Proteolytic components of serum IgG preparations.
Chemical catalysis, an effector mechanism utilized by fully assembled antibodies, can also be mediated by the isolated antibody subunits. Because trace amounts of free light chains (L chains) are present in IgG preparations, a detailed study was undertaken to identify the constituents responsible for the polyreactive proteolytic activity of IgG purified from human sera, determined as the extent of cleavage of the model peptide substrate Pro-Phe-Arg-methylcoumarinamide. Two proteolytic species with approximate mass of 50 kD and 150 kD were separated by repetitive gel filtration in a denaturing solvent (6 M guanidine hydrochloride). The activity of the renatured 50-kD fraction (in fluorescence units/microg protein) was more than 45-fold greater than of the 150-kD fraction. Both fractions lost the activity following immunoadsorption on immobilized anti-IgG antibody. Fab fragments prepared from the 150-kD IgG fraction retained the activity. Reducing and non-reducing SDS-electrophoresis suggested the 50-kD fraction isolated from the IgG preparations to be a mixture of heavy chain (H chain) monomers and disulphide bonded L chain dimers. Electrophoretically homogeneous monomers of 50-kD H chains and 25-kD L chains were prepared by gel filtration of reduced and alkylated IgG from seven human subjects. Each of the alkylated L chain preparations displayed the proteolytic activity. The activity in alkylated H chains was undetectable or only marginally greater than the background values. L chain dimers appear to be the major species responsible for the polyreactive proteolytic activity of serum IgG preparations, with a smaller contribution furnished by tetrameric IgG.
Li
L
L
Department of Pathology and Laboratory Medicine, University of Texas Medical School, Houston, TX 77030, USA.
Kalaga
R
R
Paul
S
S
D017151:Antibodies, Catalytic / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D005591:Chemical Fractionation; D019281:Dimerization; D010450:Endopeptidases / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D013379:Substrate Specificity
D017151:Antibodies, Catalytic; C029152:G-substrate; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; C025273:prolyl-phenylalanyl-arginine-4-methylcoumaryl-7-amide; D011494:Protein Kinases; D010450:Endopeptidases