Limited proteolysis of human low-molecular-mass kininogen by tissue kallikrein. Isolation and characterization of the heavy and the light chains

Abstract

The limited proteolysis of human low-molecular-mass kininogen by kallikrein from tissue sources has been studied. Porcine pancreatic kallikrein applied in catalytic amounts split the kininogen molecule (apparent mass 68 kDa) with the release of lysyl-bradykinin (1 kDa). This generated a nicked kininogen molecule with a heavy chain and light chain interconnected via disulfide bridging. Following reductive cleavage of the disulfide bonds, the heavy chain of apparent mass 62 kDa was isolated by preparative sodium dodecyl sulfate electrophoresis, and the light chain of 5 kDa by reversed-phase high-performance liquid chromatography. The light chain was found to be composed of 38 amino acids with a single half-cystine residue. Amino-terminal sequence analysis revealed that the light chain is derived from the carboxy terminus of the kininogen molecule [Lottspeich et al. (1984) Eur. J. Biochem. 142, 227-232]. Immunological characterization of the isolated L chain indicated that it harbours antigenic site(s) unique for low-Mr kininogen as well as sites common to high-Mr and low-Mr kininogen.