Analysis of human C8 with monoclonal antibodies. Characterization of a monoclonal antibody that recognizes free C8 alpha-gamma subunit

The eighth component of human C is essential for the formation of the membranolytic C attack complex. C8 has a unique structure in that two covalently linked chains, C8 alpha and C8 gamma, are associated non-covalently with the third chain, C8 beta. In order to study the structure and assembly of the C8 molecule, a panel of mAb has been produced against the C component C8. Eight of these mAb had reactivity to the C8 alpha-gamma subunit, whereas four reacted with C8 beta. One of the C8 alpha-gamma mAb, C8A2, had specificity for an epitope on the C8 alpha-chain and exhibited no cross-reactivity to any of the other terminal C components, including C8 beta. C8A2 inhibited the hemolytic activity of the C8 alpha-gamma subunit but had no effect on the activity of fluid phase whole C8 or C8 within membrane-bound C5b-8. Functional experiments suggest that C8A2 inhibits C8 alpha-gamma activity by interfering with its interaction with the C8 beta-chain. In an enzyme immunoassay using the C8A2 mAb, free C8 alpha-gamma subunit could be detected in both homozygous and heterozygous C8 beta-deficient serum. However, only low level binding was observed when homozygous C5- and C7-deficient sera were tested. Thus the mAb, C8A2, recognizes an epitope expressed on the C8 alpha-gamma subunit but not on intact C8 and can detect free C8 alpha-gamma in the presence of native C8.     

中国靴耳属分类研究概况

对已报道的我国Crepidotus属种类进行了全面汇总,并概述了其分类研究现状。文献调查结果表明:国内已报道的该属种类名称共31个,包括30个种和1个变种,分布于全国26个省区。已报道的全部记录中,28个分类单元引证了标本,其余3个记录缺乏标本引证;7个记录的描述与国外权威描述存在差异,另有4个记录的报道未提供形态描述。淡紫靴耳不是Crepidotus属的成员,已被组合到其他属中。笔者接受"靴耳属"为Crepidotus属的汉语学名,并以此为属主名对属下的分类单元进行了全面修订。     

十七种虫草的子实体培育研究

在过去的12年间,分离得到20余种虫草无性型,对其中十七种虫草无性型进行了人工诱发虫草子座的试验,结果表明:蜂头虫草Cordycepssphecocephala、亚黄蜂虫草C.oxycephala、蚂蚁草C.myrmecophila、蛹虫草C.militaris、拟细虫草C.gracilioides和布氏虫草C.brongniatii等6种人工培养的虫草观察到了成熟的子囊壳并弹射了子囊孢子;古尼虫草小孢变种C.gunniivar.minor、球孢虫草C.bassiana、戴氏虫草C.taii、蚁虫草C.formicarum、蝽虫草C.nutans、长座虫草C.longissima、台湾虫草C.formosana和双梭孢虫草近似种C.bifusisporaaff.长出了许多的未成熟的子座或孢梗束;而冬虫夏草C.sinensis、沫蝉虫草C.tricentri和细虫草C.gracilis未能培育出子实体。其中蚁虫草和亚黄蜂虫草的人工成功培育子实体为首次报道。     

Proximity of cytoplasmic and periplasmic loops in NhaA Na+/H+ antiporter of Escherichia coli as determined by site-directed thiol cross-linking

The unique trypsin cleavable site of NhaA, the Na(+)/H(+) antiporter of Escherichia coli, was exploited to detect a change in mobility of cross-linked products of NhaA by polyacrylamide gel electrophoresis. Double-Cys replacements were introduced into loops, one on each side of the trypsin cleavage site (Lys 249). The proximity of paired Cys residues was assessed by disulfide cross-linking of the two tryptic fragments, using three homobifunctional cross-linking agents: 1,6-bis(maleimido)hexane (BMH), N,N'-o-phenylenedimaleimide (o-PDM), and N,N'-p-phenylenedimaleimide (p-PDM). The interloop cross-linking was found to be very specific, indicating that the loops are not merely random coils that interact randomly. In the periplasmic side of NhaA, two patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently between the double-Cys replacements A118C/S286C, N177C/S352C, and H225C/S352C; (b) only BMH cross-links the double-Cys replacements A118C/S352C, N177C/S286C, and H225C/S286C. In the cytoplasmic side of NhaA, three patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently the pairs of Cys replacements L4C/E252C, S146C/L316C, S146C/R383C, and E241C/E252C; (b) BMH and p-PDM cross-link efficiently the pairs of Cys replacements S87C/E252C, S87C/L316C, and S146C/E252C; (c) none of the reagents cross-links the double-Cys replacements L4C/L316C, L4C/R383C, S87C/R383C, A202C/E252C, A202C/L316C, A202C/R383C, E241C/L316C, and E241C/R383C. The data reveal that the N-terminus and loop VIII-IX that have previously been shown to change conformation with pH are in close proximity within the NhaA protein. The data also suggest close proximity between N-terminal and C-terminal helices at both the cytoplasmic and the periplasmic face of NhaA.     

A phylogenetic study on galactose-containing Candida species based on 18S ribosomal DNA sequences

Phylogenetic relationships of 33 Candida species containing galactose in the cells were investigated by using 18S ribosomal DNA sequence analysis. Galactose-containing Candida species and galactose-containing species from nine ascomycetous genera were a heterogeneous assemblage. They were divided into three clusters (II, III, and IV) which were phylogenetically distant from cluster I, comprising 9 galactose-lacking Candida species, C. glabrata, C. holmii, C. krusei, C. tropicalis (the type species of Candida), C. albicans, C. viswanathii, C. maltosa, C. parapsilosis, C. guilliermondii, and C. lusitaniae, and 17 related ascomycetous yeasts. These three clusters were also phylogenetically distant from Schizosaccharomyces pombe, which contains galactomannan in its cell wall. Cluster II comprised C. magnoliae, C. vaccinii, C. apis, C. gropengiesseri, C. etchellsii, C. floricola, C. lactiscondensi, Wickerhamiella domercqiae, C. versatilis, C. azyma, C. vanderwaltii, C. pararugosa, C. sorbophila, C. spandovensis, C. galacta, C. ingens, C. incommunis, Yarrowia lipolytica, Galactomyces geotrichum, and Dipodascus albidus. Cluster III comprised C. tepae, C. antillancae and its synonym C. bondarzewiae, C. ancudensis, C. petrohuensis, C. santjacobensis, C. ciferrii (anamorph of Stephanoascus ciferrii), Arxula terrestris, C. castrensis, C. valdiviana, C. paludigena, C. blankii, C. salmanticensis, C. auringiensis, C. bertae, and its synonym C. bertae var. chiloensis, C. edax (anamorph of Stephanoascus smithiae), Arxula adeninivorans, and C. steatolytica (synonym of Zygoascus hellenicus). Cluster IV comprised C. cantarellii, C. vinaria, Dipodascopsis uninucleata, and Lipomyces lipofer. Two galactose-lacking and Q-8-forming species, C. stellata and Pichia pastoris, and 5 galactose-lacking and Q-9-forming species, C. apicola, C. bombi, C. bombicola, C. geochares, and C. insectalens, were included in Cluster II. Two galactose-lacking and Q-9-forming species, C. drimydis and C. chiropterorum, were included in Cluster III.     

Human C4 haplotypes with duplicated C4A or C4B

In the course of study of families for the sixth chromosome markers HLA-A, C, B, D/DR, BF, and C2, the two loci for C4, C4A, and C4B, and glyoxalase I, we encountered five examples of probable duplication of one or the other of the two loci for C4. In one of these, both parents and one sib expressed two different structural genes for C4B, one sib expressed one, and one sib expressed none, suggesting that two C4B alleles were carried on a single haplotype: HLA-A2, B7, DR3, BFS1, C2C, C4A2, C4B1, C4B2, GLO1. In a second case, two siblings inherited C4B*1 and C4B*2 from one parent and C4B*Q0 from the other. This duplication appeared on the chromosome as HLA-AW33, B14, DR1, BFS, C2C, C4A2, C4B1, C4B2, GLO2. In a third, very large family with 3 generations, a duplication of the C4B locus occurred which was followed in 2 generations. In one individual, there were three C4B alleles and two C4A alleles. One of the C4B alleles had a hemolytically active product with electrophoretic mobility near C4B2 and was designated C4B*22. It segregated with C4B1 in the family studied. The complete haplotype was HLA-A11, CW1, BW56, DR5, BFS, C2C, C4A3, C4B22, C4B1, GLO2. In another family with 12 siblings, one parent and eight children expressed two C4A alleles on the haplotype HLA-AW30, BW38, DR1, BFF, C2C, C4A3, C4A2, C4BQ0, GLO1.(ABSTRACT TRUNCATED AT 250 WORDS)     

The low C5 convertase activity of the C4A6 allotype of human complement component C4.

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.     

Activation of C1r by proteolytic cleavage.

C1r was unable to cleave and activate proenzyme C1s unless first incubated at 37 degrees C in the absence of calcium before the addition of C1s. The acquisition of ability to activate C1s was associated with, and paralleled by, cleavage of each of the two noncovalently bonded 95,000 dalton chains of the molecule into disulfide linked subunits of 60,000 and 35,000 daltons, respectively. Thus, C1r is converted from an inactive form into an enzyme, C1r, able to cleave and activate C1s by proteolytic cleavage in marked analogy to the activation of several other complement enzymes. Trypsin was also found to cleave C1r but at a different site, and its action did not lead to C1r activation. C1r activation was inhibited by calcium, polyanethol sulfonate, C1 inactivator, and DFP but not by a battery of other protease inhibitors. C1 inactivator inhibited C1r by forming a complex with C1r via sites located on the light chain of the molecule. In other studies, cleavage of C1r was not accelerated by the addition of C1r ot C1s. C1r and C1r were found to have the same m.w., sedimentation coefficient, and diffusion coefficients. They differed, however, in charge with C1r migrating as a Beta-globulin and C1r as a gammaglobulin on electrophoresis in agarose. The amino acid composition of C1r and of each of the two polypeptide chains of Clr was determined. Both chains contained carbohydrate. Proteolytic cleavage of the C1r molecule was found to occur on addition of aggregated IgG to a mixture of C1q, C1r, and C1s in the presence of calcium. Neither C1q, C1s nor aggregated IgG alone, not C1r nor C1s induced C1r cleavage. Liquoid, an inhibitor of C1 activation, inhibited C1r cleavage. Thus, proteolytic cleavage of C1r appears to be a biologically meaningful event occurring during the activation of C1.     

Conformational changes of the subunits C1q, C1r and C1s of human complement component C1 demonstrated by 125I labeling

C1s and C1r proenzymes and enzymes (C1s, C1r) and C1q were labeled with 125I. The distribution of the 125I label between H- and L-chain of C1s was only slightly dependent on the state of activation of C1s, and approx. 90% of the label was found in the H-chain. In the C1r proenzyme molecules 50% of the label was incorporated into the H-chain. The C1r H-chain label was reduced to 10% on activation of C1r to C1r, while the L-chain label increased to 90% of the total label. The presence of either C1s, C1q or C1qs during labeling reduced the C1r H-chain level, although C1r remained in the proenzyme form. The presence of C1s or C1rs enhanced the 125I uptake of C1q in Ca2+ or EDTA medium. This was unexpected because one would have anticipated a diminution of the C1q label due to the apposition of C1r and C1s, similarly as it occurs during C1rs complex and C1s dimer formation for the H-chain label of C1s. The results show that C1r and C1q alter their conformation during activation and C1 complex formation.     

The release of C5a in complement-activated serum does not require C6

The influence of terminal complement components on the generation and release of the complement C5a fragment was investigated by comparing the levels of C5a in complement-activated serum with the levels of C5a produced in serum depleted of complement C6. In order to investigate the release of C5a, a modified C5a assay was developed that utilizes an anti-C5b monoclonal antibody to remove C5, C5b, and C5b-C5a complexes from samples prior to C5a assay. The modified assay was developed because the standard methodology, which includes an acid-precipitation step designed to dissociate C5a and C5b, cannot distinguish free C5a from the C5a that is bound to C5b. Therefore, the standard methodology is not capable of monitoring the influence of terminal components on C5a/C5b dissociation. Levels of C5a were measured in complement-activated whole human serum, in serum depleted of C6, and in serum containing inhibitory levels of anti-C6 Fab using both the modified C5a assay and the standard methodology. Sera were complement-activated with either zymosan to activate the alternative complement pathway or with antibody-coated sheep erythrocytes to activate the classical pathway. The levels of free C5a in C6-depleted sera after activation were equivalent to the C5a levels in activated whole serum, indicating that C6 is not required for the release of C5a from C5b. In addition, the quantity of C5a detected in zymosan-activated sera using the standard acid-precipitation methodology was greater than C5a levels when assayed using the modified immunoadsorption technique, confirming that acid-treatment enhances the C5a dissociation and promotes C5a recovery. Since the other terminal components, C7, C8, and C9, bind to C5b only after C5b only after C6 is bound, these results indicate that none of the terminal components are required for the release of C5a. Although the terminal components could influence the rate of C5a release, the quantity of C5a released in serum was entirely independent of terminal components.     

The uptake of methonium compounds by isolated slices of rat cerebral cortex

Abstract— The uptakes of a series of methonium compounds, C2, C4, C6, C7, C8, C9 and C10, by isolated slices of rat cerebral cortex were measured. Analyses of the kinetics of uptake of C7, C8, C9 and C10 indicate that their initial influxes consist of two components. A Michaelis-Menten type uptake by carrier can account for the saturable fraction. C7 had the lowest affinity for the carrier, with values increasing for C8, C9 and C10, while C10 had the smallest maximum influx with values increasing for C9, C8 and C7. The unsaturable fraction corresponded to the slow continuous swelling of the tissue. The uptake of C8 was inhibited by other methonium compounds, choline, ACh, physostigmine, ouabain and morphine. C8 could be counter-transported by C7. There is evidence that the main driving force for uptake is the transmembrane potential, but the steric factor may be a restricting influence.     

An indel within the C8 alpha subunit of human complement C8 mediates intracellular binding of C8 gamma and formation of C8 alpha-gamma

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex, or MAC. It is an oligomeric protein composed of three subunits (C8alpha, C8beta, C8gamma) that are products of different genes. In C8 from serum, these are arranged as a disulfide-linked C8alpha-gamma dimer that is noncovalently associated with C8beta. In this study, the site on C8alpha that mediates intracellular binding of C8gamma to form C8alpha-gamma was identified. From a comparative analysis of indels (insertions/deletions) in C8alpha and its structural homologues C8beta, C6, C7, and C9, it was determined that C8alpha contains a unique insertion (residues 159-175), which includes Cys(164) that forms the disulfide bond to C8gamma. Incorporation of this sequence into C8beta and coexpression of the resulting construct (iC8beta) with C8gamma produced iC8beta-gamma, an atypical disulfide-linked dimer. In related experiments, C8gamma was shown to bind noncovalently to mutant forms of C8alpha and iC8beta in which Cys(164)-->Gly(164) substitutions were made. In addition, C8gamma bound specifically to an immobilized synthetic peptide containing the mutant indel sequence. Together, these results indicate (a) intracellular binding of C8gamma to C8alpha is mediated principally by residues contained within the C8alpha indel, (b) binding is not strictly dependent on Cys(164), and (c) C8gamma must contain a complementary binding site for the C8alpha indel.     

Systematics of Otarrha, a new Neotropical subgenus of Chimarra (Trichoptera: Philopotamidae)

Abstract. Subgenus Otarrha is established in genus Chimarra to include eighteen described species formerly placed either in subgenus Chimarra or unplaced to subgenus, and thirteen new species. All species are Neotropical, with collective distributions primarily in the Antilles (Greater and Lesser) and northern South America. One species occurs in southeastern Brazil and another species in Costa Rica and Panama. New species are described, recognized species redescribed (except for C. diannae and C. koki ), and a key to the identification of males in the subgenus is provided. Additionally, characters supporting monophyly of the subgenus and a phylogeny of its species are proposed. Described species transferred to this subgenus include Chimarra cubanorum Botosaneanu, C . diakis Flint, C . diannae Flint & Sykora, C . dominicana Flint, C . garciai Botosaneanu, C . guapa Botosaneanu, C . jamaicensis Flint, C . koki Botosaneanu, C . machaerophora Flint, C . patosa Ross, C . puertoricensis Flint, C . quadrifurcata Botosaneanu, C . retrorsa Flint, C . rossi Bueno-Soria, C . sensillata Flint, C . septemlobata Flint, C . septifera Flint and C . spinulifera Flint. Chimarra patosa is designated the type species of the subgenus. New species described in Otarrha include Chimarra amazonia (Peru), C . barinas (Venezuela), C . darlingtoni (Cuba), C . diaphora (Venezuela), C . incipiens (Venezuela), C . odonta (Brazil), C . parene (Peru), C . parilis (Peru), C . particeps (Peru), C . peruana (Peru), C . phthanorossi (Colombia), C . redonda (Dominican Republic) and C . tachuela (Venezuela). Two additional species are described and left incertae sedis to subgenus, Chimarra usitatissima Flint and C . angularis , sp.n. (Venezuela, Guyana).     

Variations in the enzymatic properties of human complement subcomponent C1s by treatment with human plasma kallikrein

We have investigated the effect of plasma kallikrein digestion upon hydrolytic activities of human C1s. Incubation of C1s (85 kDa) with plasma kallikrein led to progressive cleavages on the heavy chain to yield C1s-K1 (70 kDa) then C1s-K2 (53 kDa). Although these cleavages caused little change in the C2 hydrolytic and esterase activities of C1s, a marked loss in the C4 hydrolytic activity was observed. C1s-K1 and C1s-K2 were purified by DE-52 chromatography and it was found that the proteolysis of C1s into C1s-K1 was accompanied with a decrease in the C4 hydrolytic activity. Although the turnover numbers for the hydrolysis of C4 by C1s-K1 and C1s-K2 were almost the same as that of intact C1s, the Kms for C4 of C1s-K1 and C1s-K2 were found to be increased to 10 times that of intact C1s. This result suggests that the apparent decrease in the C4 hydrolytic activity upon plasma kallikrein digestion of C1s is not due to disruption in the active site but is due to decrease in the affinity between C4 and the C1s derivatives. In support of this assumption, C1s-K1 was found to be devoid of the ability to bind C4b-Sepharose. C1s is capable of forming a dimer through the C1s-binding domain in the N-terminal side of the heavy chain. Although C1s-K1 is still capable of forming a dimer, C1s-K2 fails to form a dimer, suggesting that the N-terminal C1s-binding site is released during cleavage of C1s-K1 into C1s-K2.(ABSTRACT TRUNCATED AT 250 WORDS)     

六种苏铁属植物的羽片比较解剖学研究

研究了锈毛苏铁（Cycas ferruginea),石山苏铁（C.miquelii),四川苏铁（C.szchuanensis),海南苏铁（C.hainanensis),仙湖苏铁（C.fairylakea)和贵州苏铁（C.guizhouensis)等六种苏铁属植物羽片的比较解剖学,结果显示锈毛苏铁与石山苏铁在下皮层厚壁细胞,海绵组织中含晶细胞,中脉隆起,叶缘形态,韧皮部形态及分泌道的有无等特征上具有较明显的差异,四川苏铁与海南苏铁和仙湖苏铁非常相近;海南苏铁与仙湖苏铁基本一致;贵州苏铁在中脉隆起,叶缘形态,韧皮部形态等方面与四川苏铁,海南苏铁及仙湖苏铁三者有差异。支持将锈毛苏铁,石山苏铁独立为种的观点,并认为海南苏铁与仙湖苏铁不能区分,四川苏铁与海南苏铁和仙湖苏铁具有较近的种系关系,贵州苏铁与其它种的种系关系相对较远。     

Antibody-independent activation of C1. II. Evidence for two classes of nonimmune activators of the classical pathway of complement

Nonimmune activation of the first component of complement (C1) by cardiolipin (CL) vesicles present specific features which were not demonstrated on immune complexes. CL vesicles which activate C1 in the presence of C1-inhibitor (C1-INH) were found to bind C1s in the absence of C1r, and to induce a specific C1r-independent cleavage of C1q-bound C1s. Therefore, several known natural nonimmune activators were analyzed by comparing their ability to activate C1 in the presence of C1-INH and to mediate a C1r-independent cleavage of C1s. Freshly isolated human heart mitochondria (HHM) activated C1 only in the absence of C1-INH. However, mitoplasts derived from HHM (HHMP) activated C1 regardless of the presence of C1-INH, and induced a specific cleavage of C1q-bound C1s. The same pattern was observed in the case of smooth E. coli and a semi-rough E. coli strain. DNA, known to activate C1 only in the absence of C1-INH, does not induce C1s cleavage in the absence of C1r. Thus, nonimmune activators can be classified into two distinct categories. "Strong" activators, such as CL vesicles, HHMP, or the semi-rough E. coli strain J5 can activate C1 in the presence of C1-INH. By using C1qs2 as a probe, they exhibit a specific, C1r-independent cleavage of C1s. C1s-binding to C1q is a critical factor for the activation process in this group. In the case of "weak" activators, such as E. coli smooth strains, DNA, or HHM, no C1s-binding to activator-bound C1q was detected, and C1r-independent C1s cleavage and C1 activation in the presence of C1-INH were not observed. As in the case of immune complexes, C1r activation appears to play a key role in the C1 activation by "weak" activators.     

The binding site of human C4b-binding protein on complement C4 is localized in the alpha'-chain

C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. We now demonstrate that C4b contains a binding site for C4BP, which is localized on the alpha'-chain of C4b. SDS-PAGE of C C4 and C4b both under reducing and nonreducing conditions was followed by a radiolabeled C4BP ligand blotting procedure. It was demonstrated that the C4BP binding site on C4b is localized on the alpha'-chain. In addition, we found C4BP binding to the alpha-chain of C4, which suggests that the binding site for C4BP becomes available after reduction of the C4 molecule. Direct binding of C4BP to the alpha- and alpha'-chains of C4 and C4b was demonstrated in a radio-labeled C4BP binding assay with the reduced and alkylated isolated chains. mAb against the alpha'-chain of C4b were prepared, characterized, and evaluated for their ability to block the binding of 125I-C4BP to C4b. Two mAb specific for the alpha'-chain of C4b were found that completely abolished C4BP binding to intact C4b. Other mAb recognizing both the alpha- and alpha'-chain of C4 and C4b demonstrated only minor inhibitory effect on the binding of C4BP to C4b. In conclusion, we have localized the C4BP binding site on the alpha'-chain of C4b and have demonstrated that this binding can be inhibited by mAb specific for the alpha'-chain.     

Evidence that C5b recognizes and mediates C8 incorporation into the cytolytic complex of complement

The aim of this study was to identify constituents of the intermediate C5b-7 complex of human complement that mediate binding of C8 and formation of C5b-8. Analysis of interactions between purified C8 and C5, C6, or C7 indicate that C5 and C8 associate to form a dimer in solution. This interaction is specific and involves a single C5 binding site located on the beta-subunit of C8. Simultaneous interaction of C8 with C5 and C9 in solution suggests that during assembly of the cytolytic C5b-9 complex on membranes, C8 binds to C5b-7 through association of beta with C5b, after which C9 associates through interaction with the previously identified C9-specific site on the alpha-subunit. Other evidence of interaction with C5b was provided by the fact that C8 can bind purified C5b6. Also, in situ cross-linking experiments showed that within C5b-8, the beta-subunit is in close proximity to C5b. These results indicate that C8 binding to C5b-7 is mediated by a specific C5b recognition site on beta, thus explaining the requirement for this subunit in C5b-8 formation. They also reveal that C5b contains a specific site for interaction with beta.