Assembly of the membrane attack complex of complement on small unilamellar phospholipid vesicles

Ruth E. Silversmith, Gary L. Nelsestuen

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Abstract

Light-scattering intensity was shown to be a reliable, direct, and quantitative technique for monitoring the assembly of the membrane attack complex of complement (proteins C5b-6, C7, C8, and C9) on small unilamellar phosphatidylcholine vesicles. The assembly on vesicles occurred in a simple fashion; complexes of C5b-7 bound noncooperatively to the vesicles, and final assembly of C5b-9 did not induce vesicle aggregation or fragmentation. When C5b-6 and C7 were mixed in the presence of vesicles but at molar protein/vesicle ratios of <1, there was quantitative binding of C5b-7 to the vesicles with no concomitant aggregation of C5b-7. If C7 was added at a slower rate, quantitative binding was obtained at molar C5b-7/vesicle ratios of up to 5. The latter observations (a) were consistent with the proposal that C5b-7 aggregation and membrane binding were competitive events and (b) defined conditions under which light-scattering intensity measurements could monitor C5b-9 assembly on vesicles without contribution from the fluid-phase assembly. The C8/C5b-7 ratio in the phospholipid-C5b-8 complex was 0.97 ± 0.12, and the maximum ratio of C9/C5b-8 in the final complex was 16.2 ± 2.0. One C9 molecule associated rapidly with each phospholipid-C5b-8, followed by slower incorporation of the remaining C9 molecules. The initial velocity of the slow phase of C9 addition was easily saturated with C9 and gave an activation energy of 37 kcal/mol. This was identical with the value measured for the analogous process in the fluid-phase assembly. The results indicated that the mechanism of C5b-8-dependent C9 polymerization on vesicles was indistinguishable from that of the fluid-phase assembly [Silversmith, R. E., & Nelsestuen, G. L. (1986) Biochemistry (preceding paper in this issue)]. Therefore, insertion of C9 into the membrane was not a part of the rate-limiting process.

Original languageEnglish (US)
Pages (from-to)852-860
Number of pages9
JournalBiochemistry
Volume25
Issue number4
StatePublished - 1986

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