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B F Tack

Publications and source records attributed to B F Tack.

At least 73 records · Page 4Linked to original sources
PubMed1982

Cooperative interaction of factor B and other complement components with mononuclear cells in the antibody-independent lysis of xenogeneic erythrocytes.

Synergistic cytotoxicity is a term used to describe a cytotoxic system in which xenogeneic erythrocyte target cells are lysed in the presence of nonimmune human mononuclear effector cells and antibody-depleted normal human serum. Neither the mononuclear cells nor the serum alone are cytolytic to the target erythrocytes. Previous studies have shown that the serum activity is not immunoglobulin and is heat-labile, suggesting a similarity to serum complement. In this report, sera deficient in various complement components as well as highly purified single complement components were tested with whole mononuclear cell populations and purified monocytes and lymphocytes to further characterize this cytotoxicity system. Whole mononuclear cell populations failed to mediate target cell lysis in sera deficient in C5 or factor B. However, C3-deficient serum, even in the presence of anti-C3 antibody, supported synergistic cytotoxicity normally. Purified lymphocytes were also normally cytotoxic in C3-deficient serum but failed to lyse targets in sera deficient in C5, C7, C8, or depleted of factor B. Purified monocytes failed to lyse the target cells only in factor B-depleted serum and could lyse the target cells in serum-free medium when purified factor B alone was added. Monocyte-mediated cytotoxicity induced by factor B was inhibited 73-100% by adding lymphocytes back to the purified monocytes. Thus, both lymphocytes and monocytes can serve as effector cells in this form of cytotoxicity but require cooperative interaction with different sets of complement components. In addition, lymphocytes can modulate the monocyte-mediated form of target cell lysis associated with factor B.

Animals↗
PubMed1982

Third component of human complement: localization of the internal thiolester bond.

Human complement protein C3 was inactivated by using methylamine and thereby generating a SH group from the internal thiol ester. The protein was coupled via this SH group to activated thiol-Sepharose and digested with elastase. Fragment C3d remained attached to the thiol-Sepharose and was subsequently eluted with L-cysteine. Concomitantly, the original SH group was regenerated, and it was then labeled with iodo[2-(3)H]acetic acid. Partial sequence analysis of the radiolabeled C3d fragment showed that both components of the thiol ester are located close to the amino terminus (residues 23 and 26). Specific chemical cleavage of the alpha-chain was achieved after S-cyanylation of the thiol. The two fragments obtained corresponded to the amino-terminal section (M(r) approximately 46,000) and the carboxy-terminal section (M(r) approximately 70,000). These results together indicate that fragment C3d occupies approximately positions 345-610 of the alpha-chain. The partial sequence of C3d was extended by completion of the sequence of a previously described tryptic peptide. Comparison of residues 1-49 of C3d with a peptide from alpha(2)-macroglobulin [Swenson, R. P. & Howard, J. B. (1980) J. Biol. Chem. 255, 8087-8091] shows a previously recognized identity of seven residues around the thiol ester site and a second region of identity around a known glycosylation site of alpha(2)-macroglobulin. The relationships among these proteins and protein C4 are discussed. An overall outline of the structure of C3 is presented, showing the locations of various fragments and cleavage sites. The thiol ester group places constraints on the local folding of the peptide chain; a possible conformation is suggested and discussed in relation to the mechanism of activation.

Amino Acid Sequence↗
PubMed1981

NH2-terminal structure and cleavage of guinea pig pro-C3, the precursor of the third complement component.

The NH2-terminal structures of the intracellular precursor of the third component of guinea pig complement (pro-C3), synthesized in macrophage cultures, and beta chain of C3 from guinea pig plasma were determined. Six of the first 16 residues of pro-C3 were identified by a microradiosequencing method. All six are nonpolar and are identical with the residues in beta chain of native plasma C3. This establishes beta chain as the NH2-terminal subunit in pro-C3. A comparison of primary structures of guinea pig and human C3 beta chains revealed identity of at least 8 residues within the NH2-terminal decapeptide. Incubation of plasmin with radiolabeled pro-C3 in macrophage lysate resulted in loss of pro-C3 and the generation of a two-chain molecule electrophoretically indistinguishable from native C3. These data indicate similarities between pro-C3 and the precursor of the fourth complement component, pro-C4, with respect to subunit organization, presence of phylogenetically conserved NH2-terminal regions, and the probable mechanism for generation of their respective native proteins.

Amino Acid Sequence↗
PubMed1981

Large scale isolation of functionally active components of the human complement system.

In the present work a scheme is presented for the isolation of multiple components of human complement in a functionally and biochemically pure state and with full hemolytic activity. These preparative procedures allow high recovery of milligram and gram quantities of particular complement components from a large pool (2-11 liters) of fresh EDTA plasma in no more than four chromatographic steps. Many components (C3bINA, C5, C3, C1EI, C4, and C9) are recovered functionally pure or highly purified following the first chromatographic step employing DEAE-Sephacel and may be utilized as reagents with no further purification. Prior to anion exchange, individual units of plasma are treated with inhibitors of complement activation and serum proteases, the pooled plasma is fractionated with polyethylene glycol, depleted of plasminogen on Sepharose-lysine, and rapidly ultrafiltered to low ionic strength and high protein concentration. The high degree of resolution of the components on DEAE-Sephacel subsequently obtained is demonstrated by the functional recovery and purification in a representative experiment as indicated (in their order of elution) for the following proteins: C3bINA (24%, 18-fold), C2 (74%, 12-fold), C7 (87%, 14-fold), factor B (55%, 8.7-fold),, C8 (50%, 16-fold), C6 (82%, 25-fold), beta 1H (39%, 12-fold), C5 (62%, 111-fold), C3 (99%, 64-fold), C1EI (42%, 135-fold), C9 (80%, 297-fold), and c4 (78%, 164-fold). Other components separated by these procedures include C1q and C4 binding protein. Additional steps described, which demonstrate the utility and effectiveness of this preparative scheme, have allowed isolation of C3, C5, and C7 as pure components with full hemolytic activity as judged by functional, immunochemical, and physicochemical criteria. C8, also isolated as a homogeneous protein, was recovered with partial hemolytic activity. All these components were recovered in high yield and in the purification as indicated: C3 (61%, 103-fold), C5 (24% 1350-fold), C7 (19%, 2260-fold), and C8 (32%, 547-fold). Complement components C6, beta 1H, factor B, and C2 in addition to C3bINA, C1EI, C4, and C9 are recovered partially purified with good activity and are amenable to further purification.

Complement C3↗
PubMed1981

Sequence determination of the thiolester site of the fourth component of human complement.

The fourth component of complement (C4) is inactivated by treatment with methylamine. This property is shared wit the third component (C3) and with alpha 2-macroglobulin. In each instance, the reaction with methylamine is stoichiometric, covalent, and accompanied by the appearance of a thiol group. These data are consistent with the presence of an internal thiolester bond. Incubation of C4 with [14C] methylamine in the presence of activated thiol-Sepharose resulted in immobilization of the protein via its active-site thiol. Analysis of bound C4 indicated incorporation of 1.12 mol of [14C]methylamine per mol of protein. Digestion of the immobilized protein with porcine elastase resulted in the release of C4 beta- and gamma-chains and lower molecular weight fragments. The 14C label, however, was retained on the Sepharose beads. Subsequent release of bound material with L-cysteine indicated that the radiolabel was associated with two polypeptides of Mr 25,000 [C4d(ela25)]. The released material was dialyzed and the active-site thiol was radioalkylated with iodo[2-3H]acetic acid. C4d(ela25) was further purified by chromatography on Sephadex G-100 and, after reduction and alkylation, on Sepharose CL-6B in 0.2% NaDodSO4. The C4d(ela25) pool, containing 0.83 mol of [14C]methylamine per mol of iodo[2-3H]acetic acid, was subjected to automated sequence analysis. S-carboxy-[3H]methylcysteine was released at step 21 and gamma-glutamyl-[14C]methylamide was released at step 24. The recovery of radiolabel at positions 21 and 24 confirmed the originally calculated 14C/3H incorporation ratio and further indicated that the radiolabels were present at single sites in the C4 molecule. Comparison of the derived primary structure for the thiolester site in C4 with those for the corresponding regions in C3 and alpha 2-macroglobulin has shown sequence identity. Further comparisons among these three proteins have indicated additional homologies on both the NH2- and COOH-terminal sides of the thiolester site.

Amino Acid Sequence↗
PubMed1981

Characterization of the human complement (c3b) receptor with a fluid phase C3b dimer.

The interaction of C3b receptor with C3b, the major cleavage product of C3, elicits important biologic functions, such as enhanced phagocytosis and release of cellular enzymes. We determined the binding kinetics and binding isotherm of C3b-receptor interaction by using human cells and fluid phase C3b generated by trypsin cleavage of purified native C3. 125I labeled C3b was separated into 2 molecular species, a dimer and a monomer by column chromatography. We found that dimeric C3b bound to human erythrocyte C3b receptors with an affinity that was more than 25 times that of the monomer. 125I dimeric C3b did not bind to sheep red blood cells, which lack the C3b receptor, nor to trypsinized or 2-mercaptoethanol treated normal human red blood cells, 2 methods for abrogating the immune adherence activity. Binding of 125I fluid phase C3b dimer to the C3b receptor was specific, saturable (about 90 ng of C3b dimer bound maximally per 1 X 10(9) red blood cells), reversible (in the presence of a 100-fold molar excess of unlabeled ligand), and of moderate affinity (Kd about 9.53 nM). The equilibrium binding constants were similar with the various cells tested. Binding was characterized by rapid on and off rates and did not exhibit ligand cooperativity. This specific interaction reached a steady state within 10 to 15 min at 0 degrees C; 50% of specifically bound ligand dissociated from its binding site on human red blood cells in approximately 1 min at 0 degrees C. The density of C3b receptors on human red blood cells, polymorphonuclear leukocytes, monocytes, and B lymphocyte-enriched preparations was 360, 20,000, 30,000, and 21,000 receptors/cell, respectively.

Animals↗
PubMed1980

Tritium labeling of proteins to high specific radioactivity by reduction methylation.

The third component of human complement, hemoglobin A2, and IgG have been radiolabeled with tritium to specific activities of 179, 103, and 89 Ci/mmol, respectively, by reductive methylation. The labeling procedure is mild, requiring only brief exposure to formaldehyde (10 to 12 mM) and tritiated sodium borohydride (3 to 5 mM), and specific for the alpha amino groups of NH2-terminal residues and epsilon-amino groups of lysyl residues. The extent of modification for each protein ranged between 5 and 16% at available amino groups based on a stoichiometric reactivity of 2 mol of formaldehyde/mol of primary amine. Although the degree of substitution required to obtain these specific activities was high, no significant loss of antigenic or biological activities was apparent. Each labeled protein was 90 to 95% precipitable with antiserum elicited to the unreacted protein. The hemolytic activity of C3 was reduced by only 16%; the oxygen affinity of HbA2 was minimally decreased, and the phosphorylcholine binding affinity of an IgA myeloma protein (TEPC-15) was not significantly affected. Precise double antibody radioimmunoassays were developed with each tritiated protein capable of detecting less than 10(-8) g of antigen. Labeled protein samples have been stored for time periods up to 1 year without appreciable deterioration.

Complement C3↗
PubMed1980

Third component of human complement: appearance of a sulfhydryl group following chemical or enzymatic inactivation.

Treatment of human C3 with hydroxylamine or hydrazine at physiological pH and ionic strength totally abrogates the intrinsic ability of this protein to sustain classical pathway induced hemolysis of sheep red blood cells. Concomitant with the loss of this function the appearance of a single sulfhydryl group can be followed by titration with the sulfhydryl-specific reagents p-(chloromercuri)benzoate, [1-14C]iodoacetamide, 2,2'-dipyridyl disulfide, and 5,5'-dithiobis(2-nitrobenzoic acid). These reagents have also been used to follow the appearance of a free sulfhydryl group on conversion of C3 to C3b with bovine trypsin. Autoradiography of the electrophoretogram of separated alpha-, alpha'-, and beta-polypeptide chains of inactivated, [1-14C]carboxamidomethylated C3 samples has shown that the reactive sulfhydryl group is present in the alpha chain of C3 and in the alpha' chain of C3b, respectively. Digestion of the radiolabeled protein with porcine elastase has localized this sulfhydryl group to a 28 000-dalton fragment of the alpha chain with immunochemical and functional reactivities of the C3d domain. Autoradiographic analysis of a hydrolysate prepared from radioalkylated C3 and subjected to high-voltage paper electrophoresis has shown the labeled amino acid to be [1-14C]-S-(carboxymethyl)cysteine. The susceptibility of native C3 to rapid and irreversible inactivation by nitrogen nucleophiles with the parallel appearance of a cysteinyl residue may indicate the presence of an internal thiol ester. The relationship of the proposed thiol ester to the ability of nascent C3b to acylate cell surface components and carbohydrate polymers is discussed within the context of a transesterification reaction.

Chloromercuribenzoates↗
PubMed1980

Evidence for presence of an internal thiolester bond in third component of human complement.

Treatment of the third component of human complement (C3) with methylamine results in a loss of hemolytic function and the appearance of a thiol group. Studies with [14C]methylamine have indicatd a stoichiometric and covalent reaction with the native protein. Hydrazine-inactivated C3 and C3b prepared with bovine trypsin were unreactive with [14C]-methylamine. Alkylation experiments with [1-14C]iodoacetamide have further established a 1:1 correspondence between methylamine incorporation and expression of the reactive thiol. Autoradiographic analyses of [14C]methylamine-treated C3 and methylamine-inactivated [1-14C]carboxyamidomethylated C3 after NaDodSO4/polyacrylamide gel electrophoresis have shown a specific incorporation of each radiolabel into the alpha polypeptide chain. [14C]Methylamine-treated C3 was immobilized on Sepharose 4B by reaction of the protein thiol with a mixed disulfide. Digestion with bovine trypsin in 4 M urea released 96% of the bound absorbance (at 280 nm) units; the radiolabel remained associated with the Sepharose beads. Peptide material labeled with 14C was eluted with dithiothreitol, carboxymethylated with [3H]iodoacetic acid, and chromatographed on Sephadex G-75. On Edman degradation S-[3H]carboxymethylcysteine was released at step 9 and gamma-glutamyl[14C]-methylamide was released at step 12. We interpret these data to indicate the presence of an internal thiolester bond in native C3. In addition, evidence is presented for an identical reactive site in alpha 2-macroglobulin.

Amino Acids↗
PubMed1977

Requirements for the solubilization of immune aggregates by complement: assembly of a factor B-dependent C3-convertase on the immune complexes.

During the solubilization of immune precipitates BSA-rabbit antibodies to BSA by human complement, at least three stages can be distinguished. (A) Generation of alternative pathway C3-convertase sites associated with the immune complexes. During the first minutes of interaction between the immune aggregates and serum, before any solubilization has taken place, properdin (P), factor B, and C3 moieties are incorporated into the lattice. The washed precipitates have C3-convertase activity, which can be completely inhibited by antibodies to factor B, but not to C2. The assembly of the convertase is temperature-dependent, and does not take place in the absence of Mg++. The immune complex-associated C3-convertase activity decays rapidly at 37 degrees C, but it can be restored by addition of purified factor B and properdin. (B) Amplification. When the aggregates bearing C3-convertase are incubated with purified C3, solubilization takes place. It appears that solubilization is caused by the accumulation of a large number of C3 fragments on the Ag-Ab lattice. In solubilized complexes, the molar ratios of Ab/C3 are close to one. (C) Spontaneous release. The final step in the solubilization process is a secondary reaction, during which some rearrangement of the lattice takes place. It occurs in medium devoid of serum and does not require divalent cations.

Antigen-Antibody Complex↗