Purification and Processing of Rat Liver Procathepsin B

In order to characterize the intracellular processing event of lysosomal cathepsin B, the proenzyme was purified from the rat liver microsomal contents using a Con A-Sepharose column, a Sepharose-Gly-Phe-GlySc column, and an anti-cathepsin B IgG column. The purified proenzyme gave a single protein band of 39 kDa on SDS/polyacrylamide gel electrophoresis. The proenzyme showed no appreciable enzymatic activity. When the purified proenzyme was incubated with the cathepsin B-free tritosomal contents, prepared by treatment of the tritosomal contents with anti-cathepsin B IgG Sepharose, at pH 3.0, 30°C, a remarkable increase of enzymatic activity was observed. Immunoblot analysis showed that the proenzyme was completely converted to the active intermediate form of 31 kDa after 1 h incubation. These processing and activation events were blocked in the presence of pepstatin. When the proenzyme was incubated with the cathepsins B- and Dfree tritosomal contents, prepared by treatment of the cathepsin B-free tritosomal contents with anti-cathepsin D IgG Sepharose, the processing and activation did not occur. These results indicate that cathepsin D is involved in the processing and activation of procathepsin B in rat liver lysosome. In the NH2-terminal sequence analysis of the 31 kDa form, the terminal was assigned as proline (66th residue). Since the NH2-terminus of the mature single-chain form of cathepsin B (29 kDa) ends at leucine (80th residue), the NH2-terminus of the 31 kDa form is 14 amino acid residues longer than that of the single-chain form. Therefore, we presume that procathepsin B is first processed by cathepsin D, splitting the bond between the 65th and 66th amino acid residues, and then lysosomal aminopeptidase(s) hydrolyzes the 14 amino acid residues from 66 to 79.