Biosynthesis of lysosomal cathepsins B and H in cultured rat hepatocytes

Yukio Nishimura, Jun Amano, Hiroshi Sato, Hiroshi Tsuji, Keitaro Kato

Research output: Contribution to journalArticle

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Abstract

The biosynthesis of lysosomal cysteine proteases, cathepsins B and H, was investigated by using pulse-chase experiments in vivo in primary cultures of rat hepatocytes. Cathepsins B and H were isolated from either total cell extracts or culture medium labeled with [ 35 S]methionine by immunoprecipitation and analyzed for their molecular forms. Within 60 min of chase, cellular preforms of cathepsins B of 39 kDa and H of 41 kDa were converted to single-chain form cathepsins B of 29 kDa and H of 28 kDa, respectively, and persisted as these forms even after 12-h chase periods. The proforms of cathepsins B and H derived from pulse-labeling experiments showed complete susceptibility to endoglycosidase H treatment, indicating that these proenzymes bear high-mannose-type oligosaccharides at the stage of initial events of biosynthesis. In the presence of tunicamycin, unglycosylated proenzymes of cathepsins B of 35 kDa and H of 34 kDa were found to be secreted into the extracellular medium without undergoing proteolytic processing. Furthermore, in the presence of swainsonine, a potent inhibitor of Golgi mannosidase II, considerable amounts of the proenzymes were secreted and accumulated in the medium during chasing periods. These results suggest that the oligosaccharide moiety of these enzymes would be necessary for the intracellular sorting mechanism. In monensin-treated cells, the conversion of intracellular proenzymes to mature enzymes was significantly inhibited and the proenzymes were secreted into the medium. In the presence of chloroquine or ammonium chloride, proteolytic processing of the proenzymes was completely prevented and the enhanced secretion of proenzymes was observed. These results suggest that in the presence of lysosomotropic amines the intracellular sorting of proenzymes might not occur properly during biosynthesis.

Original languageEnglish
Pages (from-to)159-170
Number of pages12
JournalArchives of Biochemistry and Biophysics
Volume262
Issue number1
DOIs
Publication statusPublished - Jan 1 1988

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Cathepsin H
Cathepsin B
Enzyme Precursors
Biosynthesis
Rats
Hepatocytes
Oligosaccharides
Sorting
Swainsonine
Tunicamycin
Monensin
Ammonium Chloride
Cysteine Proteases
Glycoside Hydrolases
Chloroquine
Enzymes
Mannose
Processing
Cell Extracts
Immunoprecipitation

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Biosynthesis of lysosomal cathepsins B and H in cultured rat hepatocytes. / Nishimura, Yukio; Amano, Jun; Sato, Hiroshi; Tsuji, Hiroshi; Kato, Keitaro.

In: Archives of Biochemistry and Biophysics, Vol. 262, No. 1, 01.01.1988, p. 159-170.

Research output: Contribution to journalArticle

Nishimura, Yukio ; Amano, Jun ; Sato, Hiroshi ; Tsuji, Hiroshi ; Kato, Keitaro. / Biosynthesis of lysosomal cathepsins B and H in cultured rat hepatocytes. In: Archives of Biochemistry and Biophysics. 1988 ; Vol. 262, No. 1. pp. 159-170.
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