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 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)
Kawabata
T
T
Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka.
Nishimura
Y
Y
Higaki
M
M
Kato
K
K
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
D004792:Enzyme Precursors; C057832:procathepsin B; D002401:Cathepsin B; D002402:Cathepsin D