May 14, 1976 - are compatible with the "doughnut" hypothesis mechanism of cytolysis by complement (4). MATERIALS AND METHODS. Complement ...
Proc. Nati. Acad. Sci. USA Vol. 73, No. 8, pp. 2852-2856, August 1976 Immunology
Increased ion permeability of planar lipid bilayer membranes after treatment with the C5b-9 cytolytic attack mechanism of complement (complement insertion into membrane/black lipid bilayer/transmembrane channel)
DAVID W. MICHAELS, AARON S. ABRAMOVITZ, CARL H. HAMMER, AND MANFRED M. MAYER Departments of Physiological Chemistry and Microbiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Communicated by Albert L. Lehninger, May 14,1976
ABSTRACT The ion permeability of planar lipid bilayers, as measured electrically, was found to increase modestly upon treatment with purified complement complex C5b,6 and complement components C7 and C8. The subsequent addition of C9 greatly amplified this change. No permeability changes occurred when components were added individually to the membrane, or when they were used in paired combinations, or when C5b,6, C7, C8, and C9 were admixed prior to addition. Thus, there is a significant parallel between the permeability changes induced in the model membrane and damage produced in biological membranes by the C5b-9 complement attack sequence. The efficiency of membrane action by C5b-9 was critically dependent on the order in which components were added to the membrane. There were also differences in the electrical properties of membranes treated with C5b-8 and C5b-9, though in both cases the enhanced bilayer permeability is best attributed to the formation of trans-membrane channels. Collectively, the data are consistent with the hypothesis that the mechanism of membrane action by complement involves the production of a stable channel across the lipid bilayer, resulting in cell death by colloid-osmotic lysis. The term "complement" refers to a group of proteins in normal blood serum that plays an important role as a mediator of both immune and allergic reactions. The sequential interaction of the nine complement proteins is illustrated in Fig. 1. One consequence of this enzymatic cascade is the drastic alteration in the permeability of biomembranes to substances of low molecular weight (1). This activity is a result of the interaction of the five terminal components (C5b-9) with the lipid moiety of the cell membrane, as has been learned from experiments in which C5b-9 released trapped marker from phospholipid liposomes concomitant with the production of lesions visible by electron microscopy (2). In studies with erythrocytes, we have found that a single membrane lesion suffices for lysis of a cell (1). In a recent study we have presented evidence which indicates that polypeptide chains from the C5b and C7 subunits of the C5b,6,7 complex become inserted in the phospholipid bilayer of the erythrocyte membrane (3). We believe that this process represents the initial stage of the cytolytic attack and that subsequent reaction of C8 and C9 with the inserted C5b,6,7 opens a channel extending through the phospholipid bilayer (4). We now present an exploration of this mechanism by use of planar lipid bilayers (black lipid membranes, BLM). This is an atrractive system because the morphology of BLM is known to be bimolecular and because changes in membrane permeability
to ions can be measured electrically. There have been several earlier studies of the effect of antigens, antibodies, and fresh blood serum as a source of complement on BLM in which changes in electrical conductance were observed (5-7). In the present experiments, we have used purified complement components and initiated the terminal reaction sequence with C5b,6 complex. This approach dispensed with the need for antigens, antibodies, and complement components C1, C2, C3, and C4. We have found that treatment of BLM with C5b,6, C7, and C8 produced a significant increase of electrical conductance. Subsequent addition of C9 generally amplified this effect. The general characteristics of these conductance changes are compatible with the "doughnut" hypothesis mechanism of cytolysis by complement (4).
MATERIALS AND METHODS Complement components C5b,6 Complex. Human sera were screened for net generation of C5b,6 complex upon activation with yeast cell-wall mannan (zymosan) (Nutritional Biochemical Corp.) in 0.75% agarose plates containing sheep erythrocytes (1 X 109/ml) and guinea pig serum (1/400) in buffer which contained 10 mM sodium barbital, 150 mM sodium chloride, 10 mM disodium EDTA at pH 7.4 (Veronal-buffered saline-EDTA), essentially as described by Lachmann and Thompson (8). Larger samples of serum were obtained from individuals whose titers were judged high in the screening assay. C5b,6 complex was isolated and purified by modification of previously reported protocols (9, 10). In brief, sera were activated with zymosan (4 mg/ml) by incubation at 370 for 10 min. C5b,6 activity was eluted from centrifugally recovered zymosan pellets with 0.75 M NaCl at 270. Subsequent manipulations were performed at 40. The eluate was dialyzed against 5 mM sodium phosphate, pH 6.0 and the euglobulin fraction was recovered by centrifugation. The precipitate was dissolved in buffer which contained 5 mM sodium phosphate and 150 mM NaCl (phosphate-buffered saline) and subjected to reverse ammonium sulfate chromatography on DEAE- cellulose (11), with a gradient ranging from 42% to 20% saturation (00) of ammonium sulfate in 0.1 M sodium phosphate, pH 7.0. The C5b,6 activity eluted at 35% ammonium sulfate saturation. All active fractions were pooled and concentrated by precipitation with 80% saturated ammonium sulfate. After centrifugation the precipitate was dissolved in phosphate-buffered saline. The product was free of C7 (-C4b,2a C2
E 240
C4b,2a,3b
C3
C5
C6
9
C5b (Labile)
0
C5b,6 (Stable)
jC7 C5b,6,7 (Labile)
E
200
w
O 160 z
O 120 S
