Kinetics of polymerization of a fluoresceinated derivative of complement protein C9 by the membrane-bound complex of complement proteins C5b-8.
The fluorescence self-quenching by energy transfer of FITC-C9, a fluoresceinated derivative of human complement protein C9 [Sims, P.J. (1984) Biochemistry (preceding paper in this issue)], has been used to monitor the kinetics of C9 polymerization induced by the membrane-associated complex of complement proteins C5b-8. Time-based measurements of the fluorescence change observed during incubation of FITC-C9 with C5b-8-treated sheep red blood cell ghost membranes at various temperatures revealed that C9 polymerization induced by the C5b-8 proteins exhibits a temperature dependence similar to that previously reported for the complement-mediated hemolysis of these cells, with an Arrhenius activation energy for FITC-C9 polymerization of 13.3 +/- 3.2 kcal mol-1 (mean +/- 2 SD). Similar measurements obtained with C5b-8-treated unilamellar vesicles composed of either egg yolk phosphatidylcholine (egg PC), dipalmitoylphosphatidylcholine (DPPC), or dimyristoylphosphatidylcholine (DMPC) revealed activation energies of between 20 and 25 kcal mol-1 for FITC-C9 polymerization by C5b-8 bound to these membranes. Temperature-dependent rates of C9 polymerization were observed to be largely unaffected by the phase state of membrane lipid in the target C5b-8 vesicles. The significance of these observations of the mechanism of C9 activation of membrane insertion is considered.
D000818:Animals; D003186:Complement C9 / Q000378:metabolism*; D003165:Complement System Proteins / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006461:Hemolysis; D011108:Polymers / Q000378:metabolism; D012756:Sheep; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates; D013997:Time Factors
D003186:Complement C9; D005452:Fluoresceins; D011108:Polymers; D013861:Thiocyanates; C042295:complement C5b-8 complex; D003165:Complement System Proteins; D016650:Fluorescein-5-isothiocyanate