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At least 19 recordsLinked to original sources
PubMed1976

beta-Galactosidase alpha complementation: properties of the complemented enzyme and mechanism of the complementation reaction.

Intracistronic alpha complementation involving Escherichia coli beta-galactosidase occurs between the cyanogen bromide peptide CB2, derived from residues 3-92 of beta-galactosidase (Langley, K.E., Fowler, A.V., and Zabin, I. (1975), J. Biol. Chem. 250, 2587), and the defective beta-galactosidase from the Z-deletion mutant strain M15. The M15 protein, a dimer, lacks residues 11-41 of beta-galactosidase (Langley, K.E., Villarejo, M.R., Fowler, A.V., Zamenhof, P.J., and Zabin, I. (1975), Proc. Natl. Acad. Sci. U.S.A. 72, 1254). The complemented enzyme formed from purified components has a molecular weight of 533 000+/-25 000, is therefore tetrameric, and has a probable stoichiometry of 1 CB2:1 M15 monomer. The complemented enzyme has the same Km for substrate as wild type enzyme, but is less stable to heat or urea treatment. The overall equilibrium constant for the complementation reaction is approximately 1-2 X 10(9) M-1. Initial velocity studies indicate saturation kinetics when either component is fixed and limiting, with an apparent Kd of about 10(-6) M. A first-order rate constant of 0.05-0.1 min-1 was estimated. The kinetics favor a model of rapid complex formation, followed by slow conformational change, as the mechanism of activation. Ultraviolet difference spectroscopy indicated an increased absorbance in the 290-300 nm region as a result of the complementation reaction. The kinetics of the increase suggest that two processes, one rapid and the other slower, could be responsible. The temperature dependence of complementation (Ea approximately 24 000 cal) is also consistent with the rate-determining step being a conformational change.

Bacterial Proteins↗
PubMed1977

Complement "specificity" and interchangeability: measurement of hemolytic complement levels and use of the complement-fixation test with sera from common domesticated animals.

The results from studies to measure lytic complement (C') in sera of different animal species were reviewed. The traditional system, using sheep red blood cells (RBC) and rabbit antibody, was confirmed as the most sensitive to measure C' levels in man, monkey, dog, guinea pig, and rat serums. Sera C' from horse, cow, and sheep were found to be best assayed using rabbit RBC, whereas C' from goat, cat, and rabbit were best assayed with human RBC. Antibodies and C' from the same species usually mediated lysis of foreign RBC, but this lysis occurred more readily with some RBC targets than with others and may be associated with the presence of natural antibodies in the test sera. The effects of the species origin of a C' source in immunologic reactions in vitro and in vivo are discussed.

Animals↗
PubMed1976

Complement origin determines lytic activity of antibodies to nucleated target cells. Comparison of common complement sources.

The effects of complement from different species of animals were measured in a variety of antibody assays by using 51Cr-labelled target cells. Sheep antibodies were measured in samples of lymph and serum obtained from animals immunised with allogeneic lymphocytes, transplanted with allogeneic kidney grafts, or immunised with mouse tumour cells. Mouse (C57BL) antibodies were measured after immunisation with allogeneic tumour (P815) or after transplantation of allogeneic thyroid grafts (BALB/c). Different species of complement gave quantitative and sometimes qualitative differences when used to assay the same samples of antibody. In all systems tested, rabbit complement caused lysis of target cells at low antibody concentration when guinea pig and rat complements gave negative results, in some antibody-target cell combinations sheep complement was as effective as rabbit complement in mediating lysis. The different complement sources showed no selective lytic affinity for either IgM or IgG1 antibody subclasses purified from immune sheep lymph. Lysis of P815 target cells occurred more quickly when mediated by rabbit or sheep complement than when mediated by guinea pig or rat complement. Complement-dependent lysis sometimes occurred in systems where antibody, target cells and complement were obtained from the same species.

Animals↗
PubMedDate not supplied

The biological role of the complement system and the clinical importance of complement measurements.

Properties of serum proteins belonging to the complement system, two pathways of the complement activation (classical and alternative pathway) as well as the physiological role of the complement system are discussed. Complement has essential importance in some physiological processes: In the induction of the humoral immune response, in the elimination of immune complexes and in the protection against bacterial and viral infections. After a short discussion of the genetics of the complement system, the principle and possibilities of clinical applications of the complement measurements are described. Finally, different approaches to the therapeutic manipulation of the complement system are discussed.

Angioedema↗
PubMed1975

Immunochemical analyses of membrane-bound complement. Detection of the terminal complement complex and its similarity to "intrinsic" erythrocyte membrane proteins.

(1) Membranes of sheep erythrocytes lysed with antibody and human or rabbit complement were solubilized in non-ionic detergents (Triton X-100 or Berol EMU-043) and analysed immunochemically using antisera directed against individual complement components. The precipitation behaviour of membrane-bound C3, C5, C6 and C9 components of complement was examined by immuno-double diffusion, rocket- and crossed immunoelectrophoresis performed in agarose gels containing 1% non-ionic detergent. (2) Membrane-bound C5, C6 and C9 are antigenically altered compared with the native (serum) components. (3) Immuno-double diffusion in the presence of non-ionic detergents reveals formation of C5-C6-C9 complexes on the membranes; these complexes are stable in non-ionic detergent. No complex formation was detected in serum between native C5, C6 and C9 components. There was also no evidence for complexing between membrane-bound C3, C4 or membrane proteins and the "late-reacting" complement components. (4) The extractability of complement components by various manipulations has been studied by use of quantitative rocket immunoelectrophoresis. Up to 65% of membrane-bound C3 is readily extracted by dialysis of membranes against 1mM EDTA, pH 8.0, 100 mM EDTA, pH 8.0, 1.2 NaCl plus or minus EDTA, by extraction in isotonic buffers at 37 degrees C, by heating at 45 degrees C over several hours, or by treating membranes with 1 mM p-chloromercuribenzoate sulfonate. In contrast, less than 6% of the terminal complement complex can be eluted by any of the described methods or combination of methods. (5) Our data suggest that the terminal complement complex associates with membrane "core" components through apolar interactions.

Animals↗
PubMed1979

Staphylococcus aureus opsonization mediated via the classical and alternative complement pathways. A kinetic study using MgEGTA chelated serum and human sera deficient in IgG and complement factors C1s and C2.

Staphylococcus aureus opsonization was studied kinetically by: (1) determination of the uptake of [3H]-thymidine labelled bacteria by human PMN's; (2) fluorescent anti-C3 and anti-IgG staining of opsonized bacteria; and (3) measuring bacterial complement consumption. Maximum opsonization in normal serum occurred within 5 min of incubation. About 80% of staphylococci were then taken up by PMN's, and IgG and C3b could be detected on the bacterial surface. In the absence of a functional classical complement pathway, as in sera deficient in C1s and C2 and in MgEGTA chelated serum, maximal opsonization was only achieved after 30--60 min incubation. Opsonization in IgG deficient serum occurred at a rate similar to that found in C2 deficient or MgEGTA chelated serum. Opsonization was greatly enhanced when sera were reconstituted. It was concluded that in IgG deficient serum Staphylococcus aureus opsonization is mediated via the alternative complement pathway. Dilution of normal serum primarily affected the classical complement pathway, resulting in a decreased rate of opsonization. In normal serum IgG did not appear to be a rate-limiting factor. S. Aureus opsonization was best studied by the phagocytosis assay and the fluorescent-antibody technique. Measuring haemolytic complement consumption was found to be an insensitive indicator of bacterial complement activation and opsonization.

Complement Activation↗
PubMed1975

Complement activation by interaction of polyanions and polycations. I. Heparin-protamine induced consumption of complement.

Interactions of heparin and protamine in fresh human serum, in amounts far below those required for complement depletion by either agent alone, were found to induce virtually complete depletion of total hemolytic complement activity. This depletion was dependent on time, temperature, pH, divalent cations, and serum concentration. The predominant complement component hemolytic activity depleted was C1; under appropriate reaction conditions C4 and C2 were depleted as well. Equivalent amounts of heparin along induced lesser but substantial depletion of C3-9, whereas equivalent amounts of protamine had no effect upon complement component activities. We conclude that interaction of heparin with protamine, like interaction of antibody with antigen, markedly enhances its ability to interact with the first component of complement and activate the classical complement pathway. It is suggested that complement activation by interactions between certain polyanions and polycations, like interactions between antigens and antibodies, may have a role in the initiation of inflammatory reactions.

Adult↗
PubMed1977

Complementable fraction and complemented enzyme of mutant M15 from Escherichia coli: partial purification by affinity chromatography.

The technique of affinity chromatography has been used in the partial purification of complementable fractions and complemented enzyme of beta-galactosidase from Escherichia coli mutant M15. The crude extract of mutant M15 was incubated with fragment CM-B. The complemented enzyme and complementable fractions were passed through a small column of p-aminophenyl-beta-D-thiogalactoside to which inhibitors had been covalently attached. A high percentage of the nonspecific protein passed directly through the affinity column while the specific enzymatic protein remained bound to the gel. Phosphate buffer with NaCl was used to elute the complementable fractions from the column. Sodium borate buffer was used to elute the bound complemented enzyme from the affinity support. The results of this study show that 100% of the complemented enzyme was bound to the column. The partially purified enzyme had the same position in disc gel electrophoresis as beta-galactosidase from E. coli.

Chromatography, Affinity↗
PubMed1978

Plasma levels of complement components and complement haemolytic activity in protein-energy malnutrition.

The plasma levels of complement haemolytic activity (CH50) of some complement components and of C3d, a C3 breakdown product, were measured in fifty-nine African children with various types of protein-energy malnutrition (PEM) including kwashiorkor, before and during recovery. A significant decrease of CH50, C3, C9 and factor B was observed in PEM without a concomitant decrease of C4 and C5. Increased plasma levels of C3d were also found in PEM patients. Two mechanisms seem to be involved in the impairment of the complement system in PEM: (1) a decreased synthesis of at least C3 and C9 as suggested by a significant correlation between C3 or C9, levels and those of albumin and cholinesterase; (2) an increased catabolism of C3 possibly due to an activation of the alternative complement pathway, as suggested by the increased level of C3d and the decreased level of factor B which are significantly correlated with C3 levels but not with albumin levels. These data support the possible role of a relative complement deficiency upon the decreased resistance to infections observed in malnourished children.

Child, Preschool↗
PubMed1977

Pleural fluid complement, complement conversion, and immune complexes in immunologic and nonimmunologic diseases.

Forty-four pleural fluids and 41 blood specimens from patients with various diseases were examined for concentration of whole complement, C4, C3, conversion products of C3 and C3PA, and immune complexes. C3 conversion was found in all eight pleural fluids from patients with rheumatoid arthritis, five of seven with lupus erythematosus, two of six with congestive heart failure, and nine of 23 with malignant diseases. Conversion of C3PA correlated closely with C3 conversion and both were significantly inversely related to whole complement, C4, and C3. Concentration of immune complexes was highest in patients with rheumatoid arthritis. Pleural fluid immune complex concentrations correlated positively with conversion of C3 and C3PA. These findings suggest that the reduced levels of pleural fluid complement in rheumatoid arthritis and lupus erythematosus may be secondary to complement conversion by immune complexes.

Antigen-Antibody Complex↗
PubMed1979

The presence of complement in human cervical mucus and its possible relevance to infertility in women with complement-dependent sperm-immobilizing antibodies.

Full-complement component lytic activity was measured in human midcycle cervical mucus, using a sensitive 51Cr release hemolytic assay. The level measured was 11.5% of the activity of complement in an equal volume of undiluted human serum. The relevance of this level of complement to complement-dependent sperm-immobilizing antibody activity was studied. After 1 hour's incubation with mucus levels of complement, immobilization of about 50% of spermatozoa occurred and after 3 hours' incubation, immobilization of about 70% of spermatozoa occurred.

Antibodies↗
PubMed1979

C5b-9 dimer: isolation from complement lysed cells and ultrastructural identification with complement-dependent membrane lesions.

The membrane attack complex (MAC) of complement was extracted from the membranes of cells lysed by human complement and its properties were compared with those of the fluid phase complex SC5b-9. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunochemical analysis, the two isolated complexes had identical subunit compositions, except that the MAC lacked the S-protein. The sedimentation coefficient and molecular weight of the extracted and isolated MAC were, respectively, 33.5 S and 1.7 x 10(6) daltons, compared to 23 S and 1.0 x 10(6) dalton for SC5b-9. Because the molecular weight of the MAC is approximately two times greater than that of C5b-0 (800,000 daltons), the MAC is considered the dimer of C5b-9. Under specified conditions, the 33.5 S dimer could be converted to the 23 S monomer without dissociation of subunits. The MAC had the electron microscopic appearance and dimensions that are characteristic for the complement produced ultrastructural membrane lesions. SC5b-9 had a different ultrastructure that is dissimilar to the morphology of the lesions. The isolated MAC could be reincorporated into phospholipid bilayers and assumed on the surface of the resultant lipid vesicles the orientation and appearance of typical complement lesions.

Animals↗
PubMedDate not supplied

Complement-dependent antibody cytotoxicity test of chicken antibody with duck complement used against cells of a Marek's disease lymphoma-derived cell line (MSB-1).

Complement-dependent antibody cytotoxicity (CDAC) against cells of a lymphoblastoid cell line (MSB-1) derived from Marek's disease lymphoma was investigated in a chicken antibody system using complements from several animal species. Cytotoxicity was seldom observed when rabbit, guinea pig, or chicken complements were used in the presence of hyperimmune chicken serum against MSB-1. With duck complement, however, cytotoxicity was always observed. Therefore, duck complement appears to be suitable for assay of hyperimmune chicken serum against MSB-1 cells by the CDAC test.

Animals↗
PubMed1977

Complement receptors and cell associated complement components.

Membrane receptors for activated complement components are widely distributed amongst tissue cells of most mammalian species. Common amongst these are receptors for C3b which mediate many of the biological functions of C3. In addition, the genetic control of certain complement components is linked to the genes which code for the major histocompatibility complex. Many of these components are also present on cell surfaces. This suggests that the function of the complement system and the major histocompatibility complex may be related.

Animals↗
PubMed1978

Experimental bovine trypanosomiasis. Changes in serum immunoglobulins, complement and complement components in infected animals.

In three calves experimentally infected with Trypanosoma congolense the amounts of IgG1 and IgG2 were little changed and similar to those of normal animals. IgM increased in amount early in the infection and the amount of the increase appeared related to the parasite burden. The amounts of IgA and IgE were both much decreased and this also appeared related to the numbers of parasites in the blood. There was a decrease in the amounts of total haemolytic complement and complement components C1, C1q and C3 in the infected calves. Furthermore the amounts of properdin fluctuated with the cyclical changes in numbers of T. congolense parasites in the individual calves. No significant change in the amount of C8 was observed. It is considered that activation of both the alternative and the classical complement pathways occurs in trypanosome infected animals but that neither pathway goes to its terminal stages.

Animals↗
PubMed1979

Augmentation of macrophage complement receptor function in vitro. I. Characterization of the cellular interactions required for the generation of a T-lymphocyte product that enhances macrophage complement receptor function.

The function of complement receptors of mouse peritoneal macrophages was converted in vitro from mediating only attachment of macrophage complement receptor function was achieved by treating freshly explanted macrophages with supernates from cultures containing T lymphocytes and appropriately triggered macrophages. Fc receptor-mediated phagocyctosis by macrophages was required for the production of active supernates, for neither ingestion via the cells' complement receptors nor ingestion via nonimmunologic means was a sufficient stimulus for the macrophages' participation in the generation of supernatant activity. Fc receptor-triggered macrophages interacted by a contact dependent, but histocompatibility independent, mechanism with T lymphocytes, thereby signalling the lymphocytes to elaborate the active product. The possible significance of enhanced macrophage complement receptor function in inflammation, host defense against microbial pathogens, immune complex disease, and neoplasia is discussed.

Animals↗