Biochemical properties and intracellular processing of lysosomal cathepsins B and H.
Lysosomal cysteine proteinases of cathepsins B and H were isolated to a homogeneous state from rat liver by employing Sephadex G-75, DEAE-Sephacel, CM-Sephadex, and Mono S column chromatography. Each of the purified cathepsins B and H was demonstrated to be composed of a mixture of a single-chain form and the processed two-chain form upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To investigate the proteolytic maturation of lysosomal cathepsins B and H, turnover kinetics of these enzymes were studied by comparing the specific radioactivities of the incorporated [3H]leucine into either the single-chain form or two-chain form in vivo. The specific radioactivity derived from each protein band of lysosomal cathepsin H in SDS-PAGE at 1, 3, 6, 12, 24 and 48 h after the injection of a radiolabel showed that the peak of specific radioactivity of the single-chain form of cathepsin H appeared at 6 h and that after 6 h, the radiolabel was sequentially incorporated into the two-chain form, while the radiolabel in the single-chain form started to gradually decrease, suggesting that the single-chain form was processed to generate the mature enzyme after the enzyme was incorporated into the lysosomes. In contrast, in the case of cathepsin B, the appearance of a radiolabel in the single-chain form or in the two-chain form was observed almost concomitantly without time lag, indicating that the processing of cathepsin B occurred very rapidly in the lysosomes.
Nishimura
Y
Y
Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Tsuji
H
H
Kato
K
K
Sato
H
H
Amano
J
J
Himeno
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002401:Cathepsin B / Q000302:isolation & purification / Q000378:metabolism*; D056655:Cathepsin H; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar
D002403:Cathepsins; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056655:Cathepsin H; C534128:Ctsh protein, rat; D007930:Leucine