Studies of the mechanism of assembly of the membrane attack complex, its regulation by inhibitors, and the relationship to cytolytic function

Complement C9 labeled with fluorescein-5-isothiocyanate (FITC-C9) displayed a fluorescence decrease upon assembly in the membrane attack complex (MAC). The fluoresence change reported a temperature-dependent transition which followed initial binding. Kinetic analysis indicated an irreversible, first order process in both the presence and absence of membranes. The rate and activation energy indicated the transition was the temperature-dependent event in complement-mediated hemolysis. Kinetic properties were different for assembly with C5b-9;Clusterin addition to C5b-8-(FITC-C9) produced a concentration-dependent fluorescence decrease suggesting an affinity of ;Fluorescence energy transfer studies between labeled MAC proteins indicated C9 polymerization was initiated by a stable C8-C9 interaction, but that a homogeneous, closed cyclic pore was not formed. C9 assembly at progressive stages of saturation exhibited kinetic and enthalpic heterogeneity. The enthalpy of assembly of the first 3 C9 equivalents with C5b-8 gave a large endothermic response that was increased in the absence of membranes. Based on its kinetic and enthalpic heterogeneity, a model of C9 polymerization was proposed in which pore expansion is principally derived from early subunits, which more fully unfold to expose cytolytic domains. Membrane interactions were exothermic, suggesting that electrostatic interactions involving phospholipid head groups may be involved.;Membrane binding properties of human and bovine forms of protein Z and other vitamin K-dependent proteins were correlated with amino acid sequences in their gamma-carboxyglutamic acid (gla)-domains. Protein Z displayed the ability to pack at high membrane densities, and exhibited the greatest affinity of the proteins in this class. From membrane affinity comparisons with 14 proteins, three positions were implicated as important to membrane contact. The differences in membrane affinity represented by these positions could account for the...