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 The purification and properties of the second component of human complement

1978 ◽  
Vol 171 (1) ◽  
pp. 99-107 ◽  
Author(s):  
M A Kerr ◽  
R R Porter
Keyword(s):  
Amino Acid ◽  
Polypeptide Chain ◽  
Alternative Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Complement Factor ◽  
Human Complement ◽  
Terminal Amino Acid ◽  
Factor B ◽  
Antigenic Properties ◽  
Terminal Amino

The second component of human complement (C2) was purified by a combination of euglobulin precipitation, ion-exchange chromatography, (NH4)2SO4 precipitation and affinity chromatography. The final product was homogeneous by the criterion of polyacrylamide-gel electrophoresis and represents a purification of about 4000-fold from serum with 15-20% yield. Component C2 comprises a single carbohydrate-containing polypeptide chain, with an apparent mol.wt. of 102000; alanine is the N-terminal amino acid. The molecule is rapidly cleaved by activated subcomponent C1s with the loss of haemolytic activity to yield two fragments with apparent mol.wts. of 74000 and 34000. These fragments are not linked by disulphide bonds and can be easily separated. A second protein isolated during the purification of component C2 was identified by its haemolytic and antigenic properties as complement Factor B, the protein serving an analogous function to component C2 in the alternative pathway. The protein, which is also a single carbohydrate-containing polypeptide chain, has an apparent mol.wt. of 95000 and threonine as N-terminal amino acid. The amino acid analyses of component C2 and Factor B are compared.

scholarly journals Factor B, the complement alternative pathway serine proteinase, is a major constitutive protein synthesized and secreted by resident and elicited mouse macrophages.

1985 ◽  
Vol 161 (2) ◽  
pp. 306-322 ◽  
Author(s):  
J S Sundsmo ◽  
J R Chin ◽  
R A Papin ◽  
D S Fair ◽  
Z Werb
Keyword(s):  
Bone Marrow ◽  
Culture Medium ◽  
Alternative Pathway ◽  
Serine Proteinase ◽  
Mononuclear Phagocytes ◽  
Secreted Protein ◽  
Complement Alternative Pathway ◽  
Factor B ◽  
Plasma Factor ◽  
Conditioned Culture Medium

Factor B, the complement alternative pathway serine proteinase, a class III gene product of the major histocompatibility complex, is a major constitutive secretion product of mouse mononuclear phagocytes. This glycoprotein was synthesized and secreted by macrophages as a doublet of Mr 90,000 and 93,000 polypeptides that were immunoprecipitable with antibodies raised to human serum factor B, and that were indistinguishable from plasma factor B by immunoreactivity, peptide mapping, and molecular weight. Macrophage factor B was cleaved and activated to factor Bb- and Ba-like fragments by factor D and cobra venom factor. Some conversion of macrophage factor B to Bb-sized fragments occurred spontaneously in the conditioned culture medium after several hours. Factor B represented approximately 0.5% of newly synthesized protein and 4-6% of the secreted protein of resident peritoneal macrophages and macrophages elicited with thioglycollate broth, pyran copolymer, NaIO4, bacillus Calmette-Guerin, or Corynebacterium parvum. We detected synthesis of factor B immediately upon explanting these macrophages in culture; synthesis continued for several days in culture. The rate of secretion of factor B, as a proportion of total protein secretion in culture, remained constant with time. By radioimmunoassay, factor B antigens accumulated in the 24-h macrophage-conditioned culture medium at 2-10 nM, and was present in cell lysates at 4-8 nmol per 10(6) cells. We detected synthesis of factor B in bone marrow-derived macrophages as early as 5 d of culture. The P388D1 macrophage line synthesized factor B, but mouse L cells did not. In contrast, apolipoprotein E, another secreted protein of macrophages, was secreted by resident and thioglycollate-elicited macrophages but not by freshly harvested pyran copolymer-activated macrophages. Its synthesis was initiated at day 9 in culture of bone marrow-derived macrophages. These data support the classification of factor B as a constitutive biosynthetic and secreted protein of immature and mature macrophages in various states of activation. Production of factor B was modulated by treatment of macrophages in vivo or in culture with bacterial lipopolysaccharide endotoxin, which increased the synthesis, secretion, and accumulation of factor B up to 11-fold.

scholarly journals The induction of macrophage spreading by factor B of the properdin system.

1979 ◽  
Vol 149 (2) ◽  
pp. 372-386 ◽  
Author(s):  
O Götze ◽  
C Bianco ◽  
Z A Cohn
Keyword(s):  
Alternative Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Serine Proteinase ◽  
Cell Spreading ◽  
Peritoneal Macrophages ◽  
Mononuclear Phagocytes ◽  
Mouse Serum ◽  
Factor B ◽  
Mouse Macrophages ◽  
Alternative Pathway Of Complement

Unstimulated mouse peritoneal macrophages attached to a glass substratum responded to activated human factor B (Bb) of the properdin system but not to native factor B with rapid spreading and a concomitant increase in their apparent surface area. Excellent correlation of the distribution of Bb protein and cell-spreading activity was found upon purification of Bb by ion-exchange and molecular seive chromatography and alkaline polyacrylamide gel electrophoresis. 1.6 microgram of purified Bb was sufficient to induce spreading in 50% of 5 x 10(4) glass attached macrophages within 1-2 h at 37 degrees C. Treatment of Bb with di-isopropyl-fluorophosphate indicated that the intact catalytic site of the serine-proteinase Bb was required for the initiation of macrophage spreading. The involvement of factor B in the induction of rapid cell spreading could also be indirectly demonstrated in an autologous system in which F(ab')2 fragments of an antiserum to mouse B prevented mouse macrophages from spreading in response to complement-activated mouse serum. These experiments suggest a role for factor B and the alternative pathway of complement fixation in the localization of mononuclear phagocytes to areas of inflammation.

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.

Structure and activation of complement components C2 and Factor B

1984 ◽  
Vol 306 (1129) ◽  
pp. 301-309 ◽  
Keyword(s):  
Amino Acid ◽  
Alternative Pathway ◽  
Serine Proteinase ◽  
Classical Pathway ◽  
Serine Proteinases ◽  
Unusual Structure ◽  
Complement Components ◽  
Factor B ◽  
And Function ◽  
Limited Sequence

The activation of complement is initiated by two independent pathways. Each leads to the formation of a complex protease, C3 convertase, with equivalent specificity and function but different composition. The convertase derived from the classical pathway is composed of complement components C4 and C2 while that from the alternative pathway consists of components C3 and Factor B. C2 and Factor B contain the catalytic site of each convertase respectively. The amino acid sequence of Factor B has been determined. Limited sequence of CNBr-peptides isolated from C2 has also been obtained. The two enzymes are shown to be homologous and to represent a novel type of serine proteinase, characterized by their unusual structure and mechanism of activation, when compared to known serine proteinases.

Plasminogen Activator and Plasminogen Activator Inhibitor Associated with Granulomatous Inflammation: A Study with Murine Leprosy

1984 ◽  
Vol 52 (03) ◽  
pp. 243-249 ◽  
Author(s):  
S Izaki ◽  
T Hibino ◽  
Y Isozaki ◽  
P S Hsu ◽  
M Izaki ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Soluble Fraction ◽  
Proteinase Inhibitors ◽  
Granulomatous Inflammation ◽  
Serine Proteinase ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Weakly Alkaline ◽  
Heat Stable ◽  
Type Plasminogen Activator

SummaryPlasminogen activator that is associated with the development of hypersensitivity granulomas (gPA) was partially purified from a saline soluble fraction of murine lepromas elicited in “resistant” mice, C57BL/6N. The gPA was shown to consist of two subspecies (23,000 and 48,000 in molecular weight) with essentially identical enzymologic properties. The gPA was found to be a relatively heat stable weakly alkaline serine proteinase with trypsin-like characteristics in the specificity for synthetic substrates and proteinase inhibitors. It showed a high affinity for H- D-Ile-Pro-Arg-pNA (Km = 1.4 × 10-4 M) H-D-Val-Leu-Lys- pNA (Km = 5.2 × 10-4 M), and L-pyroGlu-Gly-Arg-pNA (Km = 9.3 × 10-4 M). The gPA did not demonstrate antigenic cross reaction with urokinase-type or tissue-type plasminogen activator.Two distinct enzymatic regulators of the gPA were also demonstrated in the saline soluble fraction of the hypersensitivity granulomas. The gPA and its regulation are assumed to be correlated with macrophage activation in the hypersensitivity granulomas

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