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Results for “Complement Activating Enzymes”

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At least 19 recordsLinked to original sources
PubMed1977

Study of equilibration of the system involving two alternative, enzymically active complementing structures simultaneously formed from two overlapping fragments of staphylococcal nuclease.

Quantitative complementation of two overlapping fragments of staphylococcal nuclease, Nuclease-(1-126) (residues 1 to 126) and Nuclease-T-(50-149) (residues 50 to 149), simultaneously forms in 1 min, two alternative, enzymically active ordered structures (types I and II) resembling nuclease (149 residues) (Taniuchi, H., and Anfinsen, C.B. (1971) J. Biol. Chem. 246, 2291-2301). We determined the ratio of type I to type II complex formed from the two fragments as a function of time, temperature, and the presence or absence of the ligands thymidine 3',5'-diphosphate and calcium ion. The ratio of type I to type II complex was determined on the basis of the quantities of their derived complexes obtained after each experiment by removing the redundant amino acid sequences by limited digestion with trypsin in the presence of ligands. The quantity of the derived complexes was estimated by quantitative determination of the component fragments separated by gel filtration. The ratio of type I to type II complex formed in 2 min after mixing the two fragments was approximately 0.3 and appears to be independent of temperature and the presence or absence of ligands. The equilibrium of the system of type I and II complexes is attained through unfolding and folding. The ratios of type I to type II complex at the apparent equilibrium state of the system at 6 and 23 degrees were approximately 1.1 and 2.4, respectively. The observations indicate that the rate of unfolding of type II complex is greater than that of type I complex at 6 degrees and increases more than that of type I complex with increasing temperature. Thus, the change of the complementing structure from type I complex with increasing temperature. Thus, the change of the complementing structure from type I to type II causes a decrease in the activation free energy, an increase in the activation enthalpy, and thereby an increase in the activation entropy of unfolding. Since the unfolded states with which type I and II complexes are in equilibrium are the same, the distribution of the population of type I and II complexes at the equilibrium state will be determined on the basis of the respective decreases in Gibbs standard free energy from the unfolded state to type I and II complexes. On this basis type I complex has a lower energy by deltaG0 = -0.05 and -0.51 kcal mol-1 at 6 and 23 degrees, respectively, than type II complex. Nevertheless, at the initial complementation the population of type I complex formed is approximately one-third that of type II complex at both 6 and 23 degrees. That is, the probability (rate) of folding is not related to the decrease in energy from the unfolded to the folded state. Using van't Hoff's equation deltaH = 7.5 kcal mol-1 and then deltaS degrees = 27 cal deg-1 mol-1 from type II to type I complex.

Amino Acid Sequence↗
PubMed1979

The alternative pathway C3/C5 convertase: chemical basis of factor B activation.

The structural basis of activation of the alternative pathway C3 convertase was explored. For this purpose a modified isolation procedure of the activating enzyme, Factor D, was elaborated. The procedure affords a 70,000-fold purification of the enzyme with a 20% yield. A simple assay was designed for the quantitation of both Factor D and Factor B activity. On the basis of activity measurements and amino acid analysis, Factor D concentration in plasma was estimated to be 1 microgram/ml. Highly purified Factor D was used to activate Factor B in the presence of C3b and Mg++. The resulting fragments, Ba and Bb, were characterized with respect to their circular dichroism spectra, amino acid compositions, reactive sulfhydryl groups, and partial amino- and carboxy-terminal sequences. The results indicate that the Ba fragment constitutes the amino-terminal region and the Bb fragment the carboxy-terminal region of Factor B. The bond in Factor B that is cleaved by Factor D is proposed to be an arginyl-lysine bond.

Chemotaxis, Leukocyte↗
PubMed1978

The biochemistry of complement.

Current biochemical studies of the complement system are illustrated by description of the activation of complement by the classical pathway after interaction with antibody aggregates. This is described in terms of the structures of the components involved, their assembly and the mechanism of activation.

Amino Acid Sequence↗
PubMed1979

Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane.

An activity that is inhibitory to the properdin-stabilized amplification C3 convertase (C3b,Bb,P) was solubilized from human erythrocyte (E(hu)) membranes by Nonidet P-40 and purified to homogeneity. The inhibitory membrane glycoprotein had an apparent M(r) of 1-1.2x10(6) on gel filtration in the presence of Nonidet P-40. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis it presented a single stained band with an apparent M(r) of 205,000, with or without prior reduction of disulfides. The inhibitory protein of the E(hu) membrane produced a dose-related, first-order decay of C3b,Bb,P function on sheep erythrocytes (E(s)) and released (125)I-labeled Bb from these sites, indicating a mechanism of inhibition by decay-dissociation of the amplification C3 convertase. The 50% inhibitory dose of the E(hu) membrane protein was not altered by removal of sialic acid from the E(s) bearing C3b,Bb,P sites. E(hu) membrane protein also serves as a cofactor for C3b inactivator-induced cleavage of the alpha polypeptide chain of C3b. Thus, the inhibitory membrane protein can abrogate the activity of amplification convertase sites that have formed and also can prevent generation of such sites by augmenting irreversible inactivation of C3b.Discrimination between cells by the alternative complement pathway occurs after initial deposition of C3b and is related to the modulation by surface constituents of the capacity of bound C3b to function as a subunit of the amplification C3 convertase. The existence in the E(hu) membrane of a protein that can impair the functions of membrane-bound C3b and C3b,Bb,P could represent a molecular basis for preventing inappropriate self-recognition.

Binding Sites↗
PubMed1992

Complement activation in the follicular light zone of human lymphoid tissues.

A comparative immunohistochemical study of the distribution pattern of complement components and regulatory proteins within secondary lymphoid follicles was performed by the immunoperoxidase technique. Fifteen lymphoid tissues including appendices. Peyer's patches and tonsils were analysed. Sixty secondary lymphoid follicles with evident polarity, that is, the distinct coexistence of a light zone, dark zone and mantle zone in the same lymphoid follicle, were tested with single antibodies. The light zones were consistently immunostained in a dendritic meshwork pattern with all antibodies. The immunostaining patterns were classified into two major groups based on the immunoreactivity of the dark zone. One immunostaining pattern was characterized by no immunostaining of the dark zone to the majority of the antigens. The second group was characterized by a diffusely weak to moderate dendritic meshwork pattern of the dark zone to some of the immunostainings of C9 (monoclonal), S-protein, and DF-DRC1, and all immunostainings of CR1 (CD35), Ber-Mac-DRC (CD35), CR2 (CD21), and R4/23. All four complement regulatory proteins were localized by immunoelectron microscopy attached to the cell surface of the cells, including follicular dendritic cells, in the light zone. Our data indicate that there is an evident functional difference between the light zone and the dark zone, and that complete activation of the complement system occurs only in the light zone.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1992

Effects of excess factor D on early- and late-phase activation of the complement cascade.

The present study was undertaken to examine the effects of excess factor D build-up in the body of end-stage renal disease (ESRD) patients upon the activation of the alternative pathway and the terminal pathway in the fluid phase. First, to clarify the effect of excess factor D on the alternative pathway, purified factor D from an ESRD patient was added to normal serum and the changes in concentrations of C3a-des-Arg and C5a-des-Arg were investigated. The results showed that once the serum factor D level reached a concentration corresponding to 15 micrograms/ml in the serum of the ESRD patient, the C3a-des-Arg and C5a-des-Arg levels had climbed to about 1.7-fold the concentration in normal serum. Next, in order to clarify the effect of excess factor D on the terminal pathway, purified factor D was added to normal serum, and the changes in C5b6 generation were examined. The results indicated that as the factor D level increased in the serum, the C5b6 level rose gradually also; and when the factor D concentration reached 15 micrograms/ml, the C5b6 generation had risen to approximately 1.5-fold the level in normal serum. The present results therefore suggest that factor D build-up in ESRD patients provides a uremic toxin that can cause abnormal activation of the whole complement cascade.

Complement Activating Enzymes↗
PubMed1979

Activation of the classical pathway of complement by the C3NeF-stabilized cell-bound amplification convertase.

C3 nephritic factor (C3NeF) has been shown to be composed of two heavy and two light chains, like IgG; in addition it shares antigenic determinants with IgG. C3NeF, purified from the sera of eight patients by incorporation of C3NeF into the stabilized fluid phase amplification C3 convertase, C3bBb(C3NeF), followed by its release after decay of convertase function, was investigated for its ability to bind 125I-C1q and to activate 125I-C1. It was found that although fluid phase C3b,Bb(C3NeF) is fully capable of binding 125I-C1q, it is not able to activate 125I-C1 even at concentrations of 1.3 x 10(12) C3bBb(C3NeF) complexs/ml. On the other hand, cell-bound C3bBb(C3NeF) is capable of both binding 125I-C1q and activating 125I-C1. This discrepancy between fluid phase and cell-bound, C3bBb(C3NeF) was found for C3NeF preparations from eight different patients and therefore seems to apply to all C3NeF preparations.

Binding Sites↗
PubMed1979

Activation of the classical complement pathway by nephritic factor bound to the alternative pathway C3/C5 convertase.

Nephritic Factor (NF), the potent alternative pathway activator, which is occasionally found in association with certain types of nephritis has recently been identified as an IgG class autoantibody specific for the C3 convertase (C3bB) of the alternative pathway. In these studies we have examined the possibility that the cell-bound NF-stabilized C3 convertase (EC3 bBNF) binds and activates the first component of the classical pathway of complement. EC3bBNF bound C1q, and the extent of binding was dependent upon the number of NF molecules bound per cell and decreased parallel to the dissociation and release of NF from the cells. Interaction of C1 with bound NF resulted in its activation as shown by the proteolytic conversion of proenzyme C1s to its activated form C1s. As was the case with C1q binding, C1 activation was dependent on the number of NF molecules bound per cell. Thus the NF-stabilized C3 convertase binds and activates C1.

Binding Sites↗
PubMed1979

[Description of an unusual enzymic activity cleaving the third component of complement].

A large enzymatic complex cleaving C3 (C3'ase) in the absence of magnesium (EDTA) has been partially characterized in two patients. On gel filtration EDTA-C3'ase was found in an 800 000 fraction containing antigenic C4, IgA and IgG in addition to alpha-2-macroglobulin and IgM normally present. EDTA-C3'ase was specifically neutralized by antibodies to IgG, IgA and C4. The characteristics of this unusual enzymatic complex are compatible with that of a C4b2a complex stabilized by its specific binding to an auto-antibody of the IgG and/or IgA class.

Chromatography, Gel↗
PubMed1977

The activation of the C3b feedback cycle with human complement components. I. Through the classical pathway.

Reaction between the fourth, the oxidized second and the activated first components of human complement generated the stable enzyme C4oxy2 capable of cleaving the third component and depleting total complement in human serum. This enzyme was shown further to activate the C3b feedback cycle as shown by its ability to consume factor B in serum and the reduction in the extent of complement consumption in the presence of EDTA. OxyC2 on its own gave rise to C3 cleavage in normal human serum by a pathway needing classical pathway components. This unexpected finding suggests that there may be a 'C-1 tickover' in serum analogous to the 'C3b tickover'; the presence of oxyC2 allowing the 'capture' of the trivial amounts of C42 normally formed. In preliminary experiments in the rat, C4oxy2 was successfully formed in vivo, where it gave rise to cleavage of C3, consumption of C5, depletion of cobra venom factor cofactors and a biphasic change in the neutrophil count.

Animals↗