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Biomedical subjects

S B Easterbrook-Smith

Publications and source records attributed to S B Easterbrook-Smith.

At least 37 records · Page 2Linked to original sources
PubMed1990

Single-step purification of immunoglobulin M on C1q-Sepharose.

A rapid and simple affinity chromatography method for purifying IgM from myeloma serum and ascites fluid is described. Complement protein C1q is coupled to Sepharose with an efficiency of 35%, giving 1.7 mg of C1q bound/ml of gel. This C1q-Sepharose selectively binds IgM from crude samples at 5 degrees C, with a capacity of 0.4 mg of IgM/ml of gel. The bound IgM may be eluted simply and isocratically by bringing the gel to room temperature for 2 h, or by washing with buffer containing 0.5 M KI. The eluted IgM is highly pure by SDS-PAGE and double immunodiffusion analysis, although IgG may be a potential contaminant. The C1q-Sepharose is stable for at least 18 months.

Chromatography, Affinity↗
PubMed1990

Carbodiimide crosslinking of human C1q and rabbit IgG.

The water soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to covalently link carboxyl groups on rabbit IgG to lysyl groups on complement protein C1q. The interaction between C1q and IgG was disrupted by varying the pH, modifying essential residues in the IgG binding site of C1q and by reducing the interchain disulfides of IgG. Under each of these conditions the correlation found between binding and crosslinking indicated a strong requirement for the proteins to bind normally in order for crosslinking to occur. SDS-PAGE analysis of the crosslinked material showed a 210 kDa band consistent with one IgG crosslinked to two disulfide linked C1q chains. Blotting and autoradiography showed the crosslinking involved the A and/or B and C chains of C1q. The lysines flanking the intrachain half cystines are proposed as the likely candidates for crosslinking to IgG, thus delineating the immunoglobulin binding site of C1q.

Animals↗
PubMed1989

Comparison of the role of tyrosine residues in human IgG and rabbit IgG in binding of complement subcomponent C1q.

Treatment of covalently cross-linked or heat-aggregated oligomers of human IgG with 4 mM-tetranitromethane abrogated their C1q-binding activity. In contrast, tetranitromethane modification of rabbit IgG oligomers, under identical conditions, had no effect upon their C1q-binding activity. The tetranitromethane treatment led to nitration of about ten tyrosine residues per IgG molecule in both species, and the modification was specific for tyrosine residues. Reduction of the nitrated protein with Na2S2O4 did not lead to recovery of C1q-binding activity in human IgG oligomers or to loss of activity in rabbit IgG oligomers. Tryptic peptides from the nitrated proteins were isolated and a peptide containing nitrotyrosine-319 was recovered from human IgG, as well as peptides from both species corresponding to the region around nitrotyrosine-278. These data are consistent with the inactivation of C1q-binding activity in human IgG being the result of nitration of tyrosine-319; the rabbit IgG is unaffected by nitration because position 319 is phenylalanine. The evidence supports the C1q-receptor site proposed by Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek [(1980) Nature (London) 288, 338-344]: residues 316-338.

Amino Acid Sequence↗
PubMed1988

Enhancement of the binding of C1q to immune complexes by polyethylene glycol.

The binding of 125I-labelled human C1q to insoluble rabbit IgG:ovalbumin immune complexes was enhanced by polyethylene glycol (PEG, Mr 8 x 10(3)) in the concn range 0-2.5% (w/v). C1q with native immunoglobulin bindings sites rendered inactive by diethylpyrocarbonate treatment did not bind to immune complexes in the presence of PEG. The ionic strength dependence of the binding was independent of the presence of PEG. There was a linear relationship between the logarithm of the apparent affinity constant of the C1q:immune complex interaction and PEG concn.

Animals↗
PubMed1988

The role of Fc:Fc interactions in insoluble immune complex formation and complement activation.

The initial rate of formation of insoluble immune complexes from rabbit IgG and ovalbumin was approximately 12 times that for formation of F(ab')2-ovalbumin complexes. At low IgG concns, in the range 0.7-2.7 nM, the formation of insoluble immune complexes was characterised by a lag phase, especially for complexes formed in low antigen excess, compared to antibody excess. Guanidine HCl (at concns up to 0.5 M) and urea (at concns up to 1 M) decreased the initial rates of formation of IgG immune complexes more than F(ab')2 immune complexes. Pre-formed IgG immune complexes were solubilised at lower guanidine HCl concns than were F(ab')2 immune complexes. C1q enhanced the initial rate of formation of IgG immune complexes at C1q:IgG ratios up to 1:1. Higher C1q concns decreased the initial rate of formation of the complexes. Urea (1 M) blocked the C1q mediated enhancement of immune complex formation.

Animals↗
PubMed1987

High-performance liquid chromatography-based assay for the quantitation of protein maleylation at the picomole level.

A new HPLC-based assay for the direct determination of the extent of maleylation of proteins is described. The rationale is founded on the HPLC detection of maleic acid that results from cleavage of maleyl lysyls by low pH treatment. The method, employing a C-18 reverse-phase column eluted isocratically with 0.01 M acetic acid, pH 5.4, and absorbance monitored at 210 nm, is sensitive (lower limit 1 pmol) and linear in the range 1 pmol-0.5 mumol and allows recovery of the native protein.

Chromatography, High Pressure Liquid↗
PubMed1987

The presence of histidine residues at or near the C1q binding site of rabbit immunoglobulin G.

Treatment of covalently crosslinked rabbit IgG oligomers with diethylpyrocarbonate resulted in the loss of their C1q binding activity. The inactivation was a first-order process with respect to time in the range 0-8 min, and modifier concentration from 0 to 2.39 mM. Hydroxylamine treatment of diethylpyrocarbonate-treated IgG oligomers led to 80% recovery of their C1q binding activity. Diethylpyrocarbonate treatment of IgG oligomers had little effect on their absorbance at 278 nm, but led to an increase in their absorbance at 242 nm. The apparent pKa of the modified residues was 6.91 +/- 0.12. These data are consistent with diethylpyrocarbonate modification of histidine residues leading to loss of C1q binding activity in rabbit IgG oligomers. Modification of four histidine residues per IgG molecule was associated with the loss of C1q binding activity. Thus, there may be two histidine residues at or near the C1q binding sites in the CH2 domains of rabbit IgG.

Animals↗
PubMed1987

Activation of the classical complement pathway by BioRex-70.

The cation exchange resin BioRex-70 was able to activate the classical complement pathway in human serum at 37 degrees C over the resin concentration range 0-5% (v/v). Using zymosan-treated human serum, it was found that the activation proceeded as far as complement protein C3.

Cation Exchange Resins↗
PubMed1986

Conformational changes in C1q upon binding to IgG oligomers.

The interaction between C1q and immune complexes is inhibited by 1-anilino-8-naphthalenesulfonate (ANS) in the concentration range of 2-4 mM. ANS binds to Clq with a 20-fold higher affinity than to IgG [(1986) Mol. Immunol. 23, 39-44] and therefore it is possible to label only C1q with ANS in the presence of IgG. Under such conditions no inhibition is observed. Addition of monomer IgG to a solution of C1q-bound ANS did not significantly alter the fluorescence of the ANS. However when oligomeric IgG was added there was a 2-fold increase in fluorescence over the same IgG concentration range. When C1q was pretreated with diethylpyrocarbonate there was little change in the fluorescence when IgG oligomers were added to C1q:ANS solutions. These results suggest that C1q undergoes conformational changes upon binding to IgG oligomers.

Anilino Naphthalenesulfonates↗
PubMed1986

Binding of complement component C1q by spectrin.

125I-labelled human C1q was found to bind to human spectrin. Scatchard plots for the binding process were non-linear, indicating the possible presence of multiple classes of binding sites for C1q on spectrin. The binding was ionic-strength-dependent; the extent of binding decreased with increasing ionic strength. Chemical modification of arginine and histidine residues on C1q as well as pretreatment of C1q at pH 4.45 or at 56 degrees C reduced its spectrin binding activity. The amount of 125I-labelled C1q bound to immune complexes was reduced by the presence of spectrin. Spectrin was also able to deplete the complement haemolytic activity of human serum in a dose-dependent manner.

Complement Activating Enzymes↗
PubMed1986

Inhibition of serum complement haemolytic activity by lipid vesicles containing phosphatidylserine.

The effect of artificial model membranes on the complement system was investigated. Incubation of the model membranes with human serum resulted in consumption of complement haemolytic activity when phosphatidylserine-containing vesicles were used. The activation of the complement system appeared to proceed through the alternative pathway. This conclusion was supported by the failure of [125I]Clq to bind to the membranes suggesting that the classical pathway was not involved. Although always obtained when phosphatidylserine was present in the model membranes, the activation of complement was enhanced by the contemporaneous presence of phosphatidylethanolamine. Liposomes prepared from lipid extracts of red blood cells were also able to stimulate a concentration-dependent activation of complement. Fresh, intact erythrocytes, however, could not initiate the same effects unless opsonized by antibodies. When artificially aged in vitro, red blood cells were lysed if incubated with normal human serum or with Clq-depleted serum. However, no lysis was obtained if the 'aged' erythrocytes were incubated with serum pretreated with ammonia to destroy the C3 component of complement. It is suggested that one of the mechanisms of macrophage recognition of senescent erythrocytes might be provided by the activation of the alternative pathway of complement if phosphatidylserine becomes exposed on the surface of the aging cells.

Animals↗
PubMed1985

Evidence for arginine residues in the immunoglobulin-binding sites of human Clq.

The immune complex binding activity of human Clq was lost following treatment of the protein with the arginine-selective reagents cyclohexane 1,2-dione and phenylglyoxal. Both inactivations followed pseudo-first-order kinetics. The affinity of Clq for immune complexes was reduced 7-fold following cyclohexane-1,2-dione treatment, and could be substantially restored by treatment of the modified protein with hydroxylamine. Heat-aggregated IgG protected Clq against inactivation by both reagents. Incorporation of 25 molecules of [7-14C]phenylglyoxal per Clq molecule completely inactivated the protein. These data are consistent with the presence of arginyl residues in the immunoglobulin recognition sites of human Clq.

Antigen-Antibody Complex↗
PubMed1985

C1q binding to mitochondria: a possible artefact?

Mitochondrial preparations, obtained from human tonsils and from rat spleen, liver, heart, and kidney tissues, bound [125I]C1q with affinities of 10(7)-10(8)M-1. The binding of C1q was not affected by treatment of the mitochondrial preparations with pronase, trypsin, or phospholipase D, but it was lowered 5-6-fold following treatment of the mitochondria with DNase and RNase. Binding of C1q to mitochondrial preparations was also greatly diminished by limited chemical modification of C1q with cyclohexane-1,2-dione. It is suggested that the reported binding of C1q to mitochondria may have arisen from the protein binding to DNA and/or RNA contaminating the mitochondrial preparations.

Animals↗
PubMed1985

Binding of complement component C1q by rat adipocyte membranes.

Human C1q was found to bind to rat adipocyte membranes with an affinity comparable to that for aggregated immunoglobulin. The binding was ionic strength dependent, and modification of arginyl and histidyl residues in C1q abrogated its binding activity. Treatment of the adipocyte membranes with either high ionic strength buffers, EDTA or trypsin had little effect on their C1q-binding activity.

Adipose Tissue↗
PubMed1984

The effects of cleavage of the inter-chain disulphide bonds of rabbit IgG on its ability to bind C1q.

Immune complexes prepared from rabbit anti-ovalbumin IgG in which the interchain disulphide bonds had been reduced and then blocked with N-( iodoacetylaminoethyl )-8-naphthylamine-1-sulphonic acid retained the ability to bind 125I-labelled C1q. This ability was lost when a small alkylating agent (iodoacetamide) was used to block the cleaved disulphide bonds. The ability of the IgG to form insoluble immune complexes was partially compromised when iodoacetamide was used to block the disulphide bonds, but was unimpaired when N-( iodoacetylaminoethyl )-8-naphthylamine-1-sulphonic acid was used. These data are consistent with the suggestion that access to the C1q binding site in IgG in immune complexes is modulated by movement of the Fab arms, which may block access to the site.

Antigen-Antibody Complex↗
PubMed1983

Activation of the binding of C1q to immune complexes by zinc.

ZnSO4 promotes the binding of C1q to immune complexes over the same concentration range (10(-5)-10(-4) M) that it inhibits binding of C1 to cell-bound immunoglobulin [Biochem. Biophys. Res. Commun. (1981) 103, 856-862]. At higher concentrations (10(-3)-2 X 10(-2) M) ZnSO4 inhibited the binding of C1q to immune complexes, [Ki = (6 +/- 2) X 10(-3) M]. This inhibition could be correlated with a ZnSO4-induced change in the tryptophan fluorescence of C1q [delta F 25%, Kd = (9.9 +/- 1.0) X 10(-3) M].

Antigen-Antibody Complex↗
PubMed1983

Evidence for histidine residues in the immunoglobulin-binding site of human Clq.

The immunoglobulin-binding activity of subcomponent Clq of human complement is lost following treatment with diethylpyrocarbonate; the inactivation showed first-order kinetics with respect to time and modifier concentration. Soluble IgG oligomers protected Clq against diethylpyrocarbonate modification. Treatment of modified Clq with hydroxylamine resulted in an 85% recovery of its ability to bind to aggregated immunoglobulin. The inactivation process was associated with modification of 12.1 +/- 0.7 histidine residues per Clq molecule. These data are consistent with the presence of histidine residues in the immunoglobulin-binding sites of Clq; these residues may participate in ionic interactions with the carboxyl groups known to be in the Clq binding site of IgG.

Chemical Phenomena↗