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Biomedical subjects

H Kirschke

Publications and source records attributed to H Kirschke.

At least 91 records · Page 5Linked to original sources
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

Membrane sialoglycoprotein from human erythrocytes activates lysosomal proteinases.

The capacity of membrane glycoproteins to interact with proteinases was investigated in the model system: Membrane sialoglycoprotein from human erythrocytes (glycophorin) and lysosomal proteinases from rat liver. Glycophorin was found to stimulate the activity of a lysosomal proteinase mixture up to about 150% at pH 6.9. Cathepsin L was found to be the primarily stimulated proteinase. The stoichiometry in the saturation range of the dose-response curve waas about 10 to 20 molecules glycophorin per molecule cathepsin L. The mechanism of the activation is unknown. Interactions of this type may be of importance for the regulation of cell proliferation on the level of cell membranes.

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↗
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

Proteolytic and transhydrogenolytic activities in isolated pancreatic islets of rats.

In homogenates and subcellular fractions of pancreatic islets of Wistar rats we could demonstrate three groups of protein degrading enzymes. The proteinases of group 1 are characterized by both trypsin-like and carboxypeptidase B-like specificities with slightly acid pH optima (pH 5.5-6.5) and seem to play important roles in the conversion of proinsulin into insulin. The properties suggest that these enzymes localized in the secretion granule/mitochondria fraction are related to the tissue cathepsins. Group 2 enzymes are thiol-depending proteinases with a pH optimum at 7.0 occuring mainly in the cytosol and to a lesser extent in the fraction of nuclei and cell debris. Group 3 represents the thiol protein oxidoreductase with a pH optimum of 7.0. This enzyme degrading disulfide bonds could also be important in the formation of the disulfide bonds during protein folding after synthesis.

Animals↗
PubMed1977

Conversion of proinsulin into insulin by cathepsins B and L from rat liver lysosomes.

Conversion of proinsulin and intermediate forms of proinsulin into insulin were studied with rat liver cell fractions and purified lysosomal proteinases by using the technique of polyacrylamide disc-electrophoresis. Both substrates were degraded very rapidly by homogenates and crude lysosomal fractions to split products not detectable on disc-electropherograms. Neither breakdown nor conversion were detected with the cytosol and the microsomal fraction. With partially purified lysosomal fractions (mol. wt. approx. 25 000) or with highly purified cathepsin L or cathepsin B (B1) proinsulin was converted into products migrating like the intermediate forms and insulin, and the intermediates were converted into products migrating like insulin and deoctapeptide-insulin in disc-electropherograms. The mechanism of conversion seems to be different for both enzymes. The results force us to conclude that lysosomal cathepsins, especially cathepsins L and B might be involved in the process of conversion of proinsulin into insulin and perhaps also of other precursors into biologically active proteins in vivo.

Animals↗
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↗