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Biomedical subjects

S Ansorge

Publications and source records attributed to S Ansorge.

At least 163 records · Page 9Linked to original sources
PubMed1984

The insulin and glucagon degrading proteinase of rat liver. Separation of the proteinase from the thiol-proteindisulfide oxidoreductases.

Insulin degrading enzymes of rat liver cytosol, the so-called insulin and glucagon degrading proteinase (IGP, EC 3.4.23.5), and two forms of the insulin degrading thiol-protein-disulfide oxidoreductase/isomerase (glutathione-insulin transhydrogenase, TPO, EC 1.8.4.2/5.3.4.1) were separated from each other and partially purified on DEAE-Sephadex. The highly purified proteinase was obtained by polyacrylamide gel electrophoresis of the DEAE-Sephadex-purified enzyme fraction and was used to produce monospecific antibodies to the IGP in rabbits. Strong evidence is given that the insulin and glucagon degrading proteinase is an autonomous enzyme existing in addition to the TPO forms in the cytosol of the liver. Combined action of the proteinase and the TPO system on radioiodinated insulin under various conditions in vitro revealed an independent and non-sequential degradation of insulin by these two enzyme systems.

Animals↗
PubMed1984

The insulin and glucagon degrading proteinase of rat liver: a metal-dependent enzyme.

Insulin and glucagon degrading proteinase (EC 3.4.23.5) purified from rat liver cytosol was characterized using radioiodinated insulin and glucagon as substrates. Maximum activity for breakdown of both hormones was found at pH 8.1. Thiol blocking reagents as well as indole derivatives inhibit the proteinase, whereas pepstatin, leupeptin, bestatin, elastatinal, antipain, chymostatin and phosphoramidon do not have any effect. Although the Km values and maximal velocities of insulin and glucagon breakdown deviate strongly from each other, the specificity constants (kcat/Km) for both substrates are nearly identical. The insulin and glucagon degrading proteinase, known as a thiol-dependent enzyme, was found to be also a metallo enzyme. Chelating agents, such as EDTA, EGTA, bipyridine and o-phenanthroline show a concentration dependent inhibition. The strongest inhibitor found was o-phenanthroline. Zn++, Co++, Mn++, and to a smaller extent Cd++ and Fe++, are capable of preventing the o-phenanthroline mediated inhibition. Removal of the protein-bound metal(s) results in a nearly total and irreversible loss of enzymatic activity.

Animals↗
PubMed1984

Distribution of thiol-protein disulfide oxidoreductase, insulin-glucagon proteinase and cathepsin D in different cell types of the rat liver.

Cathepsin D (EC 3.4.23.5), the insulin and glucagon degrading proteinase (IGP, EC 3.4.22.-) and the thiol-protein disulfide oxidoreductase (TPO, EC 1.8.4.2, 5.3.4.1) participate in the intracellular protein degradation, the last one also in post-protein-synthetic processing. The distribution of these enzymes was determined in isolated liver parenchymal cells, Kupffer cells and endothelial cells by means of immunochemical methods in order to further characterize these cell types. The cathepsin D content, expressed as microgram enzyme per mg protein, is about 3 fold higher in endothelial cells and about 5 to 24 fold higher in Kupffer cells than in parenchymal cells. This result confirms an earlier report which is based on the activity determination. The TPO concentration is highest in parenchymal cells with half of that concentration in Kupffer cells and one third in endothelial cells. About 0.5% of the total liver protein is represented by this enzyme. The IGP has been found to be totally absent in non-parenchymal cells. It represents, therefore, together with the glucose-6-phosphatase a valuable marker enzyme for parenchymal cells of rat liver.

Animals↗
PubMed1983

Insulin- and glucagonlike peptides in the brain.

The cellular localization and regional distribution of insulin- and glucagonlike substance, C-peptide-like immunoreactivity, thiol:protein disulphide oxidoreductase, TPO (E.C.1.8.4.2.), and insulin/glucagon-specific proteinase, ISP (E.C.3.4.22.-), are studied in the CNS of man, adult and juvenile rats, mice, tortoises, and frogs by use of immunohistochemistry. Furthermore, the content of immunoreactive insulin, glucagon, and C-peptide was estimated in human cadaver brains by radioimmunoassay. It could be shown that insulinlike immunoreactive material is widely distributed in the human brain and the CNS of juvenile rats as well as in mice, whereas in the CNS of adult rats and nonmammalian animals (frogs, tortoises) the polypeptide is restricted to a few nerve cell populations. C-peptide immunoreactivity was demonstrated in human CNS in the same nerve cells as insulin. By use of two different glucagon-antisera it was revealed that gut-type glucagon occurs in many nerve cells of human and mouse brains, as well as in the CNS of juvenile rats. On the other hand, pancreas-type glucagon was less widely distributed in the human brain and nearly not detectable in the CNS of mice and rats. With the exception of neurosecretory nerve cells, there was a high degree of coincidence between the localization of insulin and TPO. The immunoreaction against the ISP antiserum was weak, but correlated well with the distribution of insulin-immunoreactivity. The occurrence of TPO and ISP in the brain demonstrates the ability of nervous tissue to degrade insulin and glucagon. By radioimmunoassay it was established that human brain contains insulin, glucagon and C-peptide at concentrations that exceed blood levels. We conclude from our data that, at least in part, cerebral insulin and glucagon are products of the brain itself.

Animals↗
PubMed1982

Neurosecretory nerve cells of rat and fish brain are rich in thiol-protein disulphide oxidoreductase (TPO) immunoreactivity.

Thio-protein disulphide oxidoreductase (TPO) was demonstrated in rat and fish brain by use of the indirect peroxidase-antiperoxidase technique. TPO immunoreactivity was found to be widely distributed throughout rat CNS. Whereas most nerve cells possessed only weak immunoreactivity to the enzyme, the neurosecretory cells of Nuc. supraopticus and Nuc. paraventricularis were heavily laden with immune products. In fishes (rudd) the neurosecret-producing neurons of the Nuc. preopticus were the only locus being positive for TPO. From our findings we conclude that thiol-protein disulphide oxidoreductase might be involved into the process of neurophysin synthesis.

Animals↗
PubMed1981

The immunochemical identification of a thiol-protein disulfide oxidoreductase (glutathione-insulin transhydrogenase) in pancreatic islets.

Pancreatic islets contained insulin-degrading activity that was completely removed by antisera to purified microsomal thiol-protein disulfide oxidoreductase from rat liver. In Ouchterlony double-diffusion experiments with these antisera, extracts of islet homogenates showed a single precipitation band of identity with the purified liver enzyme. Two dimensional immunoelectrophoresis also gave a single precipitate peak like that of the liver enzyme. The concentration of the enzyme in rat islets as determined by quantitation of the precipitates obtained in the electroimmunodiffusion analysis was in the order of 1.0% of total islet protein. The results suggest that, in vitro, cleavage of insulin into its polypeptide chains is catalyzed by the thiol-protein disulfide oxidoreductase. This enzyme promoting thiol-protein disulfide inter-change may be important for regulating the content of pancreatic insulin.

Animals↗
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

[The immunohistochemical demonstration of the Thiol: proteindisulfid oxidoreductase (TPO) in pancreas and isolated Langerhans'islets. A light-, fluorescent- and electron microscopic study (author's transl)].

Thiole: Protein disulfide Oxidoreductase (E. C. 1.8.4.2) is capable of catalyzing thiole-disulfid exchange reactions and might have an important function in both protein biosynthesis and degradation. By using histochemical methods we were able to demonstrate the localization of this enzyme in the Langerhans'islets and in the acinus cells of rat pancreas. The reaction of the acinus cells, however, was much weaker than that of the islets. Electron microscopic experiments revealed the enzyme to be located in the outer membrane of the nucleus, in the membranes of the endoplasmic reticulum and B-cell granules, and in the plasmalemma also. All these structures are known to be involved in the insulin biosynthesis and secretion. There is a good correlation between morphological and biochemical findings. In acinus cells there are a few reaction in the outer nucleus membrane, the membranes of the endoplasmatic reticulum, and the plasmalemm.

Animals↗
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

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

Presence of an endopeptidase activity in rat liver ribosomes.

Preparation of ribosomes using different procedures (treatment of postmitochondrial-postlysosomal supernatant or microsomes with 1% triton in 0.15 or 0.5 M KCl and subsequent sucrose gradient centrifugation; treatment of microsomes with 1.5% deoxycholate/2% triton) results in purified ribosomes which contain an endopeptidase activity detectable by breakdown of ribosomal proteins to trichloroacetic acid soluble split products. The proteolytic activity can be recovered also in the extracted proteins of whole ribosomes. With ribosomes the pH optimum of proteolytic breakdown is at about 7. The inhibition of the activity by leupeptin, DIFP and soya bean trypsin inhibitor suggests a serine type of the proteolytic activity.

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

[Insulin catabolism by transformed, cultivated mouse fibroblasts (L-cells)].

Cultivated, transformed mouse fibroblasts (L-cells) are endowed with enzyme systems for insulin breakdown that are comparable to those of many other types of non-dedifferentiated insulin target cells (time and substrate dependence, Km value). It cannot yet be decided, however, which of the potential enzyme systems (thiolproteindisulfide-oxidoreductase (TPO) [E.C. 1.8.4.2] or proteinase) is prevalent in insulin breakdown in intact L-cells. Despite the lack of typical target symptoms for insulin, the cultivated L-cell could provide an appropriate experimental model for the insulin metabolism.

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.

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