MOLECULAR-CLONING OF A PROTEOLYTIC ANTIBODY LIGHT-CHAIN

The cDNA for an antibody light chain raised by immunization against va soactive intestinal peptide (VIP) was cloned in a bacterial expression vector, and the recombinant light chain was purified to electrophoret ic homogeneity, The light chain catalyzed the hydrolysis of VIP effici ently owing to its comparatively high affinity for the substrate, In c ontrol experiments, the catalytic activity was preserved at a constant level after further chromatography of the light chain on anion-exchan ge and gel-filtration fast protein liquid chromatography columns, and it was removed by immunoadsorption with immobilized anti-mouse light c hain antibody. The amide bond linking methylcoumarinamide (MCA) and ar ginine in a tripeptide unrelated in sequence to VIP was cleaved by the light chain with lower affinity and kinetic efficiency (k(cat)/K-m). Hydrolysis of the peptidyl-MCA conjugate was inhibited competitively b y the alternate substrate, VIP, The K-i and K-m values for MP were in the same range, indicating that peptide-MCA and VIP hydrolysis occurs at a common catalytic site in the light chain. Molecular modeling sugg ested the presence of a serine protease-like site in the light chain, This was supported by inhibition of the hydrolytic activity by serine protease inhibitors, but not by inhibitors of other classes of proteas es, These observations suggest a poorly discriminatory catalytic site, with specificity for VIP arising chiefly by means of the antigen reco gnition function of the light chain combining site,