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At least 19 recordsLinked to original sources
PubMed1992

Channel-forming activity of the perforin N-terminus and a putative alpha-helical region homologous with complement C9.

Cytolytic lymphocytes are endowed with a pore-forming protein called perforin. Recently, a cytolytic domain was located in the first 34 residues of the perforin N-terminus. It has been proposed that the first 19 residues are composed of a 3-domain structure including a putative amphipathic beta-sheet and that the 19 residues are sufficient for cytolytic activity. This model has now been tested by synthesizing peptides covering different portions of the N-terminus, and testing their ability to lyse lipid vesicles or increase the conductance of lipid bilayers or plasma membranes. It was found that the putative beta-sheet is indispensable for lytic activity and that the first 19 residues of the N-terminus are required for optimal lytic activity but that shorter peptides, containing only 16 residues, can form pores in lipid bilayers and cell membranes. A putative amphipathic alpha-helix from the central portion of perforin, homologous to complement C9, is nonlytic to lipid vesicles, but it can form pores in lipid bilayers. Taken together, these results support the model that the perforin N-terminus is important in initial pore formation and that the putative alpha-helical domain may be involved in subsequent perforin polymerization into large pores.

Amino Acid Sequence↗
PubMed1990

Several epitopes on native human complement C9 are involved in interaction with the C5b-8 complex and other C9 molecules.

Ten monoclonal antibodies (mAb) against native human C9 exhibiting various inhibitory effects on the hemolytic activity of C9 (Bausback, J., Kontermann, R. and Rauterberg, E. W., Immunobiology 1988. 178: 58) were further analyzed regarding their reactivities with monomeric C9 (mC9), polymerized C9 (pC9), and the non-lytic SC5b-9 complex in enzyme-linked immunosorbent assay and with the membrane attack complex (MAC) generated on rabbit erythrocytes analyzed by flow cytometry. In addition, the inhibitory effects of mAb on zinc-induced C9 polymerization were investigated. One epitope of the C-terminal half of C9b exposed on the surface of pC9 and the MAC seems not to participate directly in lytic function or polymerization since no inhibitory effect of the respective mAb was observed. The nine other mAb directed against epitopes of the C9a part exhibit various inhibitory potentials. The mAb inhibit either hemolysis or polymerization, or both processes. Due to the reactivity with the tested antigens the mAb can be divided into two groups. mAb of the first group bind with nearly the same affinity to all four antigens, whereas mAb of the second group react preferentially with mC9 while their affinity to pC9, SC5b-9 and the MAC is reduced. Comparison of reaction patterns and inhibitory effects strongly suggest that different epitopes on the surface of native C9 are involved in interaction of C9 with C5b-8 and/or in C9-C9 interaction. The finding that mAb inhibiting polymerization of C9 in vitro have no inhibitory effect on hemolysis confirms that C9 polymers are no prerequisite for lysis.

Antibodies, Monoclonal↗
PubMed1992

[Purification of the ninth component of human complement (C9)].

A large amount of human C9 was purified from plasma by the following procedures: 1) Polyethylene glycol precipitation; 2) Depletion of plasminogen by passing over an L-lys-sepharose column; 3) DEAE-sephadex A-50 chromatography; and 4) Hydroxylapatite (HA) chromatography. The method of C9 purification was improved by altering the column-elution conditions and by the establishment of a novel method for preparing high-flow-rate HA. As a result, the rate of recovery of C9 was high (28.2%) and no impurities were detected either on gel electrophoretic or immunochemical examination. The hemolytic activity of purified C9 was retained.

Chromatography↗
PubMed1992

Rapid isolation of human complement component C9 to verify the specificity of a haemolytic C9 microassay.

A sensitive, haemolytic microassay of human complement component C9 was developed. The assay is based on the principle of reactive (C5b6-initiated) haemolysis and uses commercially available C9-depleted serum as reagent for C9. The specificity of the assay was verified by rapid, activity-guided isolation of the haemolytic component from human serum using high-performance liquid chromatography (HPLC) on a system for fast protein liquid chromatography. This isolation yielded a single component with characteristics of C9. The results suggest that rapid, activity-guided isolation as a new application of HPLC can be a useful tool to demonstrate the specificity of a functional assay.

Adult↗
PubMed1979

Isolation of late complement components by affinity chromatography: I. Purification of the human complement component C9 and production of a C9-defective human serum.

A new procedure for the isolation of the human complement component C9 is described. This procedure offers the possibility to prepare functionally pure C9 in a one-step procedure with a high recovery of 10-22% of the biological activity. The 125iodinated C9 had a molecular weight of 78,000 daltons and was only contaminated in traces with other proteins. Further purification by absorption with an "anti-impurity" column lead to a C9 preparation which behaved as a homogenous single polypeptide chain in SDS polyacrylamide gel electrophoresis after reduction with mercaptoethanol. It formed a single bell-shaped precipitate in crossed immunoelectrophoresis with antibodies against human serum in the gel of the second dimension. The recovery of the biological activity after the second purification step was in the order of 6-10%. Both preparative steps could be performed within a few hours 450 microgram C9 protein were isolated from 135 ml human serum. A human serum completely defective in C9 was prepared by the extensive absorption of a smaller volume of human serum proteins with the anti-C9 column.

Complement C9↗
PubMed1991

The relationship between polymerization of complement component C9 and membrane channel formation.

C9 was studied with the objective to clarify the relationship between the process of C9 polymerization and membrane channel formation. Conditions that favor C9 polymerization include low ionic strength and calcium ion in the buffer. Moreover, polymerization is dependent on the concentration of C9. Calcium ion evokes about a threefold increase in the affinity constant for C9 self-association, and at 0 degrees C it imparts reversible amphiphilic properties in the molecule. These were discerned by measuring increases in the degree of reversible nonspecific binding of C9 to hydrophobic (tyramine-zymosan) and hydrophilic (arginyl-glutamyl-zymosan) supports as well as to erythrocytes. At 0 degrees C the hydrophilic-to-amphiphilic alteration of C9 is reversible, but upon incubation at 37 degrees C this transition is rendered permanent with the formation of poly(C9). A functional relationship between C9 polymerization and cytolysis was demonstrated by showing that polymerizing C9 can lyse reduced and alkylated erythrocytes. By studying comparative radiolabeling of tyrosine side chains within thrombin-nicked C9 and its polymerized form, it was demonstrated that upon polymerization the membrane-binding site of C9 becomes exposed. It is concluded that the process of circular polymerization of C9 causes a hydrophilic-to-amphiphilic transition that is required for membrane perforation and channel formation.

Animals↗
PubMed1992

[The association between deficiency of terminal complement components and the occurrence of meningococcal meningitis].

Seventeen patients with sporadic meningococcal meningitis, registered in Fukuoka city, were studied for complement deficiency. Four of the seventeen had a selective deficiency of the ninth component of the complement (C9), four had a deficiency of the seventh component of the complement (C7), and one had a deficiency of the fifth component of the complement (C5) as determined by both hemolytic and antigenic assay. Family studies suggested that these complement deficiencies in the present study were hereditary. Based on the incidence (0.036% of homozygous C9 deficiency in Fukuoka (171 C9 deficient individuals of 475, 886 blood donors), we have concluded that patients with C9 deficiency are more susceptible to meningococcal meningitis than normal individuals (p less than 0.001). In addition, the present study further supports that complement deficiency is common in patients with sporadic meningococcal meningitis.

Adolescent↗
PubMed1992

Purification of the ninth component of the bovine complement cascade.

Conditions for purification of the ninth component of bovine complement (C9) were established. The conditions for binding and elution from diethylaminoethyl cellulose and hydroxylapatite were different than for human C9. Serum albumin, a frequent contaminant of bovine C9 preparations, was removed by chromatography on reactive-red agarose. The calculated molecular weight of bovine C9 was 66,000, and reduction with 2-mercaptoethanol affected its migration on polyacrylamide gel electrophoresis. Some preparations of bovine C9 migrated as 2 bands when partially reduced, but extensively reduced preparations had a single band.

Animals↗
PubMed1992

Molecular modeling of the domain structure of C9 of human complement by neutron and X-ray solution scattering.

C9 is the most abundant component of the membrane attack complex of the complement system of immune defense. This is a typical mosaic protein with thrombospondin (TSR) and low density lipoprotein receptor (LDLr) domains at its N-terminus and an epidermal growth factor-like (EGF) domain at its C-terminus. Between these lies a perforin-like sequence. In order to define the arrangement in solution of these four moieties in C9, high-flux neutron and synchrotron X-ray solution scattering studies were carried out. The neutron radius of gyration RG at infinite contrast is 3.33 nm, and its cross-sectional RG (RXS) is 1.66 nm. Similar values were obtained by synchrotron X-ray scattering after allowance for radiation effects. Stuhrmann analyses showed that the neutron radial inhomogeneity of scattering density alpha is 35 X 10(-5) from the RG data and 16 X 10(-5) from the RXS data. These values are typical for soluble glycoproteins and show no evidence for the existence of any large hydrophobic surface patches on free C9 that might form contacts with lipids. Indirect transformation of the neutron and X-ray scattering curves into real space showed that C9 had a maximum dimension estimated at 12 +/- 2 nm, and this suggests that the lengths of 7-8 nm deduced from previous electron microscopy studies in vacuo are underestimated. Molecular modeling of the C9 scattering curves utilized small spheres in the Debye equation, in which the analyses were constrained by the known volumes of the four moieties of C9 and the known sizes of the TSR and EGF-like domains. The most likely models for C9 suggest that these four regions of C9 are arranged in a V-shaped structure, with an angle of 10 degrees between the two arms, each of length 11.1 nm. This structure has a more hydrophobic character between the two arms. The scattering model is fully consistent with hydrodynamic sedimentation data on C9. Similar V-shaped hydrodynamic models could be developed for C6, C7, C8, and C9 of complement. Such a compact structure is atypical of other multidomain complement proteins so far studied by solution scattering and is fully compatible with mechanisms in which C9 is postulated, on activation, to undergo a drastic unfolding of its domain structure and to expose a more hydrophobic surface which can be embedded into lipid bilayers.

Complement C9↗
PubMed1975

Human complement C7 and C9 in fetal and newborn sera.

Using specific immune sera, C7, C9, and C3 activator were detected in sera from human fetuses more than 16 weeks old and in newborn samples. Levels of C9 in cord sera ranged between 10 and 30% of those present in sera from adult subjects. The mean value of Ce activator was about half that in maternal blood. The mean level of C7 in newborns was nearly 70% of the amount in normal adults.

Animals↗
PubMed1975

Determination of serum C9 level by immunodiffusion. Elevation in patients with infectious or allergic skin diseases.

The measurement of the ninth component of complement (C9) in human serum can be easily carried out by the Mancini method using antiserum raised against purified C9. The average C9 level in 29 healthy adults was 44.5 plus or minus 10.6 mug/ml. The serum C9 level was often elevated in patients with infectious or allergic skin diseases. The C9 level remained normal in most patients with collagen diseases. The availability of the monospecific antiserum has permitted identifying C9 in human serum as alpha2-globulin.

Adult↗
PubMed1991

Assembly of macromolecular pores by immune defense systems.

Immune defence systems (complement, cytolytic lymphocytes) make use of transmembrane pores assembled from up to 20 soluble monomers in a highly regulated process to induce cell death. Inhibitors of pore formation have been found which protect blood, endothelial and epithelial cells from the destructive effect of complement lesions. Recently, a pore-forming protein showing immunological crossreactivity to complement C9 has been found in the protozoan parasite Trypanosoma cruzi, thereby extending this protein family and generalizing its means of generating non-selective membrane permeability.

Animals↗
PubMed1979

Quantitation of the membrane attack complex of complement in an air-driven ultracentrifuge.

A sensitive assay of complement (C) activation via either the classical or alternative pathway was developed by evaluating assembly of the terminal complexes (C5b-9)2 or SC5b-9. Activation of serum containing [125I]C7 resulted in the formation of a stable, radiolabeled complex which was separable from its precursors by sedimentation in an air-driven ultracentrifuge. The radioactivity in the sediment was directly proportional to the amount of complex formed and assembly of the complex could be detected after C activation by aggregated IgG in concentrations as low as 10 micrograms/ml. Mild detergents such as Triton X-100 could be included in the reaction mixture, because they affected neither the assembly nor the integrity of the complexes. The assay, which detects both assembly of the membrane attack complex (MAC or (C5b-9)2) on target membranes and formation of SC5b-9 in fluid phase, measures the potential of certain substances to trigger the cytolytic phase of C regardless of whether the classical or alternative pathway was activated. However, by using serum depleted of either factor B or C1q, activation of either pathway can be assessed individually.

Cell Membrane↗