| Abstract: | Cathepsin B (EC 3.4.22.1) and an analogous thiol proteinase were isolated from mouse liver and from a transplantable tumor induced by methylcholanthrene, respectively, by a sequence of steps involving salt fractionation and ion exchange and gel permeation chromatography. Both enzymes are capable of hydrolyzing N-benzyloxycarbonyl-L-Ala-L-Arg-L-Arg-4-methoxy-2-naphthylamide but are weakly active towards N-benzoyl-DL-arginine-2-naphthylamide. The specific activity of the liver enzyme towards these substrates is approximately 14 times greater than that of the tumor enzyme. Both enzymes show a single band with slight difference in mobility when subjected to gel electrophoresis at pH 4.5, but both exhibit a multiple banding pattern when examined by isoelectric focusing. The tumor enzyme has a somewhat higher molecular weight than the liver enzyme (33,000 versus 30,000) and possesses a slightly higher helical content (48% versus 40%) based on CD spectra. Both enzymes display maximum activity in the pH range of 5.5 to 7.0 and are irreversibly denatured above pH 7 and below pH 4. Both enzymes cross-react with antiserum towards the tumor enzyme. The liver enzyme displays a higher catalytic efficiency towards a series of oligopeptide substrates than the tumor enzyme, but is only one-third as active towards N-benzoyl-L-arginine-2-naphthylamide. Both proteinases exhibit similar patterns of inhibition by iodoacetate, chloroquine, leupeptin, antipain, and several peptide chloromethylketones. Despite what appear to be subtle differences in physical properties, amino acid composition data and peptide mapping revealed significant differences between these two enzymes reflective of extensive regions of non-identity. These results suggest that the tumor thiol protease and liver cathepsin B are products of separate genes and that the tumor enzyme is not likely an immediate precursor of the liver enzyme produced by post-translational modification. |
