Purification and characterization of cathepsin J from rat liver.
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 (alpha 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 (beta 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 beta-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.
Nikawa
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T
Division of Enzyme Chemistry, University of Tokushima, Japan.
Towatari
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Katunuma
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N
D000595:Amino Acid Sequence; D000818:Animals; D020789:Cathepsin C; D002403:Cathepsins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008247:Lysosomes / Q000201:enzymology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity
D011480:Protease Inhibitors; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D002403:Cathepsins; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C; C043569:cathepsin J