Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1986 May 1;235(3):723–730. doi: 10.1042/bj2350723

The C3 convertase of the alternative pathway of human complement. Enzymic properties of the bimolecular proteinase.

M K Pangburn, H J Müller-Eberhard
PMCID: PMC1146747  PMID: 3638964

Abstract

The association of Factor B with C3b (the major fragment of complement component C3) in the presence of Mg2+ results in the formation of a bimolecular zymogen, C3b,B, which is activated by the serine proteinase Factor D, generating the C3 convertase, C3b,Bb (EC 3.4.21.47). Cleavage of native C3 by the C3 convertase was monitored by recording the increase in fluorescence associated with C3b formation in the presence of the fluorescent probe 8-anilinonaphthalene-1-sulphonate. Measurements of initial rates of C3b formation at various C3 concentrations were analysed in accordance with the Michaelis-Menten equation, yielding kcat. = 1.78 +/- 0.08 s-1, Km = 5.86 X 10(-6) M and turnover number = 107 min-1. The assay was used to measure the Ki values of a variety of proteinase inhibitors. The C3 convertase has a short half-life, owing to spontaneous dissociation of the complex. The t1/2 and kcat./Km of the enzyme were determined by fitting an equation modelling both the kinetic reaction and enzyme decay to the fluorimetrically measured progress curve. The enzyme, C3b,Bb, exhibited a t1/2 of 90 +/- 2 s and a kcat./Km of 31.1 X 10(4) +/- 0.8 X 10(4) M-1 X s-1 at physiological pH, ionic strength and temperature. The enzyme that initiates activation of the alternative pathway, C3(H2O),Bb, was also examined. It was slightly less stable (t1/2 = 77 +/- 3 s) and exhibited only half the activity of C3b,Bb (kcat./Km = 16.3 X 10(4) +/- 1.0 X 10(4) M-1 X s-1).

Full text

PDF
723

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ballow M., Cochrane C. G. Two anticomplementary factors in cobra venom: hemolysis of guinea pig erythrocytes by one of them. J Immunol. 1969 Nov;103(5):944–952. [PubMed] [Google Scholar]
  2. Caporale L. H. Leupeptin inhibits the C3/5 convertase CVFBb of the complement system. Biochim Biophys Acta. 1981 Jul 24;660(1):151–153. doi: 10.1016/0005-2744(81)90121-2. [DOI] [PubMed] [Google Scholar]
  3. Cooper N. R. Enzymatic activity of the second component of complement. Biochemistry. 1975 Sep 23;14(19):4245–4251. doi: 10.1021/bi00690a015. [DOI] [PubMed] [Google Scholar]
  4. Fearon D. T. Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5867–5871. doi: 10.1073/pnas.76.11.5867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fishelson Z., Müller-Eberhard H. J. C3 convertase of human complement: enhanced formation and stability of the enzyme generated with nickel instead of magnesium. J Immunol. 1982 Dec;129(6):2603–2607. [PubMed] [Google Scholar]
  6. Fishelson Z., Pangburn M. K., Müller-Eberhard H. J. C3 convertase of the alternative complement pathway. Demonstration of an active, stable C3b, Bb (Ni) complex. J Biol Chem. 1983 Jun 25;258(12):7411–7415. [PubMed] [Google Scholar]
  7. Fishelson Z., Pangburn M. K., Müller-Eberhard H. J. Characterization of the initial C3 convertase of the alternative pathway of human complement. J Immunol. 1984 Mar;132(3):1430–1434. [PubMed] [Google Scholar]
  8. Gutierrez J. C., Götze O., Caporale L. H. An improved fluorogenic substrate for the alternative complement pathway C3/5 converting enzyme CVFBb. Biochim Biophys Acta. 1983 May 18;744(3):276–280. doi: 10.1016/0167-4838(83)90200-5. [DOI] [PubMed] [Google Scholar]
  9. Götze O., Medicus R. G., Müller-Eberhard H. J. Alternative pathway of complement: nonenzymatic, reversible transition of precursor to active properdin. J Immunol. 1977 Feb;118(2):525–532. [PubMed] [Google Scholar]
  10. Götze O., Müller-Eberhard H. J. The alternative pathway of complement activation. Adv Immunol. 1976;24:1–35. doi: 10.1016/s0065-2776(08)60328-4. [DOI] [PubMed] [Google Scholar]
  11. Götze O., Müller-Eberhard H. J. The c3-activator system: an alternate pathway of complement activation. J Exp Med. 1971 Sep 1;134(3):90–108. [PMC free article] [PubMed] [Google Scholar]
  12. Hammer C. H., Wirtz G. H., Renfer L., Gresham H. D., Tack B. F. Large scale isolation of functionally active components of the human complement system. J Biol Chem. 1981 Apr 25;256(8):3995–4006. [PubMed] [Google Scholar]
  13. Hirata F., Iwata M. Role of lipomodulin, a phospholipase inhibitory protein, in immunoregulation by thymocytes. J Immunol. 1983 Apr;130(4):1930–1936. [PubMed] [Google Scholar]
  14. Isenman D. E. Conformational changes accompanying proteolytic cleavage of human complement protein C3b by the regulatory enzyme factor I and its cofactor H. Spectroscopic and enzymological studies. J Biol Chem. 1983 Apr 10;258(7):4238–4244. [PubMed] [Google Scholar]
  15. Isenman D. E., Cooper N. R. The structure and function of the third component of human complement--I. The nature and extent of conformational changes accompanying C3 activation. Mol Immunol. 1981 Apr;18(4):331–339. doi: 10.1016/0161-5890(81)90057-2. [DOI] [PubMed] [Google Scholar]
  16. Isenman D. E., Kells D. I., Cooper N. R., Müller-Eberhard H. J., Pangburn M. K. Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties. Biochemistry. 1981 Jul 21;20(15):4458–4467. doi: 10.1021/bi00518a034. [DOI] [PubMed] [Google Scholar]
  17. Janatova J., Tack B. F., Prahl J. W. Third component of human complement: structural requirements for its function. Biochemistry. 1980 Sep 16;19(19):4479–4485. doi: 10.1021/bi00560a015. [DOI] [PubMed] [Google Scholar]
  18. Johnson D. M., Gagnon J., Reid K. B. Factor D of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site. Biochem J. 1980 Jun 1;187(3):863–874. doi: 10.1042/bj1870863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  20. Lesavre P. H., Hugli T. E., Esser A. F., Müller-Eberhard H. J. The alternative pathway C3/C5 convertase: chemical basis of factor B activation. J Immunol. 1979 Aug;123(2):529–534. [PubMed] [Google Scholar]
  21. Lesavre P. H., Müller-Eberhard H. J. Mechanism of action of factor D of the alternative complement pathway. J Exp Med. 1978 Dec 1;148(6):1498–1509. doi: 10.1084/jem.148.6.1498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Medicus R. G., Götze O., Müller-Eberhard H. J. Alternative pathway of complement: recruitment of precursor properdin by the labile C3/C5 convertase and the potentiation of the pathway. J Exp Med. 1976 Oct 1;144(4):1076–1093. doi: 10.1084/jem.144.4.1076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Medicus R. G., Götze O., Müller-Eberhard H. J. The serine protease nature of the C3 and C5 convertases of the classical and alternative complement pathways. Scand J Immunol. 1976;5(9):1049–1055. doi: 10.1111/j.1365-3083.1976.tb03056.x. [DOI] [PubMed] [Google Scholar]
  24. Mole J. E., Anderson J. K., Davison E. A., Woods D. E. Complete primary structure for the zymogen of human complement factor B. J Biol Chem. 1984 Mar 25;259(6):3407–3412. [PubMed] [Google Scholar]
  25. Müller-Eberhard H. J., Götze O. C3 proactivator convertase and its mode of action. J Exp Med. 1972 Apr 1;135(4):1003–1008. doi: 10.1084/jem.135.4.1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nicholson-Weller A., Burge J., Fearon D. T., Weller P. F., Austen K. F. Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system. J Immunol. 1982 Jul;129(1):184–189. [PubMed] [Google Scholar]
  27. Pangburn M. K., Müller-Eberhard H. J. Kinetic and thermodynamic analysis of the control of C3b by the complement regulatory proteins factors H and I. Biochemistry. 1983 Jan 4;22(1):178–185. doi: 10.1021/bi00270a026. [DOI] [PubMed] [Google Scholar]
  28. Pangburn M. K., Müller-Eberhard H. J. Relation of putative thioester bond in C3 to activation of the alternative pathway and the binding of C3b to biological targets of complement. J Exp Med. 1980 Oct 1;152(4):1102–1114. doi: 10.1084/jem.152.4.1102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pangburn M. K., Müller-Eberhard H. J. The alternative pathway of complement. Springer Semin Immunopathol. 1984;7(2-3):163–192. doi: 10.1007/BF01893019. [DOI] [PubMed] [Google Scholar]
  30. Pangburn M. K., Schreiber R. D., Müller-Eberhard H. J. Deficiency of an erythrocyte membrane protein with complement regulatory activity in paroxysmal nocturnal hemoglobinuria. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5430–5434. doi: 10.1073/pnas.80.17.5430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pangburn M. K., Schreiber R. D., Müller-Eberhard H. J. Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3. J Exp Med. 1981 Sep 1;154(3):856–867. doi: 10.1084/jem.154.3.856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Parkes C., DiScipio R. G., Kerr M. A., Prohaska R. The separation of functionally distinct forms of the third component of human complement (C3). Biochem J. 1981 Mar 1;193(3):963–970. doi: 10.1042/bj1930963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pryzdial E. L., Isenman D. E. A reexamination of the role of magnesium in the human alternative pathway of complement. Mol Immunol. 1986 Jan;23(1):87–96. doi: 10.1016/0161-5890(86)90175-6. [DOI] [PubMed] [Google Scholar]
  34. Reeve J., Woo P. M. Steady state relations between control proteins regulating the formation of the alternative pathway C3 convertase. Immunology. 1982 Aug;46(4):859–866. [PMC free article] [PubMed] [Google Scholar]
  35. Robinson N. C., Tye R. W., Neurath H., Walsh K. A. Isolation of trypsins by affinity chromatography. Biochemistry. 1971 Jul 6;10(14):2743–2747. doi: 10.1021/bi00790a014. [DOI] [PubMed] [Google Scholar]
  36. Smith C. A., Vogel C. W., Müller-Eberhard H. J. MHC Class III products: an electron microscopic study of the C3 convertases of human complement. J Exp Med. 1984 Jan 1;159(1):324–329. doi: 10.1084/jem.159.1.324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Strunk R. C., Webster R. O. Inhibition of cleavage of the third component of human complement (C3) by its small cleavage fragment, C3a: inhibition occurs with the classical-pathway, but not the alternative-pathway, C3 convertase. Mol Immunol. 1985 Jan;22(1):37–43. doi: 10.1016/0161-5890(85)90032-x. [DOI] [PubMed] [Google Scholar]
  38. Takada Y., Arimoto Y., Mineda H., Takada A. Inhibition of the classical and alternative pathways by amino acids and their derivatives. Immunology. 1978 Mar;34(3):509–515. [PMC free article] [PubMed] [Google Scholar]
  39. Vogel C. W., Müller-Eberhard H. J. The cobra venom factor-dependent C3 convertase of human complement. A kinetic and thermodynamic analysis of a protease acting on its natural high molecular weight substrate. J Biol Chem. 1982 Jul 25;257(14):8292–8299. [PubMed] [Google Scholar]
  40. Weiler J. M., Daha M. R., Austen K. F., Fearon D. T. Control of the amplification convertase of complement by the plasma protein beta1H. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3268–3272. doi: 10.1073/pnas.73.9.3268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Whaley K., Ruddy S. Modulation of the alternative complement pathways by beta 1 H globulin. J Exp Med. 1976 Nov 2;144(5):1147–1163. doi: 10.1084/jem.144.5.1147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. von Zabern I., Nolte R., Vogt W. Treatment of human complement components C4 and C3 with amines or chaotropic ions. Evidence of a functional and structural change that provides uncleaved C4 and C3 with properties of their soluble activated froms, C4b and C3b. Scand J Immunol. 1981;13(5):413–431. doi: 10.1111/j.1365-3083.1981.tb00152.x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES