Some unexpected properties of cathepsin D

Wiederanders, Bernd; Kirschke, Heidrun; Schaper, Susanne; Valler, Martin J.; Kay, John

doi:10.1515/9783111649788-015

Cited by 3 publications

(4 citation statements)

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“…Haemoglobin and human serum albumin were labelled with ['4C]acetic anhydride as previously described (Wiederanders et al, 1989). The labelled protein was diluted with unlabelled protein to give a final concentration of 0.05 % (w/v) and about 8000 d.p.m./assay.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins

Kirschke

Wiederanders

Brὂmme

et al. 1989

Biochemical Journal

189

115

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Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis of the homogenate at pH 4.2. The isoelectric point of cathepsin S was 7.0. Cathepsin S was found to hydrolyse proteins at a similar rate to cathepsin L below pH 7.0. At pH values of 7.0-7.5 cathepsin S retained most of its activity, whereas cathepsin L was completely inactive.

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Section: Enzyme Assaysmentioning

confidence: 99%

Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins

Kirschke

Wiederanders

Brὂmme

et al. 1989

Biochemical Journal

189

115

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“…Tang and coworkers [26] found that bovine cathepsin D contains about 68% of the single-chain species, whereas the porcine kidney enzyme contains only 5%. In rat tissues, in contrast, cathepsin D exists entirely in a single-chain form [37] (Erickson, unpublished).…”

Section: Light-heavy Chain Cleavagementioning

confidence: 99%

Biosynthesis of lysosomal endopeptidases

Erickson

1989

J of Cellular Biochemistry

123

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Despite the clear differences between the amino acid sequence and enzymatic specificity of aspartic and cysteine endopeptidases, the biosynthetic processing of lysosomal members of these two families is very similar. With in vitro translation and pulse-chase analysis in tissue culture cells, the biosynthesis of cathepsin D, a aspartic protease, and cathepsins B, H and L, cysteine proteases, are compared. Both aspartic and cysteine endopeptidases undergo cotranslational cleavage of an amino-terminal signal peptide that mediates transport across the endoplasmic reticulum (ER) membrane. Addition of high-mannose carbohydrate also occurs cotranslationally in the lumen of the ER. Proteases of both enzyme classes are initially synthesized as inactive proenzymes possessing amino-terminal activation peptides. Removal of the propeptide generates an active single-chain enzyme. Whether the single-chain enzyme undergoes asymmetric cleavage into a light and a heavy chain appears to be cell type specific. Finally, late during their biosynthesis both classes of enzymes undergo amino acid trimming, losing a few amino acid residues at the cleavage site between the light and heavy chains and/or at their carboxyltermini. During biosynthesis these enzymes are also secreted to some extent. In most cells the secreted enzyme is the proenzyme bearing some complex carbohydrate. Under certain physiological conditions the inactive secreted enzymes may become activated as a result of a conformational change that may or may not result in autolysis. Analysis of the biochemical nature of the various processing steps helps define the cellular pathway followed by newly synthesized proteases targeted to the lysosome.

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“…It is difficult to differentiate these activities in homogenates using specific substrates and inhibitors [44]. However, we followed a procedure described in [45] to get some idea of their relative amounts in the lysosomal preparations used in the endocytosis experiments. The approach is based on: (a) the relative specific activities of rat liver cathepsins B, H, L and D in digesting azocasein in the presence and absence of urea (taking the specific activity of cathepsin B, which under the incubation conditions used here produces an increase at 366 nm of 0.68 A .…”

Section: Characterization Of the Proteolytic Activity In The Rat Livementioning

confidence: 99%

“…min-' . mg protein-' in the presence or absence of urea, as 1, the relative specific activities of cathepsin H, L and D are 0.15, 13 and 2 in the absence and <0.05, 24 and 14 in the presence of 3M urea, respectively) [45]; and (b) the selective inhibition of cathepsin D by pepstatin [46]. Considering these data as well as the strong inhibitory effect of leupeptin on the activities of cathepsins B and L compared to cathepsin H [47,481, equations were developed to calculate the approximate concentration of each cathepsin in the lysosomal extracts (Table 2).…”

Section: Characterization Of the Proteolytic Activity In The Rat Livementioning

confidence: 99%

Endocytosis of liposomes containing lysosomal proteins increases intracellular protein degradation in growing L‐132 cells

Vargas

Knecht

Grisolı́a

1990

European Journal of Biochemistry

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We have used a new approach to test the possible participation of lysosomes in the degradation of long-lived proteins. Rat liver lysosomal proteins were introduced, via multilamellar liposomes, into L-132 cells. Viability and protein synthesis were not impaired by this treatment. The liposomal content was released into the lysosomes of the cultured cells, as revealed by ferritin uptake and electron microscopy. Degradation rates of long-lived proteins increased with the uptake of lysosomal proteases. However, the increased rates were not affected by chloroquine and leupeptin, in contrast to the inhibition by these reagents of the increased protein degradation of cells 'starved' of serum (step-down conditions). This approach opens a new way of investigating the degradation of intracellular proteins in cultured cells.In mammalian cells, two major pathways for intracellular protein degradation, lysosomal and non-lysosomal, have been postulated on the basis of the effect of inhibitors of lysosomal functions [l -41. The relative importance of each pathway varies with the cell type, growth characteristics and nutritional and endocrine status of the cells. Thus, it has been shown in perfused liver that almost all intracellular protein degradation, in several catabolic states, occurs in lysosomes [5, 61 and that these organelles carry out the bulk of protein degradation in liver homogenates [7].In growing cultured cells, inhibitors of lysosomal functions have no effect on overall intracellular proteolysis [4, 8 -lo], while in media deficient in serum or nutrients, degradation of long-lived proteins rises, and this enhanced degradation is affected by lysosomal inhibitors [3, 4,8, 101. However, since it has been found that these inhibitors do not completely block lysosomal protein degradation in isolated rat liver lysosomes [7] and since morphological and biochemical evidence indicate that lysosomes are responsible for the degradation of many long-lived proteins [4,7,11 -131, it appeared of interest to use new approaches in the study of protein degradation in cultured cells (e. g. to increase the amounts of lysosomal proteases). To that end, multilamellar liposomes loaded with lysosomal enzymes were incorporated, via endocytosis, into exponentially growing L-132 cells. As expected, lysosomal proteases were taken up by the lysosomes. This treatment increased the degradation rate of long-lived proteins. The increase, as for basal degradation, was not affected by leupeptin and chloroquine, in contrast to the inhibitory effect of these agents on the enhanced degradation of cells starved for serum (stepdown conditions).Correspondence to E. Knecht

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Some unexpected properties of cathepsin D

Cited by 3 publications

References 0 publications

Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins

Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins

Biosynthesis of lysosomal endopeptidases

Endocytosis of liposomes containing lysosomal proteins increases intracellular protein degradation in growing L‐132 cells

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