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Biomedical subjects

P Bohley

Publications and source records attributed to P Bohley.

At least 37 records · Page 2Linked to original sources
PubMed1982

Action of rat liver cathepsin L on collagen and other substrates.

1. It has been found that cathepsin L is very susceptible to loss of activity through autolysis. When this is prevented by purification and storage of the enzyme as its mercury derivative, preparations are obtained with higher specific activity than previously. 2. Active-site titration shows, however, that even the new purification method does not give preparations in which the enzyme is 100% active. 3. Benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide has been discovered to be a very sensitive substrate for cathepsin L. Like all other known substrates for cathepsin L, however, it is also cleaved by cathepsin B. 4. Cathepsin L degrades insoluble collagen at pH 3.5 over 5-fold faster than at pH 6.0. The specific activity at pH 3.5 is 5-10-fold higher than that of cathepsin B (rat or human) or bovine spleen cathepsin N ('collagenolytic cathepsin'). 5. Qualitatively, the action of cathepsin L on collagen is similar to that of cathepsins B and N, i.e. selective cleavage of terminal peptides leads to conversion of beta- and higher components mainly to alpha-chains.

Animals↗
PubMed1982

Inhibition of cysteine proteinase activity by Z-Phe-Phe-diazomethane and of aspartic proteinase activity by pepstatin in different organs from some animals and isolated cells from rat liver.

Two methods have been developed to discriminate simultaneously between the main part of cysteine proteinase activity (cathepsin L) and all aspartic proteinase activity (mainly cathepsin D) in rat organs, using Z-Phe-Phe-CHN2 which at 5 mumol/l completely inhibits cathepsin L from rat liver and, on the other hand, pepstatin which at 0.5 mumol/l completely inhibits cathepsin D. Substrates are double-labeled cytosol proteins from rat liver at pH 3.0 or azocasein in 3 mol/l urea at pH 5.0. Several organs from rat, pigeon, frog and carp have been investigated using these methods. Especially kidneys from rat, frog and carp contain a high Z-Phe-Phe-CHN2 inhibited activity. Investigating the different liver cell types we could confirm earlier findings that Kupffer cells and endothelial cells contain more pepstatin inhibited activity than parenchymal cells.

Animals↗
PubMed1981

Surface hydrophobicity of proteins and selectivity of intracellular proteolysis.

The affinity of cytosol proteins from protein-free fed rats to hydrophobic surfaces is diminished in comparison to the affinity of cytosol proteins from normal or protein-rich fed rats. Furthermore, the affinity of these cytosol proteins to hydrophobic surfaces in the presence of ultrafiltrates from cytosol of rats fed previously a protein-rich diet or a protein-free diet is diminished. Ultrafiltration itself increases the affinity of cytosol proteins to hydrophobic surfaces (octyl-sepharose or phenyl-sepharose).

Animals↗
PubMed1981

Action of rat liver cathepsin L on glucagon.

The proteolytic specificity of cathepsin L on glucagon was determined. Major cleavages are found between Thr7 and Ser8, Asp15 and Ser16, and between Met27 and Asn28. The bonds Ser11-Lys12, Val23-Gln24, and Gln24-Trp25 are hydrolyzed to a relatively low extent only. Whereas cathepsin B hydroxyzes glucagon at the C-terminus by a peptidyldipeptidase mechanism, cathepsin L cleaves the same substrate clearly as endopeptidase.

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

[Action of proteinase inhibitors in rats. 3. Influence of leupeptin on the rate of protein synthesis and the intracellular protein degradation by use of a test system including constant infusion of labelled amino acids, estimation of 3-methyl-histidine excretion and a triple-labelling technique].

Male Wistar rats (initial body weight 90 g) were fed ad libitum a whole-egg diet containing 10,5% crude protein. The animals of the experimental group received in each case of 1 mg leupeptin per 100 g of body weight in 12 hrs-intervals by i. p.-injection (3 days of treatment). Control animals got a leupeptin free solution. In addition, lysine dihydrochloride-alpha-15N was applied during the first three days of experiment to all animals and the nitrogen balance was determined. Urine from the N-Balance collection was analysed for 3-methyl-histidine excretion in order to calculate the degradation rate of myofibrillar proteins. On the fourth day the fractional rate of protein synthesis in several organs was estimated using the continuous infusion technique with 14C-leucine and 14C-lysine. The apparent biological half-lives of tissue protein were determined by a triple labelling technique, with (14C)-guanidino-L-arginine, L-5-3H-arginine and 15N-Lysine. The short-term treatment 3 days) with leupeptin did not affect the weight gain, the apparent digestibility of nitrogen and the N-balance. The fractional rate of protein synthesis was highest in the small intestine followed by the large intestine, liver and skeletal muscle and no influence of leupeptin treatment was observed. Furthermore no differences in the degradation rates of myofibrillar proteins between treated and untreated animals were found. The 3-methyl-histidine excretion via urine was 1.44 mg . kg-1 day-1 in both groups corresponding to a fractional rate of degradation of myofibrillar proteins of 2,5% per day. Apparent half-lives of tissue proteins in the small intestine, large intestine and liver, respectively, were shortest when estimated from the decay curves for the 14C-label and longest from the curves for the 15N-label. Leupeptin treatment resulted in prolonged apparent half-lives of the proteins in the large intestine and of the slowly turning over proteins in the liver. However, this effect seems to be caused rather by an increased reutilization of labelled amino acids than by a decreased protein degradation. Before continuing this kind of work the rate of uptake of injected leupeptine into tissues has to be investigated. Studies dealing with the in vivo action of proteinase inhibitors on protein metabolism have to include estimations of N-balance, protein synthesis rate, intracellular degradation rate of proteins as well as amino acid reutilization.

Amino Acids↗
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↗
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↗
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

[Mode of action of proteinase inhibitors in rats. 2. Effect of leupeptin on N-balance and biological half-life in ad lib feeding].

During a 15-day period, growing Wistar rats (75 g) were fed ad lib. a whole-egg diet containing 10% crude protein (control group) and the same diet + a daily subcutaneous injection of 1 mg of leupeptin per animal (experimental group), respectively. To investigate the influence of the peptide aldehyde leupeptin on the M-metabolism, N-balance trials were carried out between the 1st and the 6th, and the 10th and the 15th experimental days. Leupeptine was not found to influence the true N-digestibility. However, the intermediary N-utilization, characterized by the biological value of the dietary protein, deteriorated in comparison with the control group. The peptide aldehyde under study resulted in an extension of the biological half-life of the proteins in the tissue of the small and large intestine. It was measured via the decline of radioactivity in protein following application of L-guanidino-14 C-arginine and L-(4,5-3H)-leucine. The amino acid utilization appears to have been increased as well. In connection with previous studies (SIMON et al., 1976) i, which, under somewhat different experimental conditions, leupeptine resulted in a higher intermediary N-utilization, the mentioned agent is supposed to be able to influence N-utilization. However, additional studies are required to make statements regarding the influence of dosis and metabolic situation.

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

Protein degradation in rat liver cells.

The intracellular protein catabolism in isolated hepatocytes is inhibited by pepstatin and particularly by leupeptin in the presence of liposomes. Leupeptin has an inhibitory action both in the absence and in the presence of liposomes (nearly 40%).

Animals↗
PubMed1977

Cooperation of various subcellular fractions in protein degradation in vitro.

In order to test the possibilities in protein degradation between cell organelles comparatively, [3H]- and [14C]-leucine short-time labelled subcellular fractions from rat liver were incubated with each other at pH 6.9. All fractions tested were able to degrade short-lived proteins from foreign fractions, whereby the lysosomal supernatant fraction showed the highest proteolytic activity, which declines in the sequence: lysosomes--nuclei--mitochondria--cytosol--microsomes. Short-lived cytosolic proteins were especially suited as substrate for neutral proteases from all other fractions, but also microsomal, mitochondrial and nuclear proteins were well degraded by foreign fractions in comparison with the substrate autoproteolysis. Therefore in vivo manyfold cooperations between several organelles in protein catabolism seem to be possible.

Animals↗
PubMed1977

Studies on the in vivo-action of leupeptin on the nitrogen retention in rats.

A daily s.c. injection of 0.5 mg leupeptin for 7 days does not influence the digestibility of food-N but seems to improve the N-retention. A daily injection of 1.0 mg leupeptin for 14 days is without influence on the digestibility of N, too, but the N-retention was decreased. At this leupeptin level the half-lives of intestinal proteins were prolonged. The data indicate the possibility to influence the N-retention using leupeptin.

Animals↗