Appearance of parkinsonian syndrome after administration of delta sleep-inducing peptide into the rat substantia nigra

Bilateral microinjection of delta-sleep-inducing peptide (DSIP) (10.0 nmol) into the substantia nigra provoked hypokinesia and rigidity in rats observed during 4.0 hours. Injection of DSIP in dose of 5.0 nmol into the substantia nigra or into the nuclei caudati in dose of 10.0 nmol did not induce such symptoms. The enhanced slow-wave activity was recorded in caudate nuclei during hypokinesia and rigidity which demonstrated the formation of the generator of pathologically enhanced excitation (GPEE). The systemically cyclodol administration resulted in abolishment of rigidity and increase in locomotor activity. The conclusion is that bilateral intranigral DSIP injection caused acute parkinson syndrome in rats due to the formation of cholinergic GPEE in caudate nuclei. The hyperactive caudate nuclei act as the pathologic determinant which induces the parkinson syndrome.     

Inactivation of blood plasma alpha 1-proteinase inhibitor as affected by 2 splenic thiol proteinases active in a neutral medium

It has been found that two active in neutral medium thiol proteinases from bovine spleen, cathepsin L and cathepsin H, bring about rapid and irreversible inactivation of alpha 1-proteinase inhibitor (alpha 1PI)--one of the major plasma inhibitors of serine proteinases. The activity of the enzymes studied did not change upon the interaction with alpha 1PI. With stoichiometric proteinase/inhibitor ratio, the inactivation of alpha 1PI under the effect of cathepsin L was instantaneous, while under the effect of cathepsin H it occurred within 30-60 min. The products of alpha 1PI inactivation had an inhibitory effect on the rate of its reaction with cathepsin L. alpha 1PI inactivation under the action of cathepsin L and cathepsin H was accompanied by the decrease in the molecular mass of the inhibitor from 54 kDA to 46 kDa. This was, probably, caused by the hydrolysis of the peptide bond formed by NH2 group of threonine. The 46 kDa fragment did not undergo further degradation. It did not bind to immobilized trypsin but retained antigenic properties. The results obtained show that the limited proteolysis is a mechanism of the inhibitor inactivation. It is suggested that under some conditions thiol proteinases, upon their release from the cell, participate in the control of effective alpha 1PI concentration.     

Antistressor effect of delta-sleep-inducing peptide in hypokinetic stress]

The influence of DSIP injection on the levels of neurotransmitters during hypokinetic stress was studied. It was shown that hypokinesia produced considerable changes in the levels of GABA glutamate, aspartate and homocarnosine. The injection of DSIP normalized the level of neurotransmitter amino acids. Administration of DSIP also reduced stress-induced changes in the activity of enzymes of GABA metabolism. Stress protective effect of DSIP was also demonstrated by normalising of the POL of lipids in brain.     

Elastase and cathepsin G of human monocytes: heterogeneity and subcellular localization to peroxidase-positive granules

We used antibodies to human leukocyte ("neutrophil") elastase and cathepsin G to localize the corresponding antigens in human neutrophils, monocytes, and alveolar macrophages by immunohistochemistry. Furthermore, we combined immunogold localization with enzyme histochemistry to localize proteinase antigens and endogenous peroxidase activity in the same sections. As expected, all neutrophils contained both elastase and cathepsin G, and the proteinases localized to granules with peroxidase activity. In contrast, marked heterogeneity in monocyte staining for elastase, cathepsin G, and endogenous peroxidase was found. Sixty percent or more were unstained, while the remainder varied greatly in staining intensity. The elastase and cathepsin G in monocytes were localized by immunoelectron microscopy, combined with histochemistry, to cytoplasmic granules which had peroxidase activity. Alveolar macrophages were unstained. Therefore, a subpopulation of peripheral blood monocytes contains leukocyte elastase and cathepsin G in a cell compartment from which these enzymes may potentially be released into the extracellular space. The occurrence of peroxidase and neutral proteinases in the same granules in monocytes could permit the H2O2-myeloperoxidase-halide system and the neutral proteinases to act in concert in such functions as microbe killing and extracellular proteolysis.     

Comparative study of the proteolytic products of fibronectins in the serum of adult and embryonic animals formed under the effects of proteinases of the spleen

The effects of two spleen proteinases, cathepsin D and thiol proteinase I active in neutral media--on the structural properties of fibronectins from blood plasma on adult animals and their embryos were investigated. Proteinase I and cathepsin D caused rapid fragmentation of all fibronectins under study. Fibronectin from calf embryonic serum was more sensitive to proteinase I than that from adult animal serum. The molecular weight and the correlation between the proteolytic products formed under the influence of each enzyme, on the embryonic and "adult" fibronectins, are very similar but not identical. Similar results were obtained in experiments with proteolytic products of chicken serum and embryo fibronectins. Fragmentation of embryonic fibronectin occurs more rapidly than that of chicken fibronectin; the fibronectin proteolytic products differ both qualitatively and quantitatively. However, the determination of structural differences between these fibronectins is considerably hampered by the presence of protein contaminations in chicken fibronectin preparations.     

Plasma membrane-associated cysteine proteinases in human and animal tumors

The ability of tumor cells to invade into and through normal tissue during the metastatic cascade has been attributed to tumor-associated degradative enzymes including proteinases of the metallo, serine and cysteine classes. Work from several laboratories has established that the cysteine proteinases cathepsins L and B are released from tumor cells, primarily as latent precursor forms. In addition, a cathepsin B-like cysteine proteinase has been shown to be associated with the plasma membrane fraction of several animal and human tumors. This form of the enzyme retains activity under physiologic (or pathologic) conditions including at neutral pH and in the presence of low Mr inhibitors. Since we have established that cathepsin B can degrade the basement membrane attachment glycoprotein laminin, we speculate that plasma membrane-associated cathepsin B may participate in focal dissolution of the basement membrane during tumor cell extravasation.     

Proteoglycan-degrading enzymes of rabbit fibroblasts and granulocytes.

A neutral proteinase secreted by rabbit synovial fibroblasts in parallel with specific collagenase was partially purified by ion-exchange chromatography. At pH 7.6 this proteinase degraded 35S-labelled bovine nasal proteoglycan and azo-casein. The enzymic activity was inhibited by EDTA, 1,10-phenanthroline and serum, whereas di-isopropyl phosphorofluoridate and soya-bean trypsin inhibitor had little effect. By gel filtration the apparent mol.wt. of the enzyme was 25000. The fibroblast neutral proteinase was compared with the proteoglycan-degrading neutral proteinases of rabbit polymorphonuclear-leucocyte granules. Two distinct activities were found in the granules: one was inhibited by soya-bean trypsin inhibitor and the other by EDTA. The proteoglycan-degrading proteinases of rabbit fibroblasts and polymorphonuclear leucocytes at acid pH also were examined. Both cathepsin D and a thiol-dependent proteinase contributed to the degradation of proteoglycan at pH 4.5.     

Immunochemical similarity between a gastric mucosa non-pepsin acid proteinase and neutrophil cathepsin E of the rat

Antiserum against a rat gastric mucosa non-pepsin acid proteinase precipitates rat neutrophil cathepsin E, with a precipitation curve essentially similar to that of the gastric enzyme. Taken together that the antiserum precipitates a cathepsin E-like acid proteinase from rat spleen (Muto, N., Yamamoto, M. and Tani, S. (1987) J. Biochem. (Tokyo) in press), the data indicate that the non-cathepsin D acid proteinases in rat neutrophils, gastric mucosa and spleen are immunochemically closely related. In contrast with the earlier data, cathepsin E from rabbit neutrophils exhibited a maximal activity at around pH 3.0-3.2 and preferred hemoglobin to albumin as substrate, which supports that the non-cathepsin D acid proteinases in the rat tissues are relevantly classified as cathepsin E.     

Significant accumulations of cathepsin B and prolylendopeptidase in inflammatory focus of delayed-type hypersensitivity induced by Mycobacterium tuberculosis in mice

To clarify what kinds of proteinases are secreted into the foci of allergic-inflammation involving delayed-type hypersensitivity reaction, we examined the characteristic releases of various proteinases into the foci of Mycobacterium tuberculosis (M. tuber.)-induced delayed-type allergic-inflammation in mice. The significant activities of cathepsin B and prolylendopeptidase were observed in the washing-fluids of subcutaneous inflammatory foci of M. tuber.-induced delayed-type allergic-inflammation, but not M. tuber.-induced acute-inflammation. The SDS-resistant complex of cathepsin B and a protein substrate with apparent molecular mass of 74 kDa was observed by Western blot analysis. On the other hand, no significant accumulations of other proteinases, such as matrix metalloproteinases, cathepsin D, and serine proteinases, were determined. CA-074, a specific inhibitor of cathepsin B, suppressed both swelling and cathepsin B activity in the footpad having M. tuber.-induced delayed-type allergic-inflammation in vivo. These results suggest that cathepsin B may play an important role in the formation of M. tuber.-induced delayed-type allergic-inflammation.     

Separation of acid and neutral proteinases of brain.

1. Cerebral proteinases were separated on Sephadex G-100 columns into acid and neutral fractions free from cross-contamination. Acid proteinases were more stable and were purified by additional steps with salt and pH5.0 precipitations, column chromatography on DEAE- or CM-cellulose and free-flow electrophoresis. 2. The separation made it possible to study the properties of the partially purified enzyme fractions. Some of these properties, such as K(m) with selected protein substrates, pH optima and temperature-dependence in the presence and absence of substrates, are described. 3. No requirement for metal ions or added cofactors was demonstrated. Neutral-proteinase activity was more sensitive to inhibition by heavy-metal ions; its activity could be increased by thioglycollate and glutathione, and inhibited by thiol reagents. Neutral and acid proteinases were inhibited by the chymotrypsin inhibitor chloromethyl l-2-phenyl-1-toluene-p-sulphonamidoethyl ketone. 4. In the presence of the appropriate synthetic substrates no cathepsin A activity was found, and only trace quantities of cathepsin B or C activities, which were more than 50-fold less than cathepsin D-like activity.     

Effect of pasteurization on the activity of proteinases in salmon roe

We studied the dependence of activity and stability of proteolytic enzymes in salmon roe on pH and temperature. The activity of proteolytic enzymes in roe was primarily determined by proteinases. These enzymes were active at acid pH and had an optimum of 3.6. A study of subclasses of proteolytic enzymes in salmon roe and the published data suggest that the activity of proteinases may be related to the presence of aspartyl proteinases (cathepsin D). Serine proteinases and metalloenzymes were not found in roe. The activity of cysteine proteinases was low. The proposed conditions of pasteurization favored the complete inactivation of salmon roe at pH 6.0-6.4.     

Effect of Pasteurization on the Activity of Proteinases in Salmon Roe

We studied the dependence of activity and stability of proteolytic enzymes in salmon roe on pH and temperature. The activity of proteolytic enzymes in roe was primarily determined by proteinases. These enzymes were active at acid pH and had an optimum of 3.6. A study of subclasses of proteolytic enzymes in salmon roe and the published data suggest that the activity of proteinases may be related to the presence of aspartyl proteinases (cathepsin D). Serine proteinases and metalloenzymes were not found in roe. The activity of cysteine proteinases was low. The proposed conditions of pasteurization favored the complete inactivation of salmon roe at pH 6.0–6.4.     

Fluorigenic method for the assay of proteinase activity with the use of 4-methylumbelliferyl-casein.

A method for the preparation of casein labelled with the 4-methylumbelliferyl fluorophore is described, and the product was used as a fluorigenic macromolecular substrate for a sensitive assay of the activity of proteinases. Nanogram quantities of trypsin, chymotrypsin, elastase and cathepsin D can be detected, but the substrate is unaffected by cathepsin B.     

Effect of vitamin E-deficiency on the activity of some lysosomal and non-lysosomal proteases in rabbit muscles.

The activity of different cathepsins and neutral proteinases was measured in normal and vitamin E-deficient rabbit muscles using specific substrates. Among the changes of enzyme activities in dystrophy caused by vitamin E-deficiency the increase in the activity of cathepsin B is the most striking. The activity of cathepsin H, both in the fast and slow muscles and that of MMP-ase in the slow muscle remains practically unchanged. Activities of other proteases significantly increase. The change in the activity of proteolytic enzymes in striated muscle of vitamin E-deficient rabbits seems to be selective. As a rule the increase in the activity is higher in fast than in slow muscles.     

Partial purification and some properties of a neutral sulfhydryl and an acid proteinase from Entamoeba histolytica.

The partial purification of two intracellular proteinases from the protozoan parasite Entamoeba histolytica is reported. One of these enzymes is an acid proteinase exhibiting maximum activity at pH 3.5 (hemoglobin substrate), is little affected by a range of inhibitors or activators, and is presumed to be similar to cathepsin D. Also present is a neutral proteinase exhibiting optimum activity at pH 6.0 (azocasein) but only poorly hydrolyzing either hemoglobin or serum albumen. This latter enzyme displayed no metal ion requirement, but was markedly inhibited by thiol-blocking agents and activated by free sulhydryl-containing compounds.     

Detection of procathepsin D in rat milk

The presence of procathepsin D, a zymogen of the soluble lysosomal aspartic proteinase cathepsin D, was detected in rat milk using Western blot analysis and assay of proteolytic activity in acidic buffers. No other forms of cathepsin D were found. Two different polyclonal anti-procathepsin D antibodies were used for immunochemical detection of procathepsin D. Both antibodies we found to recognize rat procathepsin D. Proteolytic activity in acidic buffers was detected using a fluorogenic substrate specific for cathepsin D and was abolished by pepstatin A, a specific inhibitor of aspartic proteinases. This study represents third demonstration of presence of procathepsin D in mammal breast milk. Potential sources and physiological functions are discussed.     

The nature of the collagenolytic cathepsin of rat liver and its distribution in other rat tissues

1. An enzyme present in rat liver extracts degraded insoluble collagen maximally at pH3.5. Collagenolytic activity was more abundant in kidney, spleen and bone marrow and was also present in decreasing concentrations in ileum, lung, heart, skin and muscle. 2. The crude collagenolytic cathepsin was activated by cysteine and dithiothreitol, but not by 2-mercaptoethanol. Iodoacetamide, p-chloromercuribenzoate and 7-amino-1-chloro-3-l-tosylamidoheptan-2-one hydrochloride inhibited the enzyme. Zn(2+), Fe(3+) and Hg(2+) ions were strongly inhibitory, but Ca(2+), Co(2+), Mg(2+) and Fe(2+) ions had little or no effect. EDTA was an activator of the enzyme. Inhibitors of cathepsin B were found to enhance collagenolysis, but phenylpyruvic acid, a cathepsin D inhibitor, inhibited the enzyme. Di-isopropyl phosphorofluoridate had no effect. 3. Collagenolysis at pH3.5 and 28 degrees C was restricted to cleavage of the telopeptide region in insoluble collagen, and the material that was solubilized consisted mostly of alpha-chains. 4. The collagenolytic cathepsin was separated from cathepsins B2 and D by fractionation on Sephadex G-100 and a partial separation from cathepsin B1 was obtained by chromatography on DEAE-Sephadex. 5. The function of the collagenolytic cathepsin in the catabolism of collagen is discussed in relation to the action of the other lysosomal proteinases and the neutral collagenase.     

Multiple acid proteinases in the cellular slime mould Dictyostelium discoideum.

Proteinase activity in the cellular slime mould Dictyostelium discoideum has been analyzed by electrophoresis on polyacrylamide gels containing denatured hemoglobin. At least eight bands due to acid proteinases have been defined using extracts of myxamoebae, four bands A-D which move faster than the fifth and major band E, a minor band E' which moves just behind E and two slow bands G and H. Fruiting body formation was accompanied by the appearance of one new proteinase band F. The proteinases were present in extracts of both axenically-grown and bacterially-grown cells. Differences between the pH dependence and stability of the individual proteinases were detected. Inhibitor studies suggested that the faster proteinases A-D may be cathepsin B-like, whilst the slower enzymes E, E' and F do not fit readily into any known group of proteinases since they were sensitive to HgCl2 but not to other inhibitors of cathepsin B and not to inhibitors of cathepsin D-like proteinases under standard conditions. None of the proteinases was apparently formed during or after preparation of extracts and the proteinases could be re-run on polyacrylamide gels to give only the band expected from the first run. The bands are believed to reflect multiple proteinase activities within the cell.     

Inhibition of aspartic proteinases by propart peptides of human procathepsin D and chicken pepsinogen

Two propart peptides of aspartic proteinases, the propart peptide of chicken pepsin and human cathepsin D, respectively, were investigated from the point of view of their inhibitory activity for a set of aspartic proteinases. These peptides display a very broad inhibitory spectrum. The strongest inhibition was observed for pepsin A-like proteinases where propart peptides can be used as titrants of active enzymes.     

Proteinase expression during differentiation of human osteoclasts in vitro

Osteoclasts are macrophage-derived polykaryons that degrade bone in an acidic extracellular space. This differentiation includes expression of proteinases and acid transport proteins, cell fusion, and bone attachment, but the sequence of events is unclear. We studied two proteins expressed at high levels only in the osteoclast, cathepsin K, a thiol proteinase, and tartrate-resistant acid phosphatase (TRAP), and compared this expression with acid transport and bone degradation. Osteoclastic differentiation was studied using human apheresis macrophages cocultured with MG63 osteosarcoma cells, which produce cytokines including RANKL and CSF-1 that mediate efficient osteoclast formation. Immunoreactive cathepsin K appeared at 3-5 days. Cathepsin K activity was seen on bone substrate but not within cells, and cathepsin K increased severalfold during further differentiation and multinucleation from 7 to 14 days. TRAP also appeared at 3-5 d, independently of cell fusion or bone attachment, and TRAP activity reached much higher levels in osteoclasts attached to bone fragments. Two proteinases that occur in the precursor macrophages, cathepsin B, a thiol proteinase related to cathepsin K, and an unrelated lysosomal aspartate proteinase, cathepsin D, were also studied to determine the specificity of the differentiation events. Cathepsin B occurred at all times, but increased two- to threefold in parallel with cathepsin K. Cathepsin D activity did not change with differentiation, and secreted activity was not significant. In situ acid transport measurements showed increased acid accumulation after 7 days either in cells on osteosarcoma matrix or attached to bone, but bone pit activity and maximal acid uptake required 10-14 days. We conclude that TRAP and thiol proteinase expression begin at essentially the same time, and precede cell fusion and bone attachment. However, major increases in acid secretion and proteinases expression continue during cell fusion and bone attachment from 7 to 14 days.