Purification and characterization of cathepsin J from rat liver

Abstract

Cathepsin J has been partially purified [Liao, J. C. R. & Lenney, J. F. (1984) Biochem. Biophys. Res. Commun. 124, 909–916], but its detailed properties are still unknown. In this study, we have purified cathepsin J completely and characterized it. It was purified to homogeneity from the mitochondrial‐lysosomal fraction of rat liver by acid treatment, followed by ammonium sulfate precipitation (20–65%), and chromatographies on S‐Sepharose, ConA‐Sepharose, Affi‐gel 501, HPLC DEAE‐5PW and HPLC TSK G3000SW. Cathepsin J was found to be a lysosomal high‐molecular‐mass cysteine protease of about 160 kDa consisted of two different subunits. One subunit (α subunit) was a glycoprotein with a molecular mass of 19–24 kDa which was reduced to 19 kDa by treatment with endoglycosidase F. It has the amino acid sequence LPESWDWRNVR at its N‐terminus, which was very similar to those at the N‐termini of rat cathepsins B, H and L. The other subunit (β subunit) was a glycoprotein with a molecular mass of 17 kDa, which was reduced to 14 kDa by treatment with endoglycosidase F. It had DTPANETYPDLLG at its N‐terminus, which had no similarity with the N‐terminal sequences of other cathepsins. Cathepsin J showed strong affinity for synthetic substrates such as N‐benzyloxycarbonyl‐phenylalanyl‐arginine 4‐methyl‐coumaryl‐7‐amide and glycyl‐arginine β‐naphthylamide. It was activated by thiol reagents and chloride ion and was inhibited by cysteine protease inhibitors. However, its initial inhibition constant Ki(initial) by N‐(L‐3‐trans‐carboxyoxirane‐2‐carbonyl)‐L‐leucine‐3‐methylbutylamide (E‐64‐c) was 1800 nM, which was 100–500 times those of cathepsins B and L. Many properties of cathepsin J were similar to those of cathepsin C (dipeptidylaminopeptidase I) reported as a lysosomal cysteine protease with dipeptidyl‐aminopeptidase activity [McDonald, J. K., Reilly, T. J. & Ellis, S. (1964) Biochem. Biophys. Res. Commun. 16, 135–140]. Furthermore, antiserum against rat liver cathepsin C reacted with rat liver cathepsin J. These findings suggested that cathepsin J is identical with cathepsin C. Copyright © 1992, Wiley Blackwell. All rights reserved