Molecular cloning of a proteolytic antibody light chain.
The cDNA for an antibody light chain raised by immunization against vasoactive intestinal peptide (VIP) was cloned in a bacterial expression vector, and the recombinant light chain was purified to electrophoretic homogeneity. The light chain catalyzed the hydrolysis of VIP efficiently owing to its comparatively high affinity for the substrate. In control experiments, the catalytic activity was preserved at a constant level after further chromatography of the light chain on anion-exchange and gel-filtration fast protein liquid chromatography columns, and it was removed by immunoadsorption with immobilized anti-mouse light chain antibody. The amide bond linking methylcoumarinamide (MCA) and arginine in a tripeptide unrelated in sequence to VIP was cleaved by the light chain with lower affinity and kinetic efficiency (kcat/Km). Hydrolysis of the peptidyl-MCA conjugate was inhibited competitively by the alternate substrate, VIP. The Ki and Km values for VIP were in the same range, indicating that peptide-MCA and VIP hydrolysis occurs at a common catalytic site in the light chain. Molecular modeling suggested 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 proteases. These observations suggest a poorly discriminatory catalytic site, with specificity for VIP arising chiefly by means of the antigen recognition function of the light chain combining site.
Gao
Q S
QS
Department of Anesthesiology, University of Nebraska Medical Center, Omaha 68198-6830.
Sun
M
M
Tyutyulkova
S
S
Webster
D
D
Rees
A
A
Tramontano
A
A
Massey
R J
RJ
Paul
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006868:Hydrolysis; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D014660:Vasoactive Intestinal Peptide / Q000276:immunology / Q000378:metabolism*
D018076:DNA, Complementary; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; D014660:Vasoactive Intestinal Peptide