Inhibitory effect of leupeptin on the intracellular maturation of lysosomal cathepsin L in primary cultures of rat hepatocytes.
To investigate the intracellular processing event for lysosomal cathespin L, we examined the effect of leupeptin, a non-covalent cysteine proteinase inhibitor, on the intracellular processing kinetics of cathepsin L as analyzed by pulse-chase experiments in vivo with [35S]methionine in primary cultures of rat hepatocytes. This revealed that cathepsin L was initially synthesized as proenzyme of molecular weight 39 kDa and the proenzyme was subsequently processed to the mature form of the enzyme, 30 and 25 kDa. In the leupeptin-treated cells, the proteolytic conversion of cellular procathepsin L, of molecular weight 39 kDa, to the mature enzyme was significantly inhibited and considerable amounts of proenzyme were found in the cell after 8 h chase periods. Furthermore, the subcellular fractionation experiment demonstrated that the intracellular processing of procathepsin L in the high density lysosomal fraction was significantly inhibited and that considerable amounts of the procathepsin L form were still observed in the dense lysosomal fraction after a 2 h chase period. These results suggest that leupeptin treatment caused significant inhibition of the intracellular maturation of cathepsin L. These findings show that cysteine proteinase plays an important role in the intracellular proteolytic processing and activation of lysosomal cathepsin L in vivo and that this processing event occurs within the lysosomes.
Nishimura
Y
Y
Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Kato
K
K
Furuno
K
K
Himeno
M
M
D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism*; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D051381:Rats; D017208:Rats, Wistar; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D013462:Sulfur Radioisotopes
D007976:Leupeptins; D015842:Serine Proteinase Inhibitors; D013462:Sulfur Radioisotopes; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534280:Ctsl protein, rat; C032854:leupeptin