title: Purification and processing of rat liver procathepsin B.
abstract: 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 degrees 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 D-free 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors: Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka.
authors: T
authors: T
authors: Kawabata
authors: Y
authors: Y
authors: Nishimura
authors: M
authors: M
authors: Higaki
authors: K
authors: K
authors: Kato
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002401:Cathepsin B / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D051381:Rats
chemical_list: D004792:Enzyme Precursors; C057832:procathepsin B; D002401:Cathepsin B; D002402:Cathepsin D