scholarly journals The separation of functionally distinct forms of the third component of human complement (C3)

1981 ◽  
Vol 193 (3) ◽  
pp. 963-970 ◽  
Author(s):  
C Parkes ◽  
R G DiScipio ◽  
M A Kerr ◽  
R Prohaska
Keyword(s):  
Initial Rate ◽  
Alternative Pathway ◽  
Complement C3 ◽  
Freezing And Thawing ◽  
Factor B ◽  
Alpha Chain ◽  
The Third ◽  
Complement Component C3 ◽  
C3 Species ◽  
Order Of Magnitude

Complement component C3 prepared by the method of Tack & Prahl [(1976) Biochemistry 15, 4513-4521] was found to contain the following trace contaminants: C3b, haemolytically inactive C3 with intact alpha- and beta-chains (C3u) and degraded C3 (apparent mol.wt. 140000) with an intact beta-chain but with a fragmented alpha-chain. The proportion of C3u in the C3 is increased on standing and by freezing and thawing. These contaminants could be separated from each other and from native C3 by chromatography on sulphated Sepharose. They have been characterized by their susceptibility to C3b inactivator in the presence of beta 1H, their ability to be cleaved by C3 convertase and their ability to form alternative-pathway C3 convertase in solution. Incubation of C3b or C3u with beta 1H and C3b inactivator resulted in cleavage of the C3 species; the alpha'-chain of C3b was cleaved to fragments of apparent mol.wts. 67000 and 43000, the alpha-chain of C3u was cleaved to fragments of apparent mol.wt. 75000 and 43000. Native C3 and degraded C3 were unaffected by incubation with beta 1H and C3b inactivator. C3u, unlike C3, was not cleaved to C3b by the classical- or alternative-pathway C3 convertase in solution. When C3b or C3 was incubated with factors B and D, forming C3 convertase, the initial rate of factor-B cleavage was several order of magnitude lower in the presence of C3 than in the presence of C3b. The slow rate observed for C3 could be decreased by preincubation with beta 1H and C3b inactivator or by rechromatography of the C3. The degraded C3 did not support factor-B cleavage by factor D.

scholarly journals The mechanism of action of the C3b inactivator (conglutinogen- activating factor) on its naturally occurring substrate, the major fragment of the third component of complement (C3b)

1975 ◽  
Vol 141 (5) ◽  
pp. 1221-1226 ◽  
Author(s):  
JD Gitlin ◽  
FS Rosen ◽  
PJ Lachmann
Keyword(s):  
Alternative Pathway ◽  
Fluid Phase ◽  
Complement C3 ◽  
Classical Pathway ◽  
Physiological Mechanisms ◽  
The Third ◽  
Immune Adherence ◽  
Normal Human ◽  
Molecular Nature ◽  
Third Component Of Complement

The fixation of the third component of complement (C3) results in many important biological phenomenon, among which are (a) immune adherence (1), (b) enhancement of phagocytosis (2,3), (c) the release of an anaphylatoxin which is a potent releaser of histamine (4), and (d) the feedback activation of the alternative pathway (5,6). The physiological mechanisms involving C3 fixation require the generation of a C3 convertase which may occur by two separate pathways. C3 convertase can be generated, in the form of C42, by the so-called classical pathway of activation or in the form C3b,B by the alternative or properdin pathway (7). In both cases, C3 is converted to C3b by cleavage of a small peptide, C3a. Normal human serum contains an inactivator of activated C3b. This C2b inactivator or conglutinogen-activating factor (KAF) has been shown to inhibit both immune hemolysis and the immune adherence properties of C3b and to cause cleavage of C3b in the fixed and fluid- phase stages (8-11). Although it is known that the C3b inactivator is not depleted during its reaction with C3b and that C3b treated with the C3b inactivator becomes extremely sensitive to proteolytic digestion by trypsin and trypsin-like enzymes (9), the exact molecular nature of the action of the C3b inactivator on C3b has not been studied. In an effort to delineate the products of this interaction, purified C3b and C3b inactivator were allowed to react for various specific lengths of time and the products of these reactions were then analyzed.

scholarly journals Binding of fluid-phase complement components C3 and C3b to human lymphocytes

1981 ◽  
Vol 198 (3) ◽  
pp. 509-518 ◽  
Author(s):  
E Sim ◽  
R B Sim
Keyword(s):  
Human Lymphocytes ◽  
Fluid Phase ◽  
Complement C3 ◽  
Human Tonsil ◽  
Complement Components ◽  
Sheep Erythrocytes ◽  
The Third ◽  
Complement Component C3 ◽  
Specific Manner ◽  
Third Component Of Complement

It is known that a population of B-lymphocytes has receptors for the third component of complement, C3, and that these lymphocytes may be identified by their ability to form rosettes with sheep erythrocytes coated with covalently bound fragments of complement component C3. Human tonsil lymphocytes, enriched for B-cells, form rosettes with sheep erythrocytes coated with antibody and complement components C1, C4b and C3b (EAC143b cells). Fluid-phase C3 will inhibit rosette formation between EAC143b and human tonsil lymphocytes over the same concentration range as fluid-phase C3b. C3 is not cleaved to C3b during incubation with lymphocytes or with lymphocytes and EAC143b cells. Fluid-phase 125I-labelled C3 and 125I-labelled C3b bind to lymphocytes in a specific manner. The characteristics of binding of both radioiodinated C3 and radioiodinated C3b are very similar, but the binding oc C3 is again not a result of cleavage to C3b. Salicylhydroxamic acid does not inhibit binding of 125I-labelled C3 to tonsil lymphocytes at concentrations that completely inhibit binding of 125I-labelled C3 to EAC142 cells via the nascent binding site of C3b. It is concluded that C3 and C3b share a common feature involved in binding to lymphocytes bearing receptors for the third component of complement.

scholarly journals Multiple sedimenting species of properdin in human serum and interaction of purified properdin with the third component of complement.

1976 ◽  
Vol 143 (2) ◽  
pp. 241-257 ◽  
Author(s):  
J Chapitis ◽  
I H Lepow
Keyword(s):  
Human Serum ◽  
Gradient Analysis ◽  
Protein Complexes ◽  
Alternative Pathway ◽  
Sucrose Density Gradient ◽  
Factor B ◽  
The Third ◽  
Sedimentation Behavior ◽  
Normal Human ◽  
Third Component Of Complement

Normal human serum subjected to sucrose density gradient analysis exhibited multiple sedimenting species of properdin antigen. Properdin antigen distribution was dependent on serum concentration, ionic strength, temperature, and the presence of C3, and was not dependent on the presence of divalent metal cations or blood coagulation. In mixtures of purified components, properdin sedimented heavier in the presence of C3, C3b, or C3c. Addition of factor B to mixtures containing C3 and properdin was without effect. These data provide insights into earlier discrepancies concerning the sedimentation behavior of partially purified properdin, indicate a propensity of some constituents of the alternative pathway to form protein-protein complexes, and suggest caution in interpretation of immunopathological studies in which properdin deposits are found in the presence of C3.

Proteolytic activity of the different fragments of factor B on the third component of complement (C3). Involvement of the N-terminal domain of Bb in magnesium binding

1990 ◽  
Vol 27 (9) ◽  
pp. 891-900 ◽  
Author(s):  
Pilar Sánchez-Corral ◽  
Luis C. Antón ◽  
JoséM. Alcolea ◽  
Guillermo Marqués ◽  
Angel Sánchez ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Complement C3 ◽  
Factor B ◽  
The Third ◽  
Terminal Domain ◽  
Magnesium Binding ◽  
Third Component Of Complement

scholarly journals The C3 convertase of the alternative pathway of human complement. Enzymic properties of the bimolecular proteinase

1986 ◽  
Vol 235 (3) ◽  
pp. 723-730 ◽  
Author(s):  
M K Pangburn ◽  
H J Müller-Eberhard
Keyword(s):  
Proteinase Inhibitors ◽  
Alternative Pathway ◽  
Serine Proteinase ◽  
Human Complement ◽  
Turnover Number ◽  
Factor B ◽  
Kinetic Reaction ◽  
Equation Modelling ◽  
Progress Curve ◽  
Complement Component C3

The association of Factor B with C3b (the major fragment of complement component C3) in the presence of Mg2+ results in the formation of a bimolecular zymogen, C3b,B, which is activated by the serine proteinase Factor D, generating the C3 convertase, C3b,Bb (EC 3.4.21.47). Cleavage of native C3 by the C3 convertase was monitored by recording the increase in fluorescence associated with C3b formation in the presence of the fluorescent probe 8-anilinonaphthalene-1-sulphonate. Measurements of initial rates of C3b formation at various C3 concentrations were analysed in accordance with the Michaelis-Menten equation, yielding kcat. = 1.78 +/- 0.08 s-1, Km = 5.86 × 10(-6) M and turnover number = 107 min-1. The assay was used to measure the Ki values of a variety of proteinase inhibitors. The C3 convertase has a short half-life, owing to spontaneous dissociation of the complex. The t1/2 and kcat./Km of the enzyme were determined by fitting an equation modelling both the kinetic reaction and enzyme decay to the fluorimetrically measured progress curve. The enzyme, C3b,Bb, exhibited a t1/2 of 90 +/- 2 s and a kcat./Km of 31.1 × 10(4) +/- 0.8 × 10(4) M-1 × s-1 at physiological pH, ionic strength and temperature. The enzyme that initiates activation of the alternative pathway, C3(H2O),Bb, was also examined. It was slightly less stable (t1/2 = 77 +/- 3 s) and exhibited only half the activity of C3b,Bb (kcat./Km = 16.3 × 10(4) +/- 1.0 × 10(4) M-1 × s-1).

scholarly journals Effect of heparin on complement activation and lysis of paroxysmal nocturnal hemoglobinuria (PNH) red cells

Blood ◽  
1977 ◽  
Vol 50 (2) ◽  
pp. 239-247 ◽  
Author(s):  
GL Logue
Keyword(s):  
Complement Activation ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Alternative Pathway ◽  
Red Cells ◽  
Complement C3 ◽  
Red Cell ◽  
The Third ◽  
Biphasic Effect ◽  
Third Component Of Complement

Abstract The effect of heparin upon the binding of the third component of complement (C3) to PNH red cells in vitro and their subsequent hemolysis is described. Heparin, in increasing concentrations, progressively inhibits membrane C3 fixation and hemolysis when the classic complement pathway is activated by anti-red cell antibodies. Heparin has a biphasic effect upon membrane C3 fixation and hemolysis when complement is activated in serum at decreased ionic strength (sucrose lysis) or in serum at decreased pH (Ham test). Heparin in concentrations above 2 U/ml inhibits C3 binding and hemolysis while lower concentrations of heparin enhance the consequences of complement activation by these two procedures. This enhanced complement activation may explain the increased hemolysis sometimes reported in PNH patients treated with heparin, and suggests that heparin may aggravate the consequences of pathologic alternative pathway complement activation in other diseases.

scholarly journals Effect of heparin on complement activation and lysis of paroxysmal nocturnal hemoglobinuria (PNH) red cells

Blood ◽  
1977 ◽  
Vol 50 (2) ◽  
pp. 239-247 ◽  
Author(s):  
GL Logue
Keyword(s):  
Complement Activation ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Alternative Pathway ◽  
Red Cells ◽  
Complement C3 ◽  
Red Cell ◽  
The Third ◽  
Biphasic Effect ◽  
Third Component Of Complement

The effect of heparin upon the binding of the third component of complement (C3) to PNH red cells in vitro and their subsequent hemolysis is described. Heparin, in increasing concentrations, progressively inhibits membrane C3 fixation and hemolysis when the classic complement pathway is activated by anti-red cell antibodies. Heparin has a biphasic effect upon membrane C3 fixation and hemolysis when complement is activated in serum at decreased ionic strength (sucrose lysis) or in serum at decreased pH (Ham test). Heparin in concentrations above 2 U/ml inhibits C3 binding and hemolysis while lower concentrations of heparin enhance the consequences of complement activation by these two procedures. This enhanced complement activation may explain the increased hemolysis sometimes reported in PNH patients treated with heparin, and suggests that heparin may aggravate the consequences of pathologic alternative pathway complement activation in other diseases.

scholarly journals Analysis of the interactions between properdin, the third component of complement (C3), and its physiological activation products

1988 ◽  
Vol 252 (1) ◽  
pp. 47-54 ◽  
Author(s):  
T C Farries ◽  
P J Lachmann ◽  
R A Harrison
Keyword(s):  
Ionic Strength ◽  
Alternative Pathway ◽  
Fluid Phase ◽  
Complement C3 ◽  
Initial Product ◽  
Native Form ◽  
Factor I ◽  
The Third ◽  
Activation Products ◽  
Third Component Of Complement

The interactions of properdin with both surface-bound and fluid-phase C3 (the third component of complement) and its activation products have been investigated by using a purified preparation of the ‘native’ form. At physiological ionic strength, a weak interaction with cell-bound C3b (the larger activation fragment of C3) could be demonstrated. In the presence of Factor B this interaction was enhanced, and further enhancement was seen when C3bBb sites were formed on the erythrocytes. The avidities of properdin for cell-bound iC3b (the initial product of Factors I and H action on C3b) and C3b were compared at low ionic strength, with that measured for iC3b being less than that for C3b. In contrast, the affinities of properdin for fluid-phase C3b, iC3b and C3c (the larger product of Factors I and H or CR1 (the C3b receptor) action on iC3b) were all very similar, and apparently much weaker than that for cell-bound C3b. No interaction with either native C3 or, more surprisingly, C3i (haemolytically inactive C3) could be detected. Properdin also inhibited Factor I binding to, and action upon, cell-bound C3b, but did not inhibit Factor I action on fluid-phase C3b. These data permit a more detailed description of the roles of properdin in the alternative pathway of complement activation, emphasizing its importance in concentrating activation at the activating surface.

scholarly journals The binding of complement component C3 to antibody-antigen aggregates after activation of the alternative pathway in human serum

1981 ◽  
Vol 195 (2) ◽  
pp. 471-480 ◽  
Author(s):  
K J Gadd ◽  
K B M Reid
Keyword(s):  
Human Serum ◽  
Heavy Chain ◽  
Alternative Pathway ◽  
Complement Component ◽  
Amide Bond ◽  
Alpha Chain ◽  
Complement Component C3 ◽  
Alternative Pathway Of Complement ◽  
Immune Aggregates ◽  
Covalently Bound

Preformed immune aggregates, containing antigen and either IgG (immunoglobulin G) or F(ab')2 rabbit antibody, were incubated with normal human serum under conditions allowing activation of only the alternative pathway of complement. Both the IgG and F(ab')2 immune aggregates bound C3b, the activated form of the complement component C3, in a similar manner, 2-3% of the C3 available in the serum being bound to the aggregates as C3b, and the rest remaining in the fluid phase as inactive C3b or uncleaved C3. It was found that the C3b was probably covalently bound to the IgG in the aggregates, since C3b-IgG complexes could be demonstrated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, after repeated washing with buffers containing high salt or boiling under denaturing conditions. Incubation of the C3b-antibody-antigen aggregates in buffers known to destroy ester linkages had little effect on the C3b-IgG complexes, which suggested that C3b and IgG might be linked by an amide bond. Two main types of C3b-IgG complexes were found that had apparent mol.wts. of 360000 and 580000, corresponding to either one to two C3b molecules respectively bound to one molecule of antibody. On reduction of the C3b-IgG complexes it was found that the beta-chain, but not the alpha'-chain, of C3b was released along with all the light chain of IgG but only about half or less of the heavy chain of IgG. These results indicate that, during activation of the alternative pathway of complement by immune aggregates containing IgG antibody, the alpha'-chain of C3b may become covalently bound at one or two sites in the Fd portion of the heavy chain of IgG.

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