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1.
Glycophorin A inhibits lysis by the complement attack phase 总被引:1,自引:0,他引:1
A glycoprotein from human erythrocyte membranes has been found to inhibit lysis of target cells by the attack-phase components C5-C9 from human complement. The inhibiting molecule was purified and identified as glycophorin A. Thus, glycophorin A may have a regulatory function in the lytic complement attack on isologous cells. 相似文献
2.
Activation of the first component of complement, C1: comparison of the effect of sixteen different enzymes on serum C1 总被引:1,自引:0,他引:1
In this study, the effect of sixteen different enzymes on serum C1 and its subcomponents was investigated. The sixteen enzymes could be divided into three groups. First, enzymes which activate native C1: trypsin (optimal concentration 2.4 x 10(-4) mM); alpha-chymotrypsin (2.3 x 10(3) mM); thrombin (1.0 x 10(-5) mM); plasmin (1.9 x 10(-5) mM); elastase (5.8 x 10(-5) mM); pronase (3.0 x 10(-6) mM). All these enzymes are serine esterase and activate native serum C1 bound to EAC4 at the given concentration within 10 min at 30 degrees C. Furthermore, native C1 inhibited by a pentosanpolysulfoester, Sp54, is unable to undergo the internal activation but can be externally activated by the serine esterases. Second, enzymes which do not activate native C1 but result in a dose and time-dependent loss of C1 activity: collagenase; pepsin; carboxypeptidase B. Third, enzymes which have no effect on C1 and C1: Lysozyme; neuraminidase; beta-galactosidase; L-amino acid oxidase; arginase; streptokinase, and acetylcholinesterase. 相似文献
3.
Native human C1 was purified from fresh human serum by affinity chromatography on protein A-bound Sepharose in the presence of 4-nitrophenyl-4-guanidinobenzoate hydrochloride (NPGB) taking advantage of the successive binding of IgG to protein A followed by C1 binding to IgG. After elution the C1 preparation contained IgG as a major contaminant as shown by SDS-PAGE. C1 was further purified by gel filtration. The yield of C1 was 12% and less than 4% of this C1 was activated during purification as assessed by a C4 consumption assay. 相似文献
4.
R J Ziccardi 《Molecular immunology》1985,22(4):489-494
The strength of interaction between the C1q and C1r2S2 subunits of C1 was studied as a function of temp. During centrifugation through sucrose density gradients at 4 degrees C, macromolecular C1 readily dissociated as it sedimented away from its free subunits. In contrast, at 20 degrees C, C1 remained associated as the 16S complex throughout centrifugation, thus indicating a stronger interaction between C1q and C1r2S2 at the higher temp. C1-inhibitor (C1-In) or nitrophenylguanidinobenzoate was present during centrifugation to prevent C1 activation. That native C1 was in fact the species being studied was confirmed by SDS-PAGE analysis. To investigate this temp dependence without using inhibitors, an alternative approach was used. Trace amounts of 125I-C1q were centrifuged through numerous sucrose density gradients, each of which contained a different concn of native C1r2S2 throughout the gradient. The s-rate of 125I-C1q increased with increasing C1r2S2 input. An association constant of 4.9 X 10(7) M-1 was calculated for this reversible interaction at 4 degrees C. However, at 20 degrees C, the data indicated a much higher affinity reaction since the addition of far less C1r2S2 was required for the s-rate of 125I-C1q to reach the 16S plateau. The presence of Cl-In did not affect these results. We have demonstrated that the association of C1q with C1r2S2 increases with increasing temp, a finding suggestive of a hydrophobic interaction. However, since we also show that C1 readily dissociates with increasing NaCl concn, the C1q-C1r2S2 interaction must, in fact, be ionic in nature. We therefore conclude that the temp dependence of the inter-subunit interaction is the result of a conformational change(s) within one of the subunits, and propose that this change may be similar to that occurring during Cl activation. 相似文献
5.
Activation of the third component of complement (C), C3, is central to the functioning of the C system in inflammation. Cleavage of C3 by the C3 convertases of both the classical and alternative pathways results in the formation of two split products, C3b and C3a. C3a inhibited cleavage of C3 by the classical-pathway C3 convertase. The inhibition varied in a concn-dependent relationship, with a concn of approximately 40 micrograms/ml yielding 50% inhibition. Removal of the carboxy terminal arginine from the C3a did not alter the inhibition. C3a did not inhibit cleavage of C3 by the alternative C pathway C3 convertase, or cleavage of C5 by C5 convertase. The C3-cleaving capacity of EAC142oxy that had been previously incubated with C3a could be recovered completely by washing the cells, indicating that the C3a binding to the EAC42oxy cell must have been reversed without having had an effect on the amount of C2 bound. Ribonuclease, a molecule of similar size and charge to C3a, did not affect C3 cleavage and C3a inhibition was not reduced by providing a surface for non-specific adsorption of the C3a, suggesting that the effect of C3a on C3 cleavage was not mediated by non-specific interaction with cell surfaces. C3a inhibited the C3-cleaving capacity of the fluid-phase enzyme, C42oxy, to the same degree as it inhibited the cell-bound enzyme, EAC42oxy, indicating that the C3a must interact with the C42 complex directly. Inhibition of C3 cleavage by C3a is the first demonstration of product inhibition of a complement enzyme. It may provide another control of C3 activation. 相似文献
6.
Doubly radiolabeled soluble complex of the late acting components (SC5b-9) was formed by activating guinea pig complement (C)2 serum mixed with C5 and C9 each labeled with either 125I or 131I with zymosan. It was isolated by sequential gel filtrations and sucrose density gradient centrifugation. Fractions of the complex obtained by centrifugation were heterogeneous in size, composition and hemolytic activity as C9. The heavier fractions contained four to six molecules of C9 per C5b molecule. The lighter fractions contained two to three C9 molecules to one C5b molecule and had stronger C9 hemolytic activity. On recentrifugation on the same sucrose density gradient, the factions moved to the same positions as on the first centrifugation.Guinea pig SC5b-9 complex bound to the surface of EA, EAC-3, EAC-5, EAC-6, EAC-7 and EAC-8 in similar amounts. Therefore, combination of the complex with these intermediate cells seems to be nonspecific. EAC-7 cells bound to the complex could be lysed by addition of C8.Lighter, hemolytically more active complex purified by recentrifugation was stable when incubated alone in buffer. But when it was incubated with free C9, it released radiolabeled C9, the amount released increasing with increase in the amount of free C9 added to a maximum of 10 to 15% of the C9 in the complex. A minute part of the complex was converted to heavier complex by incorporation of additional C9, but most of the complex did not change in size. Therefore, it did not seem to be an uncompleted complex to be saturated with more C9. The C9 molecules released from the complex were in an active form and could be reincorporatd into SC5b-9 when they were treated with zymosan in fresh guinea pig serum.It is concluded that the C9 hemolytic activity of guinea pig SC5b-9 is exerted by transfer of reversibly bound C9 molecules in the complex to C5b-8 sites on EAC-8 cells, not by the action of the complex in toto. 相似文献
7.
beta 1H-hemagglutination assay: a highly sensitive test for human beta 1H and its uses 总被引:2,自引:0,他引:2
Particle-bound human C3b (EAC14oxy23b) serves as the carrier for the detection of biologically active human beta 1 H by IgG-anti-beta 1H. This hemagglutination assay is highly sensitive (greater than or equal to 2 p of beta 1H/40 microliters), easy to perform, rapid, highly reproducible, and needs no expensive laboratory equipment. All reagents are commercially available. The correlation of the beta 1H-hemagglutination assay is applicable for the detection of beta 1H in various buffer systems and may be used for the detection of beta 1H in serum or fractions thereof. It is a very useful tool for the detection of trace contamination of beta 1H in serum fractions, particularly in complement preparations. 相似文献
8.
An efficient method is described for the isolation of highly purified, IgG-free and stable guinea-pig serum C1q. The procedure includes the chromatography of EDTA-treated serum (25 mM EDTA) on CM- and DEAE-cellulose followed by gel filtration on ACA 34-Ultrogel whereby ammonium sulfate precipitation was used for concentration. The final product stored in a glycerol containing buffer was purified 700-fold with a yield of approximately 50%. It was judged to be homogeneous by several criteria including SDS-PAGE, analytical ultracentrifugation, gel filtration and immunoprecipitation. The protein has a sedimentation rate of 11.3 S and consists of three distinct polypeptide chains A, B and C with mol. wts of 30,200, 28,200 and 24,000. Amino acid analysis revealed a content of 4.42% hydroxyproline, 1.81% hydroxylysine and 18.7% glycine. In contrast to human serum C1q a very low content of cysteine residues was detected. SDS-PAGE analysis performed in the absence of 2-mercaptoethanol but in the presence of 5-10% SDS revealed clearly that gps-C1q is dissociated in a time-dependent manner into the individual chains. 相似文献
9.
Immunoadsorbent affinity chromatography, employing a monospecific caprine anti-human C5 (IgG)-agarose bead matrix, was applied to the purification of complement attack complexes from insulin activated normal human serum (SC5b-9). Gel filtration chromatography (Biogel A-15M) of the SC5b-9 complexes eluted from the anti-C5 column by guanidine-HCl was utilized as a second step to obtain a highly purified SC5b-9 preparation. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoretic (SDS-PAGE) analysis of SC5b-9 isolated by immunoadsorbent chromatography revealed the appropriate subunit composition. Complement attack complexes extracted from complement lysed erythrocyte membranes (MC5b-9) upon solubilization with either Triton X-100 (TX-100) or deoxycholate (DOC) were also purified by anti-C5 immunoadsorbent and gel filtration chromatography, but with somewhat less than satisfactory results. Thus, an alternative purification procedure was developed based upon the ability of zwitterionic detergents to selectively extract MC5b-9 from erythrocyte membranes. Zwitterionic detergent solubilized MC5b-9 complexes were isolated in high yield in a one-step purification procedure by gel filtration column chromatography (Biogel A-15M). SDS-PAGE analysis of MC5b-9 isolated in this manner revealed a subunit composition which was similar to SC5b-9 except for the complete absence of S protein.Sucrose density gradient ultracentrifugal analysis of MC5b-9 complexes solubilized by TX-100, DOC, or zwitterionic detergents revealed similar sedimentation profiles with the major portion of MC5b-9 expressing a sedimentation coefficient of29S with minor peaks of 23S and 34S or greater. Thus MC5b-9 complexes solubilized from biological membranes by nonionic, anionic, or zwitterionic detergents form a heterogeneous population of high molecular weight, ordered, oligomeric structures with the 23S form representing MC5b-9 monomer, 29S the MC5b-9 dimer and 34S the MC5b-9 trimer or possibly tetrameric structure. These results clearly suggest a possible relationship between the observed MC5b-9 complex physical heterogeneity and the heterogeneity in the effective size of functional complement lesions. 相似文献
10.
Francesco Tedesco Maria A. Villa Peter Densen Girolamo Sirchia 《Molecular immunology》1983,20(1):47-51
Structural and functional studies were performed on a dysfunctional C8 molecule present in the serum of two siblings and an unrelated individual. The C8 in these three sera exhibited a pattern of partial immunologic identity with C8 in normal serum but was devoid of functional activity. The C8 was immunoprecipitated from the three sera and from a control serum with an antihuman C8 antiserum and analyzed by SDS-PAGE using highly purified human C8 as a reference. A selective absence of a band of 62,000 mol. wt was observed in the immunoprecipitates from the sera containing dysfunctional C8. Experiments performed with the purified α-γ and γ subunits showed that the hemolytic activity of the C8 deficient sera could be reconstituted by the addition of the β chain but not the α-γ dimer. Binding of the dysfunctional C8 to C was excluded by the following observations: (1) EAC 1–7 treated with the C8 deficient sera and then washed could not be lysed after the addition of the β subunit and C9; and (2) the abnormal molecules did not interfere with the consumption of normal C8 by the soluble complex SC5b-7. 相似文献
11.
Rapid purification of the human C3b/C4b receptor (CR1) by monoclonal antibody affinity chromatography 总被引:4,自引:0,他引:4
W W Wong R M Jack J A Smith C A Kennedy D T Fearon 《Journal of immunological methods》1985,82(2):303-313
The human C3b/C4b receptor (CR1) is a polymorphic glycoprotein that is expressed on erythrocytes, leukocytes and glomerular podocytes. Further structural analysis and molecular genetic studies would be facilitated by the availability of relatively larger amounts of purified CR1. Milligram quantities of CR1 were purified from erythrocyte membranes 10,000-fold with an average yield of 30-40% by a rapid procedure which utilized sequential chromatography on Matrex Red A and a monoclonal anti-CR1 antibody affinity column. The purified receptor was homogeneous by SDS-PAGE and consisted of the 2 most common alleles of CR1. Purified CR1 also retained its function of serving as a cofactor for the cleavage of C3b to iC3b, C3dg and C3c. The amino acid composition was typical of that of a globular protein and sequence analysis of the N-terminus of the purified CR1 revealed that it was blocked. 相似文献
12.
H.-H. Mussel T. Ehlen M. Schmitt M.D. Kazatchkine L. Neyses M.P. Dierich 《Immunology letters》1982,4(1):1-6
Applying 2 M KBr, membranes of Ehu were solubilized. By C3-affinity chromatography an activity could be isolated that inhibited the immune adherence reaction and C3b-dependent rosette formation. Since this material did not agglutinate EAC14oxy 23b we termed it monovalent C3b receptor (mC3bR). PAGE and SDS-PAGE and staining with Coomassie brillant blue and PAS reagent revealed a single glycoprotein band with a mol. wt. of 55,000–60,000 daltons and an electrophoretic mobility comparable to ovalbumin. This mC3bR proved to be antigenetically related to gp 205 [17]. The potential of mC3bR to react with C3b-carrying particles was not destroyed by heat and trypsin treatment but by neuraminidase or periodic acid treatment suggesting that mC3bR reacted by its carbohydrate moiety with C3b. As by mC3bR, immune adherence could be inhibited by D-glucose and D-galactose but not by their optical antipodes, L-glucose and L-galactose. 相似文献
13.
Guinea pig C4 (C4gp)4 was isolated from plasma by polyethyleneglycol-4000 (PEG-4000) precipitation, lysine-Sepharose affinity chromatography removal of plasminogen and plasmin, DEAE-Sephacel, SP-Sephadex chromatography, and Sepharose CL-6B gel filtration. Both phenylmethylsulfonyl fluoride (PMSF) and ethylenediamine tetraacetate (EDTA) were present in all Chromatographie buffers, and each C4 pooled concentrate was made 0.5 mM in DFP. The overall yield for three consecutive preparations averaged 3.1% of the initial plasma hemolytic C4 activity and the average specific activity rose to 139,223 U/mg, representing an average purification factor of 157-fold. The preparation of C4 gave a single band on Ouchterlony analysis with antiserum to guinea pig serum or to purified C4. Analytical alkaline discontinuous gel electrophoresis revealed a dominant protein band and functional C4 eluted from replicate gels in this region. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of unreduced C4 gave a mol. wt of 200,000, whereas SDS-PAGE of reduced and alkylated C4 demonstrated subunits of 93,000, 75,000 and 33,000 mol. wt as well as a small amount of non-reducible 200,000 mol. wt material. Elution of the 200,000 mol. wt material recognized by SDS-PAGE under reducing conditions revealed antigenic relatedness to either the α- or β-chain of C4, suggesting that it represents a pro-C4 single chain polypeptide. 相似文献
14.
Factor H, purified from mouse EDTA-plasma using a 4-step procedure, consists of a single polypeptide chain of Mr 150,000 on SDS-PAGE. Mouse H (Hmo) was required for the cleavage of fluid-phase mouse C3b by mouse I (Imo). The final product of degradation of fluid-phase mouse C3b was iC3b, consisting of fragments of the alpha'-chain (alpha'-70, alpha'-43) linked by disulfide bonds to an intact beta-chain. Imo alone was capable of cleavage of membrane-bound mouse C3b and of generating iC3b. The addition of Hmo nevertheless had an enhancing effect on Imo activity, but cleavage did not proceed beyond iC3b. These observations suggest that one important function of Hmo is to permit the inactivation of fluid-phase C3b, and to inhibit irreversibly its activity. The concentration of H in the plasma of male and female BALB/c mice was not significantly different. Among different inbred strains of mice, large differences were observed in the plasma levels of H, and plasma H levels were positively correlated with the plasma levels of C3. This observation, taken together with the well known role of H in the control of the activation of the alternative pathway, suggests that the turnover of C3 is controlled to some extent by H. 相似文献
15.
In vitro synthesis of a 28 kilodalton antigen present on the surface of the schistosomulum of Schistosoma mansoni 总被引:5,自引:0,他引:5
J M Balloul R J Pierce J M Grzych A Capron 《Molecular and biochemical parasitology》1985,17(1):105-114
Adult Schistosoma mansoni proteins were fractionated on polyacrylamide slab gels, recovered by electrophoretic elution and used for immunization of Fischer rats. Three antisera recognizing, respectively, 28, 78 and 85 kDa antigens were obtained. The 28 kDa antigen was found among the in vitro translation products from adult worm RNA, and among the 125I-labelled surface antigens of S. mansoni schistosomula. The isoelectric point of the 28 kDa antigen was 6.3-6.5. The 28 kDa antiserum mediated a cytotoxic activity against schistosomula when used in an in vitro assay in the presence of a purified eosinophil cell population. 相似文献
16.
A comparison of knobby (K+) and knobless (K-) parasites from two strains of Plasmodium falciparum 总被引:9,自引:0,他引:9
T J Hadley J H Leech T J Green W A Daniel M Wahlgren L H Miller R J Howard 《Molecular and biochemical parasitology》1983,9(3):271-278
Erythrocytes infected with Plasmodium falciparum develop knob-like protrusions on their membranes. Knobby (K+) parasites of the FCR-3 (Gambian) strain have been shown to possess a histidine-labelled protein of apparent molecular weight 80 000 which is absent from knobless (K-) variants of the same strain. Here we report similar findings with K+ and K- parasites of another strain, the Malayan Camp strain, and also with cloned K+ and K- parasites of the FCR-3 strain. A histidine-labelled protein unique to the two K+ parasites was identified as a broad band with an apparent molecular weight of 89 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of this protein in both K+ Malayan Camp parasites and K+ FCR-3 (Gambian) parasites and its absence from K- parasites of both strains is consistent with this protein being a major component of knobs. 相似文献
17.
Paul J. Durda Robert J. Pagano Norman Bauman John A. Brockman 《The Journal of allergy and clinical immunology》1982,70(5):353-360
The effects of iodipamide on C3 and factor B in normal human serum and in purified form have been examined by immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Temperature-dependent changes in immunoelectrophoretic profile's have been observed; however, these are not the same as those obtained after treatment of normal human serum (NHS) with cobra venom factor Naja naja. Analyses of iodipamide-treated NHS and purified C3 and factor B by reducing SDS-PAGE indicate that no macromolecular changes have occurred in C3 and factor B that can be ascribed to proteolysis (i.e., activation). The changes observed in C3 and factor B, including loss of hemolytic activity, appear to be due to direct interactions between iodipamide and C3 and factor B. In the case of factor B, iodipamide treatment at 37° C induces aggregation, which is reversible upon reduction with β-mercaptoethanol. 相似文献
18.
T A Gaither C H Hammer J E Gadek K Katusha M Santaella M M Frank 《Molecular immunology》1983,20(6):623-635
Cleavage of C3 by purified leukocyte enzymes and crude extracts of human polymorphonuclear leukocyte (PMN) granules has been reported. We demonstrate that viable PMN mediate the cleavage of erythrocyte-bound C3b and C3bi via cell-associated proteases. Greater than 50% of 125IC3(x) was released from EAC43bix during a 5-min incubation with viable PMN at 37 degrees C. More than a 30-min incubation was required for substantial release from EAC43bx. Culture fluids from PMN suspensions had limited cleaving ability; cleavage of cell-bound C3bx and C3bix was only partially reduced when PMN were preincubated with high levels of soluble C3 which completely blocked EAC43b rosettes. Thus, cell-to-cell contact between opsonized erythrocytes and viable PMN with surface-associated proteases are responsible for cleavage of these opsonic sites. The effect of defined protease inhibitors on PMN cleaving activity as well as on purified leukocyte elastase was examined. Phenylmethylsulfonyl fluoride (PMSF) and the leukocyte elastase inhibitor, methoxy-succinate-alanine-alanine-valine-chloromethyl ketone (MeO) each inhibited cleavage of C3b by 90% and C3bi by 60%. In contrast, the cathepsin-G inhibitor, benzyloxy-carbonyl-glycine-leucine-phenylalanine-chloromethyl ketone (Z) inhibited C3b and C3bi cleavage by less than 20 and less than 5%, respectively. Ethylenediaminetetra-acetate (EDTA), which had a minimal effect on soluble leukocyte elastase, also inhibited PMN-related release. Thus, elastase appeared to be the principle but not the only enzyme responsible for cleavage of C3b and C3bi. PMSF and MeO had a minimal effect on the activity of purified C3bINA (Factor I); and PMN-mediated release of C3b fragments was not inhibited by anti-Factor I and anti-beta 1H (Factor H) IgG and Fab. Thus, these control proteins are not involved in the PMN-mediated cleavage under study. PMN-mediated cleavage of C3b was also inhibited when PMSF- and MeO-treated PMN were washed to remove the fluid phase phase protease inhibitor before adding EAC43b. This suggests that proteases localized in the PMN membrane, prior to the adherence of EAC43b, are responsible for C3b cleavage. Normal human serum was effective in blocking PMN-mediated release activity, while serum from alpha 1 antitrypsin-deficient patients was minimally effective. This suggests a mechanism for the in vivo regulation of PMN-mediated release of C3b and C3bi from opsonized particles by the natural plasma protease inhibitors. 相似文献
19.
C. Juarez J.P. Albar F. Vivanco-Martínez R. Bragado F. Ortíz 《Molecular immunology》1981,18(10):935-943
The structural features of rabbit IgG F(ab')2 fragments required for complement activation through the alternative pathway have been investigated by means of chemical modification of specific groups. These included ionic (amino and carboxyl) and indole groups.Potassium cyanate, glycine-ethyl ester in the presence of EDC,2 and NBB were used as modifying agents of the amino, carboxyl and indole groups respectively. Three different aspects of the modified protein were analysed: the conformational integrity of the protein (by CD and antigenic reactivity), the antibody activity (by the haemaglutination test and radiobinding to the erythrocyte surface) and the capacity to activate complement through-the alternative pathway (by direct haemolysis in conditions in which the classical pathway was blocked).Changes in protein conformation caused by the chemical treatment applied were not detected by the techniques used. An enhancement of the antibody activity of the F(ab')2 fragment against the sheep erythrocyte membrane was observed when it was amidated by glycine-ethyl ester and a decrease when it was carbamylated or two tryptophanyl residues were altered per molecule. The complement-activating capability was preserved after tryptophan modification. On the contrary, modification of amino groups produced a partial decrease in the complement-activating ability and almost total loss of the ability when 34 carboxyl groups were amidated. 相似文献
20.
A H Hale 《Immunology letters》1981,3(3):179-181
The involvement of cell surface components associated with anti-viral cytotoxic T-lymphocytes (CTLs) in the specific lysis of virus-infected target cells was analyzed by the ability of different monoclonal antibodies to specifically inhibit CTL lytic activity. Utilizing monoclonal anti-Lyt antibodies, we showed that Lyt-2 antigens are closely associated with the CTL lytic event. The nature and limitations of this association are discussed. 相似文献