Interaction of human beta-endorphin with the terminal SC5b-9 and "preterminal" SC5b-7 and SC5b-8 complexes of human complement

Human beta-endorphin (beta H-EP) is demonstrated to bind to the "preterminal" SC5b-7 and SC5b-8 complexes and to the terminal SC5b-9 complex of human complement. Detailed binding studies revealed saturability, reversibility and structural specificity of the beta H-EP interaction with high or low affinity non-opiate binding sites on SC5b-7 and SC5b-9 complexes. The high affinity binding sites seem to be located predominantly on C5b, C6 or C7 subunits of the complexes.     

Cytolysis of nucleated cells by complement: inhibition of membrane-transmethylation enhances cell death by C5b-9

Inhibition of transmethylation, i.e., enzymatic transfer of methyl groups to phosphatidyl ethanolamine resulting in generation and translocation of phosphatidyl choline, enhances the killing of nucleated cells by complement. Furthermore, under complement attack, transmethylation measured as incorporation of [3H]methyl groups into phosphatidyl choline is enhanced, suggesting that transmethylation functions as a membrane defense mechanism either by increasing the phosphatidyl choline synthesis or by transducing a signal that might trigger another membrane repair process.     

Inhibition of homologous complement by CD59 is mediated by a species-selective recognition conferred through binding to C8 within C5b-8 or C9 within C5b-9.

The capacity of the human complement regulatory protein CD59 to interact with terminal complement proteins in a species-selective manner was examined. When incorporated into chicken E, CD59 (purified from human E membranes) inhibited the cytolytic activity of the C5b-9 complex in a manner dependent on the species of origin of C8 and C9. Inhibition of C5b-9-mediated hemolysis was maximal when C8 and C9 were derived from human (hu) or baboon serum. By contrast, CD59 showed reduced activity when C8 and C9 were derived from dog or sheep serum, and no activity when C8 and C9 were derived from either rabbit or guinea pig (gp) serum. Similar specificity on the basis of the species of origin of C8 and C9 was also observed for CD59 endogenous to the human E membrane, using functionally blocking antibody against this cell surface protein to selectively abrogate its C5b-9-inhibitory activity. When E bearing human CD59 were exposed to C5b-8hu, CD59 was found to inhibit C5b-9-mediated lysis, regardless of the species of origin of C9, suggesting that the inhibitory function of CD59 can be mediated through recognition of species-specific domains expressed by human C8. Consistent with this interpretation, CD59 was found to bind to C5b-8hu but not to C5b67hu or C5b67huC8gp. Although CD59 failed to inhibit hemolysis mediated by C5b67huC8gpC9gp, its inhibitory function was observed for C5b67huC8gpC9hu, suggesting that, in addition to its interaction with C5b-8hu, CD59 also interacts in a species-selective manner with C9hu incorporated into C5b-9. Consistent with this interpretation, CD59 was found to bind both C5b67huC8gpC9hu and C5b-8huC9gp, but not C5b67huC8gpC9gp. Taken together, these data suggest that the capacity of CD59 to restrict the hemolytic activity of human serum complement involves a species-selective interaction of CD59, which involves binding to both the C8 and C9 components of the membrane attack complex. Although CD59 expresses selectivity for C8 and C9 of human origin, this "homologous restriction" is not absolute, and this human complement regulatory protein retains functional activity toward C8 and C9 of some nonprimate species.     

The membrane attack complex of complement: C5b-8 complex as accelerator of C9 polymerization

Polymerization of C9 occurs spontaneously or can be induced by the tetramolecular complex C5b-8. Spontaneous C9 (0.15 mg/ml) polymerization required more than 3 days at 37 degrees C. In the presence of C5b-8, C9 polymerization was complete within 10 min. The molar C9:C5b-8 ratio determined the extent of tubular poly C9 formation by C5b-8-bearing phospholipid vesicles. When this ratio was 9:1 or 12:1, 72% of complex-bound C9 was present as SDS resistant tubular poly C9 (Mr = 1.1 X 10(6]. At lower C9:C5b-8 ratios, poly C9 was bound primarily in nontubular form. Tubular poly C9, as part of C5b-9, could also be generated on rabbit erythrocytes by using whole human serum as a complement source. At limiting serum concentration (molar C9 to C8 ratio approximately 2), no SDS-resistant tubular poly C9 was detected. At high serum concentration or when using serum that was supplemented with C9, up to 40% of the C9 was SDS-resistant tubular poly C9, and the rest was poly C9, which was incompletely polymerized. It is suggested that the C5b-8 complex acts as an accelerator of C9 polymerization, and that its relative concentration to C9 determines the ultrastructure of the C5b-9 complex.     

Monoclonal antibodies against neoantigens of the terminal C5b-9 complex of human complement

Assembly of the terminal C5b-C9 complement components into the cytolytic C5b-9 complex is accompanied by exposure of characteristic neoantigens on the macromolecule. We report the production and characterization of mouse monoclonal antibodies to C9-dependent neoantigens of human C5b-9. Binding-inhibition assays with EDTA-human plasma and micro-ELISA assays with purified C9 showed that the antibodies did not react with native complement components and thus confirmed the specificity of the antibodies for the neoantigens. The monoclonal antibodies did, however, cross-react with cytolyticaIly inactive, fluid-phase C5b-9 complexes, Thus, expression of the neoantigenic determinants was not dependent on the formation of high molecular weight C9 polymers with the complex, since these are absent in fluid-phase C5b-9. Radioiodinated antibodies could be utilized in immunoradiometric assays for the detection and quantitation of C5b-9 on cell membranes. Cross-reactivities of the antibodies with C9-dependent neoantigens of several other animal species were examined and antibody clones cross-reacting with rabbit (clones 3BI, 3Dg, and 2F3), sheep (clones 3Dg and 2F3) and guinea-pig (clone 3D8) neoantigens were identified . Three of four tested clones (3D8, 2F3, IA12) precipitated C5b-9 complexes in double-diffusion assays, probably due to their interaction with multiple and repeating C9-epitopes on the terminal complexes. The monoclonal antibodies will be of value for definitive identification and quantitation of C5b-9 on cell membranes and in tissues, and for establishing immunoassays for detection and quantitation of terminal fluid-phase C5b-9 complexes in plasma.     

Molecular composition of the terminal membrane and fluid-phase C5b-9 complexes of rabbit complement. Absence of disulphide-bonded C9 dimers in the membrane complex

The terminal membrane C5b-9(m) and fluid-phase SC5b-9 complexes of rabbit complement were isolated from target sheep erythrocyte membranes and from inulin-activated rabbit serum respectively. In the electron microscope, rabbit C5b-9(m) was observed as a hollow protein cylinder, a structure identical with that of human C5b-9(m). Monodispersed rabbit C5b-9(m) exhibited an apparent sedimentation coefficient of 29 S in deoxycholate-containing sucrose density gradients, corresponding to a composite protein-detergent molecular-weight of approx. 1.4 X 10(6). Protein subunits corresponding to human C5b-C9 were found on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. By densitometry, there were consistently six molecules of monomeric C9 present for each monomeric C5b-8 complex. Fluid-phase rabbit SC5b-9 was a hydrophilic 23 S ma macromolecule that differed in subunit composition from its membrane counterpart in that it contained S-protein and only two to three molecules of C9 per monomer complex. The data are in accord with the previous report on human C5b-9 that C5b-9(m) contains more C9 molecules than SC5b-9 [Ware & Kolb (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6426-6430]. They corroborate the previous molecular-weight estimate of approx. 10(6) for C5b-9(m) and thus support the concept that the fully assembled, unit lesion of complement is a C5b-9 monomer [Bhakdi & Tranum-Jensen (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1818-1822]. They also show that C9 dimer formation is not required for assembly of the rabbit C5b-9(m) protein cylinder, or for expression of its membrane-damaging function.     

Complement lysis: evidence for an amphiphilic nature of the terminal membrane C5b-9 complex of human complement

The terminal, membrane-derived C5b-9 complex of human complement (C) is an apparently hollow, cylindrical macromolecule vertically oriented on the target membrane. In the present study, an antiserum to the complex has been used to probe its immunobiochemical properties. "Neoantigenic" determinants characteristic of the complex have been detected, which are absent on native C5-C9 molecules. Evidence that the C5b-9 complex is an amphiphilic molecule that possesses apolar, detergent-binding surfaces has been obtained by using charge-shift crossed immunoelectrophoresis, and by direct demonstration of Triton X-100 binding to the complex in quantitative immunoelectrophoresis. By the same criteria, serum C5, C6, and C9 are hydrophilic molecules. The results indicate that assembly of C5-C9 into the terminal membrane C5b-9 complex is accompanied by conformational changes in the individual C components that lead to the exposure of apolar molecular regions in the complex. It is proposed that this constitutes the basis for the lipid-binding properties of the macromolecule, which enable it to become inserted into biologic and artificial lipid membranes with apparent generation of a transmembrane pore.     

Deposition of the terminal C5b-9 complement complex in infarcted areas of human myocardium

Poly- and monoclonal antibodies to neoantigens of the human C5b-9 complement complex, as well as polyclonal antibodies to C5, C8, and C9, were used to detect and identify C5b-9 deposits in human myocardial tissue. Immunocytochemical studies were performed on fresh-frozen autopsy material derived from patients with myocardial infarctions; in addition, in 17 of these patients, paraffin sections of formalin-fixed tissue were investigated. Sixteen autopsies from patients with noncardiac diseases were analyzed as controls. Without exception, C5b-9 positivity was registered selectively and exclusively on and in myocardial cells located within the zones of infarction. The selectivity of staining was confirmed by control reactions for succinic dehydrogenase activity performed in adjacent, respective double-stained sections. Most intensive staining with anti-neoantigen antibodies was observed in the peripheral areas of the infarctions. Weak staining for C3d, rather strong staining for C5 and C9, and intermediate staining with anti-C8 antibodies were observed in the same localizations. Stainings for C4 and IgA were negative, whereas immunocytochemical reactions for IgG and IgM revealed an irregular and very weak staining. Only very weak staining was also observed with a monoclonal antibody to complement S-protein, indicating that the terminal complement components were deposited mainly in the form of membrane-damaging C5b-9 complexes. Immunocytochemical staining for C5b-9 was found to represent a most sensitive tool for detection of ischemic myocardial lesions, permitting easy detection even of single cell necroses. As a working hypothesis, we suggest that initial ischemia may cause loss of the ability of the heart muscle cells to regulate complement turnover at the membrane level. The resulting deposition of C5b-9 on the cell membranes may contribute to functional disturbance and irreversible damage of myocardial cells during the infarction process.     

The terminal membrane C5b-9 complex of human complement. Evidence for the existence of multiple protease-resistant polypeptides that form the trans-membrane complement channel

C5b-9(m) complexes were incorporated into lecithin liposomes and subjected to proteolysis in the presence of DTT to remove the externally oriented annulus. Liposomes were recovered that selectively carried the membrane-bound, thin-walled cylindrical portion of the C5b-9(m) complex. The presence of DTT during proteolysis enhanced peptide bond cleavage in the C5b-9(m) complex. All C5-C9 components were degraded to lower m.w. fragments. A protease-resistant, but hydrophilic 85 to 86,000-dalton polypeptide derivative of C5, possibly representing the C5 beta-chain, was recovered in the fluid phase. This component is not intimately associated with the target lipid bilayer. Immunochemical analyses yielded evidence for the existence of minor C5-C9 antigenic determinants on the membrane-bound C5b-9(m) residue. SDS polyacrylamide gel electrophoreses of liposomes carrying the C5b-9(m) residues revealed the persistence of at least six major polypeptides of approximately m.w. 50,000, 45,000, 40,000, 38,000, 20,000, and 16,000. The data are interpreted to indicate that multiple protease-resistant polypeptide chains derived from several terminal C components participate in formation of the trans-membrane C channel.     

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.     

Assembly of complement components C5b-8 and C5b-9 on lipid bilayer membranes: visualization by freeze-etch electron microscopy

We have visualized by freeze-etch electron microscopy the macromolecular complexes of complement, C5b-8 and C5b-9, respectively, assembled on synthetic phospholipid bilayers. These complexes were formed sequentially by using purified human complement components C5b-6 followed by C7, C8, and C9. Complexes of C5b-8 were observed on the external surface (ES) of vesicles as 12-nm particles that tended to form polydisperse aggregates. The aggregates were sometimes of a regular chainlike structure containing varying numbers of paired subunits. Etching of vesicles containing C5b-9 complexes revealed on the ES large rings of approximately 27-nm outer diameter. One or two knobs usually were attached to the perimeter of the rings. Splitting of the membrane resulted in partitioning of the C5b-9 with the outer leaflet. Thus, round holes of approximately 17-nm diameter were present in the protoplasmic face (PF), and raised circular stumps of a matching size were present on the exoplasmic face (EF) of C5b-9 vesicles. C5b-9 complexes were frequently localized in regions of the lowest lipid order. That is, in micrographs of the EF and ES, single C5b-9 complexes were located where the ripples of the P beta' phase bend or reach a dead end, and linear arrays of C5b-9 complexes outlined disclination-like structures in the lattice; the holes in the PF mirrored this distribution. The membrane immediately surrounding C5b-9 rings was often sunk inwardly over an area much larger than that of the ring itself.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacterial killing and inhibition of inner membrane activity by C5b-9 complexes as a function of the sequential addition of C9 to C5b-8 sites

The assembly of the C5b-9 complex on the outer membrane of C-sensitive cells of Escherichia coli results in a rapid inhibition of inner membrane function and ultimately a loss of cell viability. Cells bearing C5b-8 sites suffer no deleterious effects; however, the addition of C9 results in a rapid inhibition of inner membrane function and cell death. An attempt was made to examine the relationship between the toxic effects of the C5b-9 complex and the number of C9 molecules per C5b-8 site. Cells bearing C5b-8 sites were exposed to excess C9 at 0 degrees C and washed three times at 4 degrees C. The number of C9 molecules bound to each cell was equivalent to the number of C5b-8 sites present on each cell, and no additional C9 molecules could be bound when the cells were maintained at 4 degrees C. These cells were then incubated at 37 degrees C for 3 min and returned to 0 degrees C, a technique which exposed additional C9-binding sites equivalent to the number of C9 molecules previously bound to the cells. This technique was repeated and demonstrated that the sequential build-up of a C5b-9 site with two C9 molecules per C5b-8 site was capable of inhibiting both inner membrane function (respiration and amino acid transport) and cell viability. Three C9 molecules per complex had effects that approached the inhibitory effects of complexes formed in the presence of excess C9.     

The structure of human complement component C7 and the C5b-7 complex

The molecular architecture of human complement component C7 was elucidated at several structural levels. The complete primary structure of C7 was derived from the cDNA sequence of clones isolated from a human liver library. C7 is a mosaic protein that consists of 821 amino acids. The amino-terminal two-thirds of C7 has 23-30% homology with complement components C8 and C9. In addition, the carboxyl-terminal third contains four cysteine-rich segments that have overlapping internal homology. The protein is a single polypeptide chain with 28 disulfide bonds and is glycosylated at two sites. Virtually all the cysteines are found in small units of 35-77 amino acids that exhibit homology with those of various proteins including the low density lipoprotein receptor, epidermal growth factor precursor, thrombospondin, and blood coagulation factors IX and X. The secondary structural analysis, estimated by circular dichroism, suggested a high content of beta-sheet (38%) and beta-turns (24%). The tertiary structure, visualized by transmission electron microscopy, indicated a flexible elongated molecule with dimensions of 151 X 59 X 43 A. The quaternary structure of the C5b-7 complex bound to lipid vesicles was observed to be in the form of monomers or dimers. The monomer C5b-7 consists of a leaflet and a long flexible stalk, and the dimer has two leaflets linked through a supercoiled stalk. Membrane binding is mediated by the stalk part of the complexes. Using a radioiodinated photoreactive cross-linking reagent bound to the polar head group of phosphatidylethanolamine, the stalk part of the C5b-7 complex could be labeled preferentially, and it was found to consist mainly of C6 and C7. Thus, C7 plays a major role in bringing about the hydrophilic-amphiphilic transition during the formation of the membrane attack complex, and it serves as a membrane anchor for the C5b-7 complex.     

Elimination of terminal complement complexes in the plasma membrane of nucleated cells: influence of extracellular Ca2+ and association with cellular Ca2+

Nucleated cells, unlike erythrocytes, are able to survive limited complement attack by eliminating potentially cytolytic complement channels from the plasma membrane (PM) by processes that involve, plasma membrane (PM) by processes that involve, but may not be limited to, endocytosis. The observation that C5b-9 channels, as well as C5b-8 and C5b-7 intermediates, are rapidly eliminated from the cell surface of nucleated cells has prompted us to examine whether terminal complement complexes stimulate membrane events that lead to accelerated elimination of these complexes. We have suggested previously that ion flux through terminal complement complexes might influence the rate of elimination on the basis of our finding that terminal complement complexes with larger functional channel sizes are more rapidly eliminated. In this study, we examined the role of Ca2+ on the elimination rate of terminal complement complexes in the PM of Ehrlich cells, because changes in Ca2+ flux across the PM are known to influence many metabolic activities including endocytosis. To determine the elimination rate for terminal complement complexes by functional analysis, cells bearing C5b-7 or C5b-8 complexes with or without a sublytic dose of C9 were incubated at 37 degrees C for various time intervals before converting the remaining complexes to lytic C5b-9 channels. The initial elimination rates for the terminal complement complexes were compared in the presence of 0.015, 0.15, and 1.5 mM CaCl2 in the medium. Sufficient lowering of the extracellular Ca2+ concentration, (Ca2+)o, resulted in prolonging the elimination of each of the terminal complement complexes to a different extent. The effect of (Ca2+)o on the elimination rate was most pronounced for C5b-8 in the presence of a sublytic number of C5b-9, with less of an effect on C5b-8 alone, and the least effect with C5b-7. The elimination rates for terminal complement complexes were also determined by measuring the persistence of C5b antigen on the cell surface at 37 degrees C in the presence of various (Ca2+)o by using fluorescence-activated cell sorter analysis and were comparable with that obtained by functional analysis. Examination of the effect of terminal complement complexes on the cellular Ca2+ concentration, (Ca2+)i, revealed that these complexes increased the (Ca2+)i in proportion with the known functional pore size of the terminal complement complex in the PM. In addition, Quin 2, which can buffer internal Ca2+ transients, was found to increase the susceptibility of Ehrlich cells to lysis by C5b-9, further suggesting a relationship between the (Ca2+)i and the elimination process.(ABSTRACT TRUNCATED AT 400 WORDS)     

The membrane attack complex of complement: relation of C7 to the metastable membrane binding site of the intermediate complex C5b-7

Isolated C7 (m.w. 120,000) in 1% deoxycholate (DOC) forms dimers with an apparent m.w. of 230,000 and a DOC-binding capacity of 82 mol per mol of dimer. Dimerization of C7 also occurs in the presence of DOC-phospholipid mixed micelles and eventuates in the insertion of C7 dimers into the lipid bilayer upon the removal of the detergent. C5b-7 complex formation in the fluid phase or on lipid vesicles likewise involves polymerization. C5b-7 sedimented with 17-40S, which suggests a dimeric to hexameric composition. In avidin-biotin binding experiments in which two differentially labeled forms of C5b,6 (biotinyl 125I-C5b,6, and 131I-C5b,6) were used in equimolar amounts to assemble C5b-7, more than 50% of the biotinyl 125I-C5b,6-containing complexes also contained 131I label; again suggesting that C5b-7 consisted of oligomers rather than monomers. The conformation of C7 in C5b-7 and in dimeric C7 appeared similar by the following criteria. On formation of C5b-7 from C5b,6 and C7, a 20% increase in beta-pleated sheet structure was observed by circular dichroism spectroscopy, and a similar change occurred on dimerization of isolated C7. Tryptic and thermolytic digests of C5b-7 and C7 dimers containing 125I-C7 were analyzed by autoradiography after SDS-polyacrylamide gel electrophoresis and were found to contain similar peptides that were distinct from those in the digests of monomeric C7. Direct evidence showing that the metastable membrane binding site of the C5b-7 complex resides in the C7 subunit was obtained by using the conjugates of C5b,6 and colloidal gold. Viewed in the electron microscope, these conjugates were aggregated upon the addition of isolated C7. In contrast, when conjugates of C7 and colloidal gold were treated with soluble C5b,6, no such aggregates occurred, but instead, individual C5b-7 complexes were observed arranged around single gold particles, resulting in star-like structures. The results strongly suggest that structures of C7 are responsible for the expression of the membrane binding site of metastable C5b-7.     

Transfer of preformed terminal C5b-9 complement complexes into the outer membrane of viable gram-negative bacteria: effect on viability and integrity

An efficient fusion system between Gram-negative bacteria and liposomes incorporating detergent-extracted C5b-9 complexes has been developed that allows delivery of preformed terminal complexes to the cell envelope (Tomlinson et al., 1989b). Fusion of Salmonella minnesota Re595 and Escherichia coli 17 with C5b-9-incorporated liposomes resulted in the transfer of 1900 C5b-9 complexes to each target bacterial cell. No loss in viability of bacteria was observed following fusion, even though the deposotion of 900 complexes onto the envelope following exposure to lysozyme-free serum effected a greater than 99% loss of viability. Increased sensitivity to antibiotics normally excluded from the cell by an integral outer membrane (OM), as well as the ability of the chromogenic substrate PADAC to gain access to periplasmically located beta-lactamase, indicated that transferred C5b-9 complexes functioned as water-filled channels through the OM. A similar conclusion was drawn from measurements demonstrating the uptake by cells of the lipophilic cation tetraphenylphosphonium (bromide), a result further indicating that the membrane potential across the cytoplasmic membrane was maintained following C5b-9 transfer to the OM. Examination of S. minnesota Re595 by electron microscopy revealed no obvious difference between cells exposed to lethal concentrations of lysozyme-free serum and cells following fusion with C5b-9-incorporated liposomes. These data suggest either that there are critical sites in the OM to which liposome-delivered C5b-9 complexes are unable to gain access or that bacterial cell death is related to events occurring during polymerization of C9 on the cell surface.     

Penetration of C8 and C9 in the C5b-9 complex across the erythrocyte membrane into the cytoplasmic space

We have developed a technique in which transglutaminase is used to measure the penetration of terminal complement proteins across the erythrocyte membrane into the cytoplasmic space. Penetration of a given terminal complement protein into the cytoplasmic space was assessed by labeling the protein of interest with radioactive iodine, forming the complement channel using the labeled protein, adding transglutaminase to only one side of the membrane, and allowing the enzyme to cross-link the susceptible proteins on that side of the membrane. Cross-linking was assessed by measuring the increase in molecular weight of the appropriate molecule on sodium dodecyl sulfate gels under reducing conditions. The results of these experiments indicate that C8 and C9 are rapidly cross-linked to high molecular weight from either the interior or the exterior of the membrane. In order to determine whether the cross-linking mediated by enzyme on the interior was occurring from within the ghosts and not via enzyme that had leaked into the extracellular medium, experiments were performed with dimethylcasein in the extracellular medium. In the presence of this protein, cross-linking of C8 and C9 from outside was negligible. Hence, if cross-linking occurs when transglutaminase is trapped inside the ghosts, it cannot be due to leakage of enzyme, but must be attributable to cross-linking from the inside. The results show that C9 definitely penetrated across the membrane into the intracellular space. With respect to C8, statistical evaluation indicates that C8 probably penetrated into the intracellular space.     

C5b-9 assembly: average binding of one C9 molecule to C5b-8 without poly-C9 formation generates a stable transmembrane pore

Membrane attack by serum complement normally results in the formation of C5b-9 complexes that are heterogeneous with respect to their C9 content. We here report that an apparently homogeneous population of C5b-9 complexes can be generated through treatment of C5b-7-laden sheep erythrocytes with C8 and C9 for 60 min at 0 degree C. Experiments performed by using radioiodinated C8 and C9 components have indicated that binding of C8 to these target cells is essentially temperature independent. In contrast, when a surplus of C9 molecules is offered to C5b-8 cells, an approximately fourfold to 4.5-fold higher number of C9 molecules become cell bound at 37 degrees C as opposed to 0 degree C. C5b-9 complexes isolated from target membranes treated with C9 at 0 degree C contain no polymerized C9 and do not exhibit the ring structure characteristic of the classical complement lesion. Nevertheless, these complexes generate stable transmembrane channels and cause hemolysis at 37 degrees C. The pores have been sized to 1 to 3 nm effective diameter by osmotic protection experiments. SDS-PAGE of the isolated complexes indicates an average stoichiometry of only one molecule C9 bound per C5b-8 complex. The results show that oligomerization of C9 with formation of ring lesions is not a basic requirement for the generation of stable transmembrane complement pores in sheep erythrocytes. They indirectly support the contention that terminal complement components other than C9 contribute to the intramembrane domains of C5b-9 pores.