [Google Scholar]
  30. Humphrey J. H. 1957; International standard for hyaluronidase. Bull. WldHlth Org. 16:291
    [Google Scholar]
  31. Ikezawa H., Yamamoto A., Murata R. 1964; Gel filtration of Clostridium perfringens α-toxin (phospholipase C). J. Biochem. Tokyo: 56:480
    [Google Scholar]
  32. Ispolatovskaya M. V. 1964; The possible existence of several exolecithinases in Clostridium welchii. Biochemistry, N. Y 29:744 (Translated from Biokhimiya, 29, 869.)
    [Google Scholar]
  33. Isopolatovskaya M. V. 1967; Zinc as a cofactor metal of lecithinase C of Clostridium perfringens. Dokl. (Proc.) Acad. Sci. U.S.S.R., Biological Sciences 173:113 (Translated from Dokl. Akad. Nauk. SSSR, 173, 1445.)
    [Google Scholar]
  34. Ispolatovskaya M. V. 1970; Role of metals in the activity of Clostridium perfringens lecithinase. Biochemistry, N. Y. 35:394 (Translated from Biokhimiya, 35, 434.)
    [Google Scholar]
  35. Ispolatovskaya M. V. 1971; Type A Clostridium perfringens toxin. In Microbial toxins— bacterial protein toxins. edited by Kadis S., Montie T. C., Ajl S. J. New York and London: vol. 2A, p 109
    [Google Scholar]
  36. Ispolatovskaya M. V., Klimacheva L. V. 1966; Effects of certain chemical compounds on lecithinase of Cl. perfringens. Biochemistry, N. Y 31:429 (Translated from Biokhimiya, 31, 491.)
    [Google Scholar]
  37. Ispolatovskaya M. V., Levdikova G. A. 1962; Further purification and immunochemical properties of lecithinase, the lethal factor of B. perfringens toxin. Biochemistry, N.Y. 27: 533 (Translated from Biokhimiya, 27, 631.)
    [Google Scholar]
  38. Ispolatovskaya M. V., Levdikova G. A., Larina I. A. 1961; Separation of the lecithinase and collagenase activities of the Cl. perfringens toxin by electrophoresis on starch. Biochemistry, N. Y 26:66 (Translated from Biokhimiya, 26, 77.)
    [Google Scholar]
  39. Ispolatovskaya M. V., Levdikova G. A., Larina I. A. 1962; The separation of lecithinase, collagenase and hyaluronidase activity of B. perfringens toxin by ion exchange cellulose. Biochemistry, N. Y 27:65 (Translated from Biokhimiya, 27, 82.)
    [Google Scholar]
  40. Ito A. 1968; Alpha toxoid of Clostridium perfringens. I. Purification and toxoiding of alpha toxin of Cl. perfringens. Jap. J. med. Sci. Biol 21:379
    [Google Scholar]
  41. Josephson R. V., Maheswaren S. K., Morr C. V., Jenness R., Lindorfer R. K. 1971; Effect of urea on pis of ampholytes and casein in isoelectric focusing. Analyt. Biochem. 40:476
    [Google Scholar]
  42. Kunitz M. 1950; Crystalline desoxyribonuclease. I. Isolation and general properties. Spectrophotometric method for the measurement of desoxyribonuclease activity. J. gen. Physiol 33:349
    [Google Scholar]
  43. Kushner D. J. 1957; An evaluation of the egg-yolk reaction as a test for lecithinase activity. J. Bact. 73:297
    [Google Scholar]
  44. Levy M. 1958; Titration of bovine serum albumin in urea and in formaldehyde solutions. C. r. Trav. Lab Carlsberg: (chim.) 30:301.
    [Google Scholar]
  45. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. 1951; Protein measurement with the Folin phenol reagent. J. biol. Chem 193:265
    [Google Scholar]
  46. Lynch K. L., Moskowitz M. 1968; Relationship of route of inoculation and the nature of toxin preparation to bioassay of Clostridium perfringens α-toxin in mice. J. Bact. 96:1920
    [Google Scholar]
  47. Macchia V., Pastan I. 1967; Action of phospholipase C on the thyroid. Abolition of the response to thyroid-stimulating hormone. J. biol. Chem 242:1864
    [Google Scholar]
  48. Macchia V., Pastan I. 1968; Effect of sphingomyelinase from Clostridium perfringens on the metabolic activity and phospholipid composition of thyroid slices. Biochim. biophys. Acta 152:704
    [Google Scholar]
  49. Macchia V., Bates R. W., Pastan I. 1967; The purification and properties of a thyroid-stimulating factor isolated from Clostridium perfringens. J. biol. Chem 242:3726
    [Google Scholar]
  50. Macchia V., Tamburrini O., Pastan I. 1970; Role of lecithin in the mechanism of TSH action. Endocrinology 86:787
    [Google Scholar]
  51. Macfarlane M. G. 1942; The specificity of the lecithinase present in Cl. welchii toxin. Biochem. J 36:iii
    [Google Scholar]
  52. Macfarlane M. G. 1948; The biochemistry of bacterial toxins. 2. The enzymic specificity of Clostridium welchii lecithinase. Biochem. J 42:587
    [Google Scholar]
  53. Macfarlane M. G., Knight B. C. J. G. 1941; The biochemistry of bacterial toxins. 1. The lecithinase of Clostridium welchii toxins. Biochem. J 35:884
    [Google Scholar]
  54. Macfarlane R. G., Oakley C. L., Anderson C. G. 1941; Haemolysis and the production of opalescence in serum and lecithovitellin by the α-toxin of Clostridium welchii. J. Path. Bact 52:99
    [Google Scholar]
  55. McNiven A. C., Owen P., Arbuthnott J. P. 1972; Multiple forms of staphylococcal alpha-toxin. J. med. Microbiol 5:113
    [Google Scholar]
  56. Matsumoto M. 1961; Studies on phospholipids. II. Phospholipase activity of Clostridium perfringens toxin. J. Biochem Tokyo: 49:23
    [Google Scholar]
  57. Maurer H. R. 1971; The polyacrylamide gel. In Disc gel electrophoresis and related techniques of polyacrylamide gel electrophoresis. edited by Fischbeck K. 2nd ed Berlin and New York: p 1
    [Google Scholar]
  58. Meduski J., Volkova M. S. 1957; Radiation determination of molecular weight of phospholipase C—Cl. perfringens, welchii. Dokl. Akad. Nauk SSSR 116:266 (in Russian)
    [Google Scholar]
  59. Meduski J., Volkova M. S. 1958; Determination of the molecular weight of phospholipase C in Clostridium perfringens by means of gamma-ray inactivation. Medycyna dosw. Mikrobiol. 10:246 (in Polish) (abstracted in Biol. Abstr., 1960, 35, no. 38506)
    [Google Scholar]
  60. Mollby R., Nord C.-E., Wadstrom T. 1973; Biological activities contaminating preparations of phospholipase C (α-toxin) from Clostridium perfringens. Toxicon 11:139
    [Google Scholar]
  61. Murata R., Yamada T., Kameyama S. 1956; Production of alpha toxin of Clostridium perfringens. I. Preparation of the reproducible peptone medium for the production of toxin of high potency. Jap. J. med. Sci. Biol 9:81
    [Google Scholar]
  62. Murata R., Yamamoto A., Soda S., Ito A. 1965; Nutritional requirements of Clostridium perfringens PB6K for alpha toxin production. Jap. J. med. Sci. Biol 18:189
    [Google Scholar]
  63. Nagler F. P. O. 1939; Observations on a reaction between the lethal toxin of Clostridium welchii (type A) and human serum. Br. J. exp. Path 20:473
    [Google Scholar]
  64. Pastan I., Macchia V., Katzen R. 1968; A phospholipase specific for sphingomyelin from Clostridium perfringens. J. biol. Chem 243:3750
    [Google Scholar]
  65. Pejaudder L., Audran R., Steinbuch M. 1971; Molecular modification of ceruloplasmin by isoelectric focusing. Biochim. biophys. Acta 229:437
    [Google Scholar]
  66. Roth F. B., Pillemer L. 1953; The separation of alpha toxin (lecithinase) from filtrates of Clostridium welchii. J. Immunol 70:533
    [Google Scholar]
  67. Roth F. B., Pillemer L. 1955; Purification and some properties of Clostridium welchii type A theta toxin. J. Immunol 75:50
    [Google Scholar]
  68. Saito K., Mukoyama K. 1968; Enzymatic hydrolysis of phosphosphingolipids by Clostridium perfringens phospholipase C. Biochim. biophys. Acta 164:596
    [Google Scholar]
  69. Seiffert G. 1939; Eine Reaktion menschlicher Sera mit Perfringenstoxin. Z. ImmunForsch. exp. Ther 96:515
    [Google Scholar]
  70. Shapiro A. L., Vinuela E., Maizel J. V. Jr 1967; Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. biophys. Res. Commun 28:815
    [Google Scholar]
  71. Shemanova G. F., Vlasova E. V., Tsvetkov V. S. 1965; Isolation and properties of purified lecithinase C of Cl. perfringens. Biochemistry, N. Y 30:634 (Translated from Biokhimiya, 30, 739.)
    [Google Scholar]
  72. Shemanova G. F., Vlasova E. V., Tsvetkov V. S., Logunov A. I., Levin F. B. 1968; Extraction and properties of Clostridium perfringens lecithinase. Biochemistry, N.Y 33:110 (Translated from Biokhimiya, 33, 130.)
    [Google Scholar]
  73. Smyth C. J. 1972; Toxins and enzymes of Clostridium perfringens type A. Ph.D. Thesis. University of Glasgow
    [Google Scholar]
  74. Smyth C. J., Arbuthnott J. P., Freer J. H. 1972; Purification, properties and biological characteristics of Clostridium perfringens type A α-toxin. J. gen. Microbiol 73:xxvi
    [Google Scholar]
  75. Stephen J. 1961; The isolation of the α-toxin of Clostridium welchii type A by zone electrophoresis in vertical columns. Biochem. J 80:578
    [Google Scholar]
  76. Sugahara T., Ohsaka A. 1970; Two molecular forms of Clostridium perfringens α-toxin associated with lethal, hemolytic and enzymatic activities. Jap. J. med. Sci. Biol 23:61
    [Google Scholar]
  77. Teodorescu G., Bittner J., CeacAreanu A. 1970; Donnees preliminaries sur la purification et la structure de l’alpha-toxine Clostridium perfringens. Archs roum. Path, exp. Microbiol 29:541
    [Google Scholar]
  78. Ui N. 1971a; Isoelectric points and the conformation of proteins. I. Effect of urea on the behaviour of some proteins in isoelectric focusing. Biochim. biophys. Acta 229:567
    [Google Scholar]
  79. Ui N. 1971b; Isoelectric points and the conformation of proteins. II. Isoelectric focusing of a-chymotrypsin and its inactive derivative. Biochim. biophys. Acta 229:582
    [Google Scholar]
  80. Vesterberg O. 1967; Isoelectric fractionation, analysis and characterisation of ampholytes in natural pH gradients. V. Separation of myoglobins and studies on their electrochemical differences. Acta chem. scand. 21:206
    [Google Scholar]
  81. Wadstrom T. 1967; Studies on extracellular proteins from Staphylococcus aureus. II. Separation of deoxyribonucleases by isoelectric focusing. Purification and properties of the enzymes. Biochim. biophys. Acta 147:441
    [Google Scholar]
  82. Zamecnik P. C., Brewster L. E., Lipmann F. 1947; A manometric method for measuring the activity of the Cl. welchii lecithinase and a description of certain properties of this enzyme. J. exp. Med 85:381
    [Google Scholar]
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