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Biomedical subjects

H Kirschke

Publications and source records attributed to H Kirschke.

At least 55 records · Page 3Linked to original sources
PubMed1989

The properties of peptidyl diazoethanes and chloroethanes as protease inactivators.

Earlier work has demonstrated the irreversible inactivation of serine and cysteine proteinases by peptides with a C-terminal chloromethyl ketone group. With a C-terminal diazomethyl ketone, on the other hand, peptides become reagents specific for cysteine proteinases. We have now synthesized and examined the properties of reagents with an additional methyl side chain near the reactive grouping with the goal of diminishing side reactions in a cellular environment. Derivatives of neutral amino acids as well as of lysine and arginine have been prepared. The chloroethyl ketones are about 60% less reactive to chemical nucleophiles than the chloromethyl ketones. However, the susceptibilities of the proteases examined varied remarkably. Cathepsins B and L of the papain family of cysteine proteinases were much less susceptible (about 2 orders of magnitude less) to both peptidyl diazoethyl and chloroethyl ketones. In marked contrast, clostripain, a cysteine proteinase of a separate family was decisively more susceptible to chloroethyl ketones. The serine proteinases showed a drop in susceptibility to the chloroethyl ketones generally, and this was similar to the drop in chemical reactivity in proceeding from the chloromethyl to the chloroethyl ketone.

Amino Acid Chloromethyl Ketones↗
PubMed1989

Peptide methyl ketones as reversible inhibitors of cysteine proteinases.

Peptide methyl ketones represent a new class of reversible, competitive cysteine proteinase inhibitor with little or no effect on serine proteinases. The affinity of the inhibitors to papain (EC 3.4.22.3), cathepsin B (EC 3.4.22.1) and cathepsin L (EC 3.4.22.15) depends on the peptide chain length and on side-chain effects. Variations in the P1 and P4 positions (terminology of Schechter and Berger) and their influence on the efficiency of the inhibitors have been investigated. The most effective inhibitors display inhibition constants in the micromolar range. In contrast to the endopeptidases papain and the cathepsins B and L, the aminoendopeptidase cathepsin H (EC 3.4.22.16) is not inhibited by N-acylated peptide methyl ketones but only by amino methyl ketones containing a free alpha-amino group. The endopeptidases are not affected by amino methyl ketones.

Amino Acid Sequence↗
PubMed1989

Cathepsin B immunoreactive neurons in rat brain. A combined light and electron microscopic study.

The regional distribution and cellular localization of the lysosomal proteinase cathepsin B was studied by use of monospecific antiserum. The application of the peroxidase-antiperoxidase technique at the light microscopic level revealed cathepsin B immunoreactive neurons in many brain areas. A strong immunoreaction was found in pyramidal cells of the cortex, large neurocytes of the septal region, some hippocampal neurons and magnocellular nerve cells of the hypothalamus. Immunogold labeling on ultrathin cryosections of rat neocortex revealed the enzyme protein to be associated with lysosomes.

Animals↗
PubMed1989

Isolation and some properties of a cathepsin E type proteinase from rat spleen.

Cathepsin E (EC 3.4.23.--) has been isolated from rat spleen. The procedure included autolysis at pH 4.2 which was probably the reason why we isolated a polypeptide of Mr 42 kDa instead of 90 kDa. The latter is reported in the literature to be the Mr of native cathepsin E. The enzyme dissociates under reducing conditions in two identical monomers. In our preparation a mechanism different from reduction must be active producing the 42 kDa polypeptide. This enzyme was hard to distinguish from cathepsin D (EC 3.4.23.5.) which shows similar properties such as size, substrate specificity, stability in 6 M urea, and dependence of the activity on pH. The clear distinction between the two enzymes was proven on the basis of immunochemical reactions. Antibodies to both cathepsins, D and E, did not show any crossreaction with the nonrelated antigen.

Animals↗
PubMed1988

Active center differences between cathepsins L and B: the S1 binding region.

The substrate peptide bond cleaved by cathepsins B and L is determined not by the amino acid contributing the carboxyl group to this bond as in the case of serine proteases but rather by the presence of a neighboring amino acid with a large hydrophobic side chain. From a study of the inhibitory potency in a series, Cbz-Phe-X-CHN2, in which Phe promotes binding at S2 (terminology of [(1968) Biochem. Biophys. Res. Commun. 32, 898-902]) while the amino acid X probes S1, it is shown that this region of cathepsin L also has the ability to accommodate large hydrophobic side chains. In this respect cathepsin L differs from cathepsin B. Thus Cbz-Phe-Tyr(O-t-Bu)CHN2 inactivates cathepsin L with a rate 2.5 x 10(4) greater than that for cathepsin B.

Amino Acids↗
PubMed1988

Cathepsin B immunoreactivity is widely distributed in the rat brain.

The cellular localization and regional distribution of cathepsin B within rat CNS was revealed by immunohistochemistry using a monospecific antiserum. Cathepsin B protein was found to be widely but unevenly distributed throughout rat brain. Neurons were always cathepsin B immunoreactive. Glial elements were only occasionally immunostained. The distribution of the enzyme resembles largely that of cathepsin D.

Animals↗
PubMed1987

Action of rat liver cathepsin B on bradykinin and on the oxidized insulin A-chain.

Rat liver cathepsin B was tested for its peptide-bond specificity against bradykinin and the oxidized insulin A-chain. Bradykinin was shown to be resistant to the action of cathepsin B. One possible reason for this resistance is the proline content of the peptide and the discrimination against proline residues at three or four subsites of cathepsin B. Oxidized insulin A-chain was degraded by a peptidyl dipeptidase activity. Three dipeptides were cleaved from the C-terminal part of the insulin A-chain after having been incubated for 2 h (molar ration E:S = 1:2800) and six dipeptides were released after a longer digestion (10 h, E:S = 1:575).

Amino Acid Sequence↗
PubMed1987

Enzyme-substrate interactions in the hydrolysis of peptides by cathepsins B and H from rat liver.

The number of possible subsites of the rat liver cysteine proteinases cathepsin B and cathepsin H was determined in the N-terminal direction from the scissile bond. An elongation of the substrate peptide chain of up to four amino acid residues enhances the hydrolysis rate of both cathepsins. The greatest increase in activity was observed by elongation to the dipeptide substrate for cathepsin B and to the tetrapeptide substrate for cathepsin H. Both proteinases discriminate proline from their subsites S1 and S2, but accept it well in S3. A quantitative distinction between the endopeptidase and the peptidyl dipeptidase activity of cathepsin B was feasible by using two model peptides: (Formula: see text) (Z = benzyloxycarbonyl; X = NH2 or OH; the arrow shows the cleavage site). Whereas the peptide acid, representing the peptidyl dipeptidase substrate, was hydrolysed by cathepsin B twice as fast as the peptide amide as an endopeptidase substrate, cathepsin H clearly had a preference for the amide substrate.

Animals↗
PubMed1987

Lysosomal proteinases.

A characteristic of lysosomal cysteine proteinases is given by their kinetic constants with specific substrates, their sequence homology, and their reactivity with monospecific polyclonal antibodies.

Animals↗
PubMed1986

Cathepsin S. The cysteine proteinase from bovine lymphoid tissue is distinct from cathepsin L (EC 3.4.22.15).

Cathepsin S was purified from bovine spleen by acid autolysis, (NH4)2SO4 fractionation and chromatography on CM-Sephadex C-50, CM-cellulose and activated-thiol-Sepharose. Cathepsin L was isolated from lysosomal fractions of rat liver, rat kidney and bovine liver. Generally, cathepsin L was bound tightly to CM-Sephadex C-50. Preparations of cathepsin L from rat liver, rat kidney and bovine liver were shown to have kinetic constants for the substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide in the same range (Km 2-3 microM). Benzyloxycarbonyl-Phe-Phe-diazomethane proved to be a sensitive irreversible inhibitor of cathepsin L from different species. Cathepsin S differed in all these characteristics from cathepsin L. A polyclonal antibody to cathepsin L from rat reacted with bovine cathepsin L but not with bovine cathepsin S.

Animals↗
PubMed1986

[Age dependent changes in the collagen content of the human oviduct].

On the basis of present investigations, namely on account of the hydroxyproline determinations and the biomathematical analysis of data, the ageing of the human Fallopian tube with regard to its collagen content can be characterized as follows: Collagen concentration of the oviduct increases slightly and almost linearly up to the 5th decade of woman life. In the 2nd half of the fifth decade, i.e. already in the premenopausal years, this increase is accelerated, then changing into a yet almost linear but stronger rise of the collagen content.

Adult↗
PubMed1986

Cathepsins B and D in rat brain glia during experimentally induced neuropathological defects. An immunocytochemical approach.

The lysosome-associated cathepsins B and D were localized by means of immunocytochemistry (peroxidase-antiperoxidase technique) in glial cells of rat brain. A specific reaction pattern of hippocampal neuroglia could be observed after intracerebroventricular application of the neurotoxin kainic acid. After the induction of a focal epilepsy in rats by the implantation of cobalt pellets there was a pronounced immunoreaction of glia near the primary focus as well as the mirror focus. It is concluded that both cathepsins are useful immunocytochemical markers to trace functionally activated glia.

Animals↗
PubMed1986

Are there species differences amongst the lysosomal cysteine proteinases?

Kinetic constants for the hydrolysis of synthetic substrates by cathepsin B, cathepsin L or cathepsin H show only small variations between various species. The species variants purified from human, beef and rabbit reacted with antisera raised against the enzymes from rat. With regard to these criteria we could show that cathepsin I from rabbit lung is a species variant of cathepsin H, whereas cathepsin S is a separate enzyme from cathepsin L.

Animals↗
PubMed1986

Immunohistochemical analysis of the distribution of cathepsin D throughout human nervous system with reference to developmental aspects.

The aspartic endopeptidase cathepsin D was demonstrated in post mortem human brain by means of the peroxidase-antiperoxidase technique. The enzyme protein was found to be present in multiple neurons as well as in some glial cells. In embryonic nervous tissue cathepsin D was detected as early as at the 12th gestational week. During neuroontogenesis there was a continuous increase in cathepsin D immunoreactivity.

Aged↗
PubMed1986

Demonstration of cathepsins H and L in rat tissues.

Using immunohistochemical techniques, cathepsins H and L were shown to be widely distributed in rat tissues. The intracellular location of the enzymes in the parenchymatous tissues was granular under light microscopy and was shown to be lysosomal by immunoelectron microscopy of the kidney proximal tubule epithelium. In the lymphatic tissue both cathepsins H and L were seen in the reticular cells and not in the lymphoid cells. The locations of the two enzymes differed from each other in some tissues suggesting that the enzymes may have specialized functions in those tissues.

Animals↗