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B Wiederanders

Publications and source records attributed to B Wiederanders.

14 recordsLinked to original sources
PubMed1992

Phylogenetic conservation of cysteine proteinases. Cloning and expression of a cDNA coding for human cathepsin S.

A 1.8-kilobase full-length cDNA of human cathepsin S, a lysosomal cysteine proteinase, has been isolated. The single long open reading frame encodes a polypeptide of 331 amino acids consisting of a 15-amino acid NH2-terminal signal peptide, a propeptide of 99 amino acids, and a mature polypeptide of 217 amino acids. The deduced amino acid sequence contains only one potential N-glycosylation site located in the propeptide. The NH2-terminal amino acid sequence of the mature polypeptide was confirmed by sequencing cathepsin S purified from human spleen. The cDNA detects a 1.9-kilobase transcript in poly(A)+ RNA from human fibroblasts. Expression of human cathepsin S in transfected baby hamster kidney cells resulted in up to more than 300-fold cathepsin S activity as compared to untransfected controls. In the expressing baby hamster kidney cells, human cathepsin S is transported to the lysosomes via the mannose 6-phosphate receptor pathway as shown by density gradient centrifugation, immunofluorescence, and detection of the 37-kDa cathepsin S precursor in the medium in the presence of NH4Cl. The deduced amino acid sequence of human cathepsin S exhibits a substantial degree of similarity with other human cysteine proteinases and papain indicating that they have a common ancestral gene and are members of a gene family.

Amino Acid Sequence↗
PubMed1979

Lysosomal cysteine proteinases.

Cathepsin B has so far been the most investigated cysteine (thiol) proteinase of lysosomes. The use of cytosol proteins as substrates has allowed the detection of two new lysosomal cysteine proteinases from rat liver: the endoaminopeptidase cathepsin H and cathepsin L, which splits almost no synthetic substrates but has a more than 10-fold higher specific activity with proteins as substrates than other mammalian cysteine proteinases. The properties of cathepsin L are compared with those of other cysteine proteinases (cathepsin B,H,N,S and others) from different tissues in relation to substrate specificity and sensitivity to inhibitors. A new test system for determining cathepsin L allows us to investigate the distribution of this enzyme between different cell types and to speculate about the special role of cysteine proteinase in intracellular protein degradation.

Animals↗
PubMed1979

The ribosomal serine proteinase: cathepsin R.

As has been known for several years, thoroughly purified ribosomes contain a firmly bound serine proteinase with an optimum of activity at neutral pH. The present paper shows that the activity is found in free cytoplasmic ribosomes as well as in ribosomes detached from the membranes of the endoplasmic reticulum of rat liver. After ribosome dissociation, the proteinase activity is found only on the 40 S subunits. Recovery of the proteinase in the proteins of whole ribosomes or of 40 S subunits amounts to 44 and 65%, respectively. Ribosomes purified both from plant (Euglena) and bacterial (Acinetobacter) cells contain a serine proteinase having an activity quite comparable to that of rat liver ribosomes. In view of the recommendations of BARRETT et al. ( in REICH, RIFKIN and SHAW (eds).: Proteinases and Biological Control, Cold Spring Harbour Lab., 1975, p. 481), who no longer restrict the name "cathepsin" to acid or even lysosomal proteinases, we propose the name " ccathepsin R" for this ribosomal serine proteinase.

Animals↗
PubMed1978

The age dependence of intracellular proteolysis: changes of the substrate proteins.

Liver cytosol proteins of young (4--6 months) and old (18--27 months) rats were degraded in vitro by papain, pronase, trypsin, pepsin, cathepsin D from rat liver and a soluble lysosomal enzyme mixture from rat liver. We could demonstrate the capability of the latter enzyme mixture to degrade proteolytically the cytosol proteins of young animals about 20% faster than those of the older animal group. Digesting radioactive labelled "young" cytosol in the presence of unlabelled "old" cytosol the possibility could be excluded, that this effect was due to an inhibitor of macromolecular size present in the "old" cytosol.

Aging↗
PubMed1978

[Age dependent structural changes in non collagen proteins and their consequences in protein turnover degradative rate (author's transl)].

During the aging process the occurence of proteins with physico-chemical properties different from their juvenile counterparts are observed in many organisms. The probable molecular mechanisms of such variations are reviewed. Although no direct evidence could be found in the literature for an altered breakdown rate of such a single "old" protein, the possible influence of this kind of modifications in protein molecules on their over all degradative rate is discussed.

Aging↗
PubMed1977

Cathepsin L. A new proteinase from rat-liver lysosomes.

1. Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondrial pellet, gel filtration of the lysosomal extract and chromatography on CM-Sephadex. 2. Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate its molecular weight was found to be 23000-24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8-6.1 ascertained by analytical isoelectric focusing. 3. Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6-7. In contrast to cathepsin B1, its capability of hydrolyzing N-substituted derivatives of arginine is low and it does not split esters. 4. Greatest activity is obtained close to pH 5.0 with 70-90% of maximal activity at pH 4.0 and pH 6.0 and 30-40% at pH 7.0. 5. The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl-lysine. Leupeptin acts as a pseudo-irreversible inhibitor. 6. The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep-frozen state.

Animals↗
PubMed1977

Aging changes in intracellular protein breakdown.

Using radioactively labelled cytosol proteins as substrates we were able to exclude the possible accumulation of any specific inhibitor for the lysosomal proteases in rat liver cytosol during the aging process. There were also no gross changes in the molecular weight patterns of these proteins during the aging process. The percentage of more hydrophobic proteins seems to be identical in both the "old" and "young" cytosol proteins. From immunological experiments we suppose a qualitative change in the composition of rat liver cytosol proteins or of their properties during the aging process.

Aging↗
PubMed1977

Cathepsin H: an endoaminopeptidase from rat liver lysosomes.

1. Cathepsin H is an endoaminopeptidase belonging to the group of thiol enzymes. It was purified from rat liver lysosomes by gel filtration on Sephadex G-75, chromatography on CM-Sephadex C-50, on DEAE-Cellulose DE-52 and subsequently on an organomercurial absorbent. 2. The molecular weight of cathepsin H was found to be 28,000 and the isoelectric point was estimated to be at pH 7.1 by analytical isoelectric focusing. 3. Cathepsin H has to be designated as endoaminopeptidase, because it catalyzes the hydrolysis of proteins, N-terminal substituted proteins and amino acid derivatives, respectively, as well as of peptides of various chain length and N-terminal free amino acid derivatives. Cathepsin H shows amidase and esterase activity, but it does not show carboxypeptidase activity. The finding of the amino- and endopeptidase nature of cathepsin H has been revealed mainly by the results obtained with inhibitors and by the rather high temperature stability of the enzyme. The chlormethyl ketone of leucine proves to be the strongest inhibitor of the aminopeptidase as well as of the endopeptidase activity, whereas leupeptin endopeptidase activity and endopeptidase substrates inhibit competitively the aminopeptidase activity. 5. Cathepsin H shows highest activity at pH 6.0 in the presence of 1--5 mM GSH and EDTA. 6. The enzyme is stable for several months at slightly acid pH values in a deep frozen state.

Animals↗
PubMed1976

[On the age dependence of the intracellular proteolysis (author's transl)].

We report the age dependent changes of the proteolytic capacity of the rat liver at pH 3.0 and pH 6.0. The total proteolytic activity increases during the whole cycle. During the weanling period the specific activity (mug substrate split x min-1 X mg liver protein-1) rises up to values about 30% higher than those of the remaining lifetime. We found the specific activity in old male animals (18 months) to be lower than in younger ones (5 months). These findings are correct for the liver homogenate as well as for the cell fractions.

Aging↗
PubMed1976

[Intracellular protein breakdown. VIII. The use of double-labeled proteins as substrates].

Double-labeled proteins from rat liver cytosol (14C in long-lived, 3H in short-lived proteins after in-vivo-labeling) are used as substrates for unlabeled proteinases in vitro. Differences in the degradation rates of short-lived and long-lived proteins in vitro by different proteinases and after addition of different effectors allow conclusions concerning their importance for the in-vivo-turnover of substrate proteins. The main activity (greater than 90%) of soluble-lysosomal proteinases at pH 6,1 and pH 6,9 is caused by thiolproteinases, which degrade preferentially short-lived cytosol proteins. These proteinases are inhibited by leupeptin. Autolysis of double-labeled cell fractions shows a remarkably faster breakdown of short-lived substrate proteins only in the soluble part of lysosomes. Microsomal fractions degrade in vitro preferentially long-lived substrate proteins.

Animals↗
PubMed1976

[Intracellular protein breakdown. VI. Isolation, properties and biological significance of cathepsin D from rat liver].

The preparation and properties of cathepsin D from rat liver are reported. The enzyme is an endopeptidase of lysosomal origin. The molecular weight was estimated to be 49000 by sodium-dodecylsulfate electrophoresis. We did not find any dissociation into subunits under reducing conditions, in contrast to some other authors. We found the enzyme to occur in at least 4 forms with the isoelectric points 5.87, 5.65, 5.41 and 5.13. Strong -SH-blocking reagents inhibit the activity, but the most powerful and specific inhibitor was pepstatin (Ki=38 nM). The substrate specificity is discussed. There was no proof for any zymogen activation in a great number of experiments. Since the cathepsins B1, B3 and L obviously seem to play the major role in the intracellular protein breakdown within the rat liver, the main task of cathepsin D is the degradation of extracellular proteins in this organ.

Animals↗
PubMed1976

[Intracellular protein breakdown. VII. Cathepsin L and H; two new proteinases from rat liver lysosomes].

Some properties (molecular weight, pI, temperature stability, action of selected inhibitors, substrate specificity and pH-activity dependence) of two not yet known cathepsins from rat liver lysosomes are compared with the properties of the known cathepsin B1. Cathepsin L is a thiolproteinase, has a molecular weight of 23--24000 and a pI of 5,8--6,1. By disc electrophoresis and isoelectric focusing there appear several protein bands which all have enzymatic activity. Leupeptin behaves as a strong inhibitor. The pH-optimum for digestion of proteins is close to 5,0. Cathepsin L does not hydrolyse esters and splits synthetic low molecular substrates only to a low degree. Cathepsin L stored in presence of glutathion and EDTA in liquid nitrogen kept its activity for some months. Cathepsin H is an aminopeptidase as well as an endopeptidase. An enzyme with these bifunctional properties was detected up to now only in E. coli but not in animal cells. Cathepsin H is a thiol-enzyme with a molecular weight of 28000 and a pI of 7,1. Strong inhibitors are leucyl-chlormethan and SH-blocking substances. Leupeptin shows only a weak inhibitory effect to this enzyme compared to its action on cathepsins L and B1. The pH-optimum for hydrolysis of all substrates is 6.0. Cathepsin H splits proteins, amino acid derivatives and selected N-protected amino acid derivatives. Cathepsin H compared to cathepsin L and B1 is quite temperature stable.

Animals↗