RESUMO
Clusterin prepared from human serum by monoclonal antibody affinity chromatography was devoid of the ability to increase the rates of formation of insoluble immune complexes associated with clusterin preparations obtained by polyclonal IgG affinity chromatography. Clusterin did not bind to AMP-Sepharose but the protein responsible for increasing the rates of formation of insoluble immune complexes did bind to this affinity matrix. This protein was identified as complement protein C1q on the basis of its behaviour on SDS/PAGE and reactivity in sandwich ELISA with monoclonal antibodies specific for C1q. C1q (identified from its behaviour on SDS/PAGE, immunoreactivity with C1q-specific monoclonal antibodies and N-terminal sequencing data) was purified from serum by AMP-Sepharose chromatography. The binding of C1q to AMP-Sepharose was inhibited by adenine nucleotides.
Assuntos
Nucleotídeos de Adenina/farmacologia , Complexo Antígeno-Anticorpo/metabolismo , Complemento C1q/metabolismo , Glicoproteínas/farmacologia , Chaperonas Moleculares , Monofosfato de Adenosina/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Cromatografia de Afinidade , Cromatografia em Agarose , Clusterina , Proteínas Inativadoras do Complemento/farmacologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/imunologia , Glicoproteínas/isolamento & purificação , Humanos , Imunoglobulinas/metabolismo , Ligação Proteica , Análise de SequênciaRESUMO
Treatment of anti-ovalbumin rabbit IgG with diethylpyrocarbonate (DEPC) at concentrations up to 100 microM led to a progressive decrease in the rates of formation of insoluble immune complexes, without affecting the final extent of immune complex formation. DEPC concentrations approximately 10-fold higher were needed to give comparable decreases in the rates of immune complex formation by F(ab')2. Treatment of DEPC-treated IgG with hydroxylamine led to substantial restoration of the rates of formation of insoluble immune complexes. Carbethoxylation of two histidine residues per IgG molecule had little effect on rates of formation of insoluble immune complexes, but these rates were markedly decreased in samples of IgG with four to five histidines per molecule modified. There were parallel decreases in the protein A-binding activity and in the rates of formation of insoluble immune complexes in IgG treated with increasing concentrations of DEPC. The presence of complement protein C1q restored the rates of formation of insoluble immune complexes of DEPC-treated IgG.
Assuntos
Complexo Antígeno-Anticorpo/biossíntese , Dietil Pirocarbonato/farmacologia , Histidina/imunologia , Imunoglobulina G/efeitos dos fármacos , Imunoglobulina G/imunologia , Animais , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/efeitos dos fármacos , Complemento C1q/farmacologia , Histidina/química , Histidina/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/química , Cinética , Testes de Precipitina , Coelhos , Proteína Estafilocócica A/imunologiaRESUMO
Clusterin was purified from human serum by sequential affinity chromatography over IgG-, protein A- and Con A-Sepharose. The protein was approximately 70 kDa by SDS/PAGE under nonreducing conditions and was resolved into approximately 35 kDa bands under reducing conditions. The protein reacted with clusterin-specific Mabs in ELISA and in Western blots. Its N-terminal sequences agreed with those published for clusterin. An antiserum specific for clusterin made by the above method detected it in complement membrane attack complexes on rabbit erythrocyte membranes. The interaction of clusterin with IgG was physiologically relevant because it was found to increase the rate of formation of insoluble immune complexes.
