Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.23.5 (cathepsin D)
4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using hepatectomized rats, it was shown that immediately after a partial liver removal the Kupffer macrophages were accumulated. At the maximal mitotic activity (36 hours following partial hepatectomy), the relative amount of Kupffer cells keeps low, but 72 hours later turns out to be higher again. The periodic changes of the Kupffer cell amount in hepatectomized rats are accompanied by remarkable increase (by 1.5--3 times) of free and total lysosomal enzymes (acid DNAase, DNAase, cathepsin D). The activation of the Kupffer macrophage lysosomes goes ahead of labilization of hepatocyte lysosomal membranes. The blockade of mononuclear phagocyte system by means of carbonate iron in an early prereplicative period leads to an as long as 10--12 hours retardation of hepatocyte proliferation. The role of the Kupffer macrophages in reparative liver regeneration is discussed.
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PMID:[Change in the state of the lysosomes in isolated Kupffer cells and hepatocytes in the process of liver reparative regeneration]. 72 76

To learn more about pathological iron storage in the liver, two sorts of lysosomes were isolated from rat livers in Percoll - sucrose or sucrose gradients: siderosomes (= iron-loaded terminal lysosomes) and light lysosomes (secondary and terminal). Such cell fractions were obtained from acutely iron-loaded and control rat livers. After lysis with Triton X-100 the preparations were assayed for proteolytic activity against rat liver ferritin (RLF) and denatured bovine hemoglobin (DBH), for buffer-soluble ferritin protein content, total protein and non-heme iron. At pH 3.6 both fractions displayed considerable proteolytic activity (cathepsin D activity) against DBH and endogenous proteins but little activity against RLF. By contrast, proteolytic activity against RLF was maximal at the highest pH tested, 6.5, at which DBH was practically insusceptible. The behavior of proteolytic activity against ferritin at pH 6.5 makes it likely that a single enzyme was involved that acted by Michaelis-Menten kinetics. However, no more than 2.5% of endogenous ferritin protein in the organelles was buffer-soluble. 41 to 89 hours after an intramuscular dose of 50 mg Fe, given as iron dextran, the non-heme iron content of light lysosomes and siderosomes had increased markedly and the ratio of non-heme Fe to buffer-soluble ferritin protein also became much elevated in the organelles; but the ratio of buffer-soluble ferritin to total protein did not rise significantly. The rise in organellar non-heme Fe exceeded iron saturation of rat liver ferritin and thus reflected conversion of ferritin to hemosiderin, which is buffer-insoluble.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Studies of iron overload. Lysosomal proteolysis of rat liver ferritin. 373 72

The conventional assay procedure for cathepsin D (E.C.3.4.23.5) activity in tissue homogenates and subcellular fractions requires incubation with hemoglobin as substrate. Cathepsin D (CD) activity is calculated by determining the increase in absorbance at 280 nm after precipitation of all proteins with trichloroacetic acid. This increase in absorbance (presumably due to the release of tyrosine residues from hemoglobin) is converted to arbitrary CD activity units. Homogenization and fractionation of cardiac tissue frequently requires that ethylenediamine tetraacetic acid (EDTA) be included in the homogenization medium. We have observed that subcellular fractions of cardiac tissue prepared in the presence of EDTA demonstrate residual CD activity despite either quantitative removal of all CD protein by immunoprecipitation or complete inhibition of CD by pepstatin. The present study demonstrates that this 'apparent' CD activity (residual increase in absorbance at 280 nm) is due to the formation of an Fe-EDTA complex which absorbs at 280 nm. Data are presented which demonstrates that the EDTA of the medium complexes with non-heme iron which contaminates commercially available hemoglobin. A method for preparing hemoglobin free of contaminant non-heme iron is described for use in studies of CD metabolism when EDTA is present in the homogenization buffer.
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PMID:Identification of artifactual cathepsin D activity in cardiac subcellular fractions related to formation of an iron-EDTA complex. 393 91

To investigate storage of ferritin and its transition to hemosiderin under conditions of iron overload, rats were either given multiple injections of iron dextran over 4 to 5 weeks or fed a diet containing 1.3% Fe as ferric ammonium citrate for 60 days. Then, preparations of liver siderosomes (heavily iron-laden lysosomes) were examined for content of buffer-soluble ferritin and buffer-insoluble, ferritin-related protein, total nonheme iron and protein, cathepsin D activity, and ability to incorporate 14C-leucine into ferritin. Total liver nonheme iron, ferritin protein and iron, and cathepsin D activity were also determined. Although parenteral iron loading produced higher total nonheme iron in livers than dietary loading, the iron content of ferritin was approximately 20% in both groups, reflecting saturation of ferritin with iron. Siderosome nonheme iron content was greater than 40% in relation to protein. The siderosomes contained little buffer-soluble ferritin; on isoelectric focusing this was composed of isoferritins present also in cytosol ferritin. Buffer-insoluble ferritin protein, identified in siderosomes by immunofluorescence, was solubilized and found to contain immunoreactive material corresponding to L and H subunits of buffer-soluble ferritin. Transmission electron microscopy indicated the presence of relatively large quantities of "ferritin" in siderosomes, and it is argued that this was mostly buffer insoluble (denatured) or represented ferritin [FeOOH]x cores divested of protein shells. Although siderosomes had substantial cathepsin D activity, the known resistance of ferritin to this and other proteases makes it unlikely that proteolysis is an early event in the decomposition of ferritin in siderosomes. Heavily iron-laden siderosomes did not take up newly labeled ferritin or ferritin protein or 14C-precursor within 24 hours of labeling, when 14C-labeled ferritin was abundant in cytosol. The author proposes a sequence of steps leading from sequestration of buffer-soluble cytosol ferritin to storage of insoluble "hemosiderin."
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PMID:Studies of iron overload. Rat liver siderosome ferritin. 669 50

Using hepatectomized rats it was shown that immediately after partial liver removal (PLR) the Kupffer macrophages were accumulated in liver remnant. At the maximal mitotic activity (36 hours following PLR) the relative amount of Kupffer cells keeps low, but 72 hours later turns out to be higher again. The periodic changes of the Kupffer cell amount in hepatectomized rats are accompanied by remarkable increase (1.5-3 fold) of free and total lysosomal enzyme activity (acid DNA-ase, acid RNA-ase, cathepsin D). The activation of the Kupffer macrophage lysosomes goes ahead of labilization of hepatocyte lysosomal membranes. The blockade of mononuclear phagocyte system by means of carbonate iron overloading in the early prereplicative period leads to an as long as 10-12 hours retardation of hepatocyte proliferation. The role of Kupffer macrophages in reparative liver regeneration is discussed.
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PMID:Lysosomal enzyme activity in hepatocytes and Kupffer cells from intact and partially hepatectomized rats. 727 93

Lysosomal enzymes can, under certain circumstances, be secreted in large amounts. One example is uteroferrin (Uf), an iron-containing, purple-colored acid phosphatase secreted by the uterus of the pig during pregnancy. Uf is identical to the intracellular tartrate-resistant acid phosphatase of pig spleen, yet is the major protein component of uterine secretions. To investigate possible regulatory mechanisms that might direct Uf along a secretory pathway, we expressed Uf in Chinese hamster ovary (CHO) cells under the control of the SV40 early promoter using an expression construct, pX/Uf. The proportion of Uf secreted into the medium relative to the amount retained intracellularly increased as total Uf expression was increased. At transfection doses of 15 micrograms pX/Uf per 10(6) cells, over 80% of the Uf produced in 48 h was secreted. A parallel situation was observed when human cathepsin D was overexpressed in CHO cells. Thus, high production of Uf, as occurs in the uterus in response to progesterone, may overwhelm the intracellular enzymatic and receptor systems that are normally employed to target acid hydrolases to lysosomes, resulting in secretion. Both Uf and cathepsin D secreted by CHO cells possess N-linked, phosphorylated high-mannose oligosaccharide chains. However, the phosphate groups on the oligosaccharide chains of Uf, unlike those on cathepsin D, cannot be readily removed by alkaline phosphatase treatment. These results suggest that the phosphate groups on Uf are masked at least partially by covering N-acetylglucosamine residues and that two mechanisms may contribute to hypersecretion of Uf in the uterus: 1) very high rates of synthesis and 2) partial masking of the mannose 6-phosphate recognition signal.
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PMID:Overexpression of uteroferrin, a lysosomal acid phosphatase found in porcine uterine secretions, results in its high rate of secretion from transfected fibroblasts. 828 14

To test whether the possibly enhanced sensitivity of aged cells to oxidative stress may depend on their content of ceroid/lipofuscin, AG-1518 human fibroblasts with various amounts of the pigment accumulated due to prolonged cultivation under normobaric hyperoxia were exposed to acute oxidative stress (2.5 microM naphthazarin, 15 min) and then returned to standard culture conditions. Twenty-four hours after the naphthazarin treatment, 37% of the cells were still vital, whereas others had undergone oxidative stress-induced apoptosis with ensuing postapoptotic necrosis. The average amount of ceroid/lipofuscin within the surviving cells was only about half of that of the initial population of cells, as measured before the naphthazarin exposure. This finding suggests that ceroid/lipofuscin-rich cells have an increased sensitivity to oxidative stress. The ceroid/lipofuscin quantity strongly positively correlated with the size of the acidic compartment (as evaluated by uptake of the weakly basic lysosomotropic fluorochrome acridine orange) and with its content of the lysosomal protease cathepsin D, as assayed by immunocytochemistry. We hypothesize that the enhanced sensitivity of ceroid/lipofuscin-loaded cells to oxidative stress may be caused by the increased amounts of lysosomal enzymes, known as mediators of oxidative damage, and/or by catalysis of intralysosomal oxidative reactions by lipofuscin-associated iron.
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PMID:Ceroid/lipofuscin-loaded human fibroblasts show increased susceptibility to oxidative stress. 1057 36

Adverse effects of doxorubicin (adriamycin) have been reported to be due to iron-catalyzed free radical formation, which can be prevented with the cytoprotective chelating agent [(+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)]propane (dexrazoxane; ICRF-187). Affected tissues include the heart, gastrointestinal tract, and kidney. However, there is very little information on the effects of adriamycin on skeletal muscle, despite the fact that there is direct and indirect evidence to show that both adriamycin and ICRF-187 are myotoxic. To investigate the mechanisms of cytotoxicity of these agents in skeletal muscle, we have conducted a systematic investigation of the activities of the major lysosomal (dipeptidyl aminopeptidase I and II and cathepsins B, D, H, and L) and cytoplasmic (alanyl-, arginyl-, and leucyl aminopeptidase, dipeptidyl aminopeptidase IV, tripeptidyl aminopeptidase, and proline endopeptidase) muscle proteases. These enzymes play an important role in normal cellular function and represent potential targets for toxic and protective agents. Male Wistar rats (approx. 0.2 kg) were subjected to a pretreatment phase of 30 min followed by a treatment stage of either 2.5 or 24 h. The pretreatment involved injection of a single bolus of either saline (0.15 mol/l NaCl; 5 ml/kg ip) or ICRF-187 (100 mg/kg; 5 ml/kg ip). After 30 min, rats were injected again with a single bolus of either adriamycin (5 mg/kg; 10 ml/kg ip) or saline (0.15 mol/l NaCl; 10 ml/kg ip) in the treatment phase. At either 2.5 or 24 h after the last adriamycin or saline injection, rats were killed for subsequent dissection of the gastrocnemius muscle for analysis. In the 2.5-h study, there were significant reductions in cathepsin D activities of adriamycin-treated rats compared to saline injected control (p = 0.02). In both 2.5- and 24-h studies there were also significant differences (p = 0.05) in cathepsin H activities between rats treated with adriamycin and ICRF-187, although these differences were not significant when data were compared with corresponding saline-injected rats. There were no other overt effects for any of the other proteases at either 2.5 or 24 h. We conclude that both adriamycin and ICRF-187 have very little effect on the activities of muscle proteases and that altered proteolysis is not involved in the reported pathological reactions induced by these agents.
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PMID:Effects of Doxorubicin (Adriamycin) and [(+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)]propane (ICRF-187) on skeletal muscle protease activities. 1124 12

Nramp2 (DMT1) is a pH-dependent divalent cation transporter that acts as the transferrin-independent iron uptake system at the intestinal brush border and also transports iron released from transferrin across the membrane of acidified endosomes. In this study, RAW264.7 macrophages and 2 independently derived murine Sertoli cells lines, TM4 and 15P-1, were used to further study the subcellular localization of Nramp2/DMT1 in phagocytic cells, including possible recruitment to the phagosomal membrane. Nramp2/DMT1 was localized primarily to the EEA1-positive recycling endosome compartment, with some overlapping staining with Lamp1-positive late endosomes. After phagocytosis, immunofluorescence analysis and in vitro biochemical studies using purified latex bead-containing phagosomes indicated Nramp2/DMT1 recruitment to the membrane of Lamp1, cathepsin D, and rab7-positive phagosomes. Nramp2/DMT1 was also found associated with erythrocyte-containing phagosomes in RAW macrophages and with the periphery of sperm-containing phagosomes in Sertoli cells. These results suggest that, as for the macrophage-specific Nramp1 protein, Nramp2/DMT1 may transport divalent metals from the phagosomal space.
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PMID:Iron transporter Nramp2/DMT-1 is associated with the membrane of phagosomes in macrophages and Sertoli cells. 1223 76

Hydrogen peroxide, the major oxidoradical species in the central nervous system, has been involved in neuronal cell death and associated neurodegenerative diseases. In this study, we have investigated the involvement of the lysosomal pathway in the cytotoxic mechanism of hydrogen peroxide in human neuroblastoma cells. Alteration of lysosomal and mitochondrial membrane integrity was shown to be an early event in the lethal cascade triggered by oxidative stress. Desferrioxamine (DFO), an iron chelator that abolishes the formation of reactive oxygen species within lysosomes, prevented lysosome leakage, mitochondrial permeabilization and caspase-dependent apoptosis in hydrogen peroxide-treated cells. Inhibition of cathepsin D, not of cathepsin B, as well as small-interference RNA-mediated silencing of the cathepsin D gene prevented hydrogen peroxide-induced injury of mitochondria, caspase activation, and TUNEL-positive cell death. Cathepsin D activity was shown indispensable for translocation of Bax onto mitochondrial membrane associated with oxidative stress. DFO abolished both the cytosolic relocation of Cathepsin D and the mitochondrial relocation of Bax in hydrogen peroxide-treated cells. siRNA-mediated down-regulation of Bax expression protected the cells from oxidoradical injury. The present study identifies the lysosome as the primary target and the axis cathepsin D-Bax as the effective pathway of hydrogen peroxide lethal activity in neuroblastoma cells.
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PMID:Cathepsin D-Bax death pathway in oxidative stressed neuroblastoma cells. 1739 4


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