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Fluid-phase assembly of the membrane attack complex of complement.

R E Silversmith, G L Nelsestuen

    Biochemistry
    |February 25, 1986
    PubMed
    Summary

    The study characterizes the fluid-phase assembly of the membrane attack complex (MAC) of complement, revealing stable intermediates and precise protein stoichiometry. Light-scattering measurements confirm ordered assembly and quantify the incorporation of C8 and C9 proteins into the MAC structure.

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    Area of Science:

    • Immunology
    • Biochemistry
    • Molecular Biology

    Background:

    • The membrane attack complex (MAC) of complement is crucial for cell lysis.
    • Understanding the assembly dynamics and protein stoichiometry of MAC is essential for elucidating its function.

    Purpose of the Study:

    • To characterize the fluid-phase assembly dynamics and protein stoichiometry of the membrane attack complex (MAC) of complement.
    • To investigate the ordered assembly process and identify stable intermediates.

    Main Methods:

    • Light-scattering intensity measurements were used to analyze the assembly process.
    • Sucrose gradient ultracentrifugation was employed to assess particle heterogeneity.
    • Protein stoichiometry was determined by quantifying the incorporation of C8 and C9.

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    Main Results:

    • Fluid-phase assembly of C5b-7 formed stable, reproducible intermediates with a weight-average molecular weight of (4.1 +/- 0.2) X 10(6), corresponding to an average of nine C5b-7 complexes per particle.
    • C8 incorporation into C5b-7 occurred with a ratio of 0.98 +/- 0.03 C8 per C5b-7 moiety.
    • C9 incorporation into C5b-8 showed a biphasic pattern, with an initial rapid phase suggesting limited C9 binding sites and a slow phase with an activation energy of 37 kcal/mol, similar to C9 polymerization alone.

    Conclusions:

    • The fluid-phase assembly of the membrane attack complex is an ordered process yielding stable intermediates.
    • Protein stoichiometry of the assembled MAC was precisely determined, revealing specific incorporation ratios for C8 and C9.
    • Light-scattering intensity measurements are a reliable method for quantitatively characterizing complement complex assembly.