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)

Phenobarbital was given to male rats as a single injection and as repetitive injections for 7 days. The effects of treatment on the lysosomal hydrolases acid phosphatase, cathepsin D, and aryl sulfatase were analyzed at different intervals ranging from 1 to 15 days after seven injections, and from 1 to 48 h after a single injection. In both cases, microsomal protein and NADPH-cytochrome c reductase were measured to ensure proper induction. After a single injection, a slight decrease in hydrolytic activities was observed. Repetitive administration of phenobarbital gave rise to a marked decrease of lysosomal enzyme activities 1 day after cessation of treatment. This decrease was followed by a continuous increase in activity up to day 3 and 4. One or 2 weeks after treatment, enzyme activities declined to control values. The increase in activity of lysosomal hydrolytic enzymes was correlated with the onset of induced autophagy of endoplasmic reticulum membranes described as occurring in liver upon cessation of phenobarbital exposure. It is concluded that phenobarbital treatment per se decreases lysosomal enzyme activities, whereas the induced autophagy following cessation of exposure is associated with enhanced levels of lysosomal hydrolases in rat liver.
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PMID:Induction of liver lysosomal enzymes during the autophagic phase following phenobarbital treatment of rat. 40 31

Homogenates of HTC cells have been fractionated by differential centrifugation (in four particulate fractions: N, M, L, P, and a supernatant S) or isopycnic banding in linear sucrose gradients. On this basis, the following subcellular organelles may be characterized: (i) Mitochondria, detected by cytochrome oxidase and succinodehydrogenase, are collected in the M and L fractions, and equilibrate, as a narrow band, at a median buoyant density of 1.18 g/cm3. (ii) Lysosomes, detected by the latent hydrolases beta-glycerophosphatase and N-acetyl-beta-glucosaminidase, are largely sedimented in the M and L fractions, and display a broad density distribution pattern with a median value of 1.17 g/cm3. This density is decreased or increased after cultivation of the cells in presence of Triton WR-1339 or Dextran 500, respectively. The behavior of cathepsin D is somewhat at variance with that of the two other hydrolases. (iii) Plasma membrane is tentatively detected by alkaline phosphodiesterase I. Largely recovered in the P fraction, this enzyme equilibrates at a median density close to that of the lysosomal hydrolases; the bulk of cholesterol and about half of the leucyl-2-naphthylamidase are closely associated with alkaline phosphodiesterase I; HTC cells do not contain typical 5'-nucleotidase. (iv) Catalase-bearing particles, of high buoyant density (1.22 g/cm3) are present, but 30-40% of the catalase is also found readily soluble. NADPH- and NADH: cytochrome c reductase, and RNA show more complex distributions. It is suggested that the former enzyme is associated with the endoplasmic reticulum; as in liver, NADH reductase activity is shared between the endoplasmic reticulum and the mitochondria; half of the RNA is associated with free ribosomes of polysomes. True glucose-6-phosphatase could not be detected.
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PMID:Analytical fractionation of cultured hepatoma cells (HTC cells). 56 43

The propeptides of lysosomal enzymes have been implicated in membrane association and mannose 6-phosphate-independent sorting to the lysosome (Rijnboutt, S., Aerts, H., Geuze, H. J., Tager, J. M., and Strous, G. J. (1991) J. Biol. Chem. 266, 4862-4868; McIntyre, G. F., and Erickson, A. H. (1991) J. Biol. Chem. 266, 15438-15445). In this report, the function of the propeptide of procathepsin D in sorting to the lysosome was directly assessed using a cathepsin D deletion mutant lacking the propeptide, and using a chimeric cDNA encoding the cathepsin D propeptide fused to the secretory protein alpha-lactalbumin. Proteins encoded by these cDNAs were expressed in mouse Ltk- cells and in human hepatoma Hep G2 cells, and then immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. The deletion mutant was glycosylated but was rapidly degraded in a chloroquine-independent fashion and did not assume an active conformation. Thus the propeptide appeared to be necessary for correct folding. The chimeric protein was glycosylated and secreted. The coincidence of complex oligosaccharide modification and secretion of the chimeric protein suggested that it was slowly released from the endoplasmic reticulum and rapidly passed through the cell to the extracellular compartment. This was confirmed by immunofluorescent localization of the proteins. The data indicated that the propeptide appeared to be necessary for folding of cathepsin D but, unlike the yeast vacuolar propeptides, was not sufficient to direct a secretory protein to the lysosome in fibroblasts or in epithelial cells.
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PMID:The role of the cathepsin D propeptide in sorting to the lysosome. 140 Apr 84

We investigated the cellular and subcellular distribution of surfactant protein D (SP-D) by immunogold labeling in lungs of adult rats that had been given bovine serum albumin coupled to 5-nm gold (BSAG) for 2 hr to visualize the endocytotic pathway. Specific gold labeling for SP-D was found in alveolar Type II cells, Clara cells, and alveolar macrophages. In Type II cells abundant labeling was observed in the endoplasmic reticulum, whereas the Golgi complex and multivesicular bodies were labeled to a limited extent only. Lamellar bodies did not seem to contain SP-D. Gold labeling in alveolar macrophages was restricted to structures containing endocytosed BSAG. In Clara cells labeling was found in the endoplasmic reticulum, the Golgi complex, and was most prominent in granules present in the apical domain of the cell. Double labeling experiments with anti-surfactant protein A (SP-A) showed that both SP-A and SP-D were present in the same granules. However, SP-A was distributed throughout the granule contents, whereas SP-D was confined to the periphery of the granule. The Clara cell granules are considered secretory granules and not lysosomes, because they were not labeled for the lysosomal markers cathepsin D and LGP120, and they did not contain endocytosed BSAG.
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PMID:Immunocytochemical localization of surfactant protein D (SP-D) in type II cells, Clara cells, and alveolar macrophages of rat lung. 152 77

We have studied the intracellular transport of the pulmonary surfactant SP-C precursor in vitro and in vivo. In order to monitor the route of the SP-C precursor, we constructed various C-terminally truncated forms of SP-C, which were tagged with a sequence derived from the C-terminus of the human c-myc gene (aa 409-419). Expression of these constructs under the control of the SV40 enhancer and the huMT-II promoter in stably transformed Chinese hamster ovary (CHO) cells revealed that the complete precursor molecule is localized mostly in vesicular structures, probably of lysosomal origin. The truncated precursor lacking the last 22 amino acids at the C-terminus (SP-C/Ctag), however, was restricted to the endoplasmic reticulum as shown by immunofluorescence, using antibodies directed against the tag-sequence, the lysosomal enzyme cathepsin D, the enzyme disulfide isomerase, and the Golgi zone. The intracellular localization was substantiated by subcellular fractionation analysis, suggesting that the last 22 amino acids are necessary for intracellular targeting. Furthermore, Triton X-114 extractions from CHO cells revealed a modification of the SP-C precursor. In vitro translation and pulse-chase experiments in the absence or presence of microsomes showed that the modification occurs post-translationally and in a time-dependent manner. Membrane association studies using an SP-C precursor lacking the mature peptide indicated that the modification is of hydrophobic nature but not a thioester-linked fatty acid.
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PMID:The C-terminal domain of the pulmonary surfactant protein C precursor contains signals for intracellular targeting. 159 Oct 9

We describe the use of a Xenopus laevis egg extract for the in vitro translation and post translational modification of membrane and secretory proteins. This extract is capable of the translation and segregation into membranes of microgram per millilitre levels of protein from added mRNAs. Signal sequences of segregated proteins are efficiently cleaved and appropriate N-linked glycosylation patterns are produced. The extract also supports the quantitative assembly of murine immunoglobulin heavy and light chains into tetramers, and two events which take place beyond the endoplasmic reticulum, mannose 6 phosphorylation of murine cathepsin D and O-linked glycosylation of coronavirus E1 protein, also occur, but at reduced efficiency. The stability of the membranes allows protease protection studies and quantitative centrifugal fractionation of segregated and unsegregated proteins to be performed. Conditions for the use of stored extract have also been determined.
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PMID:A highly efficient, cell-free translation/translocation system prepared from Xenopus eggs. 175 76

Immunocytochemical localization of two distinct intracellular aspartic proteinases, cathepsins E and D, in human gastric mucosal cells and various rat cells was investigated by immunogold technique using discriminative antibodies specific for each enzyme. Cathepsin D was exclusively confined to primary or secondary lysosomes in almost all the cell types tested, whereas cathepsin E was not detected in the lysosomal system. The localization of cathepsin E varied with different cell types. Microvillous localization of cathepsin E was found in the intracellular canaliculi of human and rat gastric parietal cells, rat renal proximal tubule cells, and the bile canaliculi of rat hepatic cells. The immunolocalization of each enzyme in gastric cells were essentially the same in humans and rats. In the gastric feveolar epithelial cells and parietal cells, definite immunolabeling for cathepsin E was observed in the cytoplasmic matrix, the cisternae of the rough endoplasmic reticulum, and the dilated perinuclear envelope. In rat kidney, cathepsin E was detected only in the proximal tubule cells, while cathepsin D was found mainly in the lysosomes of the distal tubule cells but not in those of the proximal tubule cells. These results clearly indicate the distinct intracytoplasmic localization of cathepsins E and D and suggest the possible involvement of cathepsin E in extralysosomal proteolysis that is related to specialized functions of each cell type.
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PMID:An immunocytochemical study on distinct intracellular localization of cathepsin E and cathepsin D in human gastric cells and various rat cells. 179 85

The membrane association of the lysosomal enzymes cathepsin D and glucocerebrosidase and its naturally occurring sphingolipid activating protein was studied in HepG2 cells. We differentially permeabilized cells with low concentrations of saponin, at which secretory proteins rinsed out completely, whereas integral membrane proteins were not released. All relevant intracellular compartments were shown to be permeabilized by saponin. Metabolic labeling showed that early precursors of cathepsin D, sphingolipid activating protein, and glucocerebrosidase were completely released from the cells, whereas more than 80% of the high molecular mass intermediates were retained by the cells. Treatment of permeabilized cells with 10 mM mannose 6-phosphate released only 50% of the cell-associated cathepsin D. Glucocerebrosidase remained membrane-associated, but cathepsin D and sphingolipid activating protein were released from the cells after proteolytic processing. Sphingolipid activating proteins and cathepsin D behaved similarly during biosynthesis and showed similar sensitivity to mannose 6-phosphate. The membrane association of the intermediate form of cathepsin D was independent of the presence of N-linked oligosaccharides. Subcellular fractionation on sucrose gradients showed that the lysosomal proteins became membrane-associated probably in the Golgi complex, and that both mannose 6-phosphate-dependent and mannose 6-phosphate-independent membrane association occur in the same compartments. We conclude that, in HepG2 cells, cathepsin D, sphingolipid activating protein, and glucocerebrosidase exhibit MPR-independent membrane association which is acquired in the same compartments beyond the rough endoplasmic reticulum.
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PMID:Mannose 6-phosphate-independent membrane association of cathepsin D, glucocerebrosidase, and sphingolipid-activating protein in HepG2 cells. 184 27

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulatory enzyme of cholesterol biosynthesis and is located in the endoplasmic reticulum (ER). A fusion protein, HMGal, consisting of the membrane domain of HMG-CoA reductase fused to Escherichia coli beta-galactosidase and expressed in Chinese hamster ovary (CHO) cells from the SV40 promoter, was previously constructed and was found to respond to regulatory signals for degradation in a similar fashion to the intact HMG-CoA reductase. Degradation of both HMG-CoA reductase and HMGal in CHO cells was enhanced by addition of mevalonate or low density lipoprotein (LDL). In this report we show that 2 cysteine protease inhibitors, N-acetyl-leucyl-leucyl-norleucinal (ALLN) and N-acetyl-leucyl-leucyl-methioninal (ALLM), completely inhibit the mevalonate- or LDL-accelerated degradation of HMG-CoA reductase and HMGal and also block the basal degradation of these enzymes. It has been shown that in vitro these protease inhibitors inhibit the activities of Ca(2+)-dependent neutral proteases as well as lysosomal proteases, including cathepsin L, cathepsin b, and cathepsin D. However, the mevalonate-accelerated degradation of HMG-CoA reductase and HMGal is not affected by lysosomotropic agents, suggesting that the site of action of these inhibitor peptides in preventing the degradation is not the cathepsins. In brefeldin A-treated cells, where protein export from the ER is blocked, ALLN is still effective in inhibiting the degradation of HMG-CoA reductase and HMGal. These results indicate the involvement of non-lysosomal Ca(2+)-dependent proteases in the basal and the accelerated degradation of HMG-CoA reductase and HMGal. Enzymatic assays in vitro and immunoblot analyses have revealed calpain- and calpastatin-like proteins in CHO cells. The activities and the amount of these proteins do not change under conditions of enhanced degradation, indicating that the levels of these proteins are not subject to mevalonate regulation.
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PMID:Inhibition of degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vivo by cysteine protease inhibitors. 190 66

The nature and levels of hemoglobin (Hb)-hydrolyzing acidic proteinases including cathepsin D and cathepsin E, which were most active at pH 3.5-4.0, were enzymatically and immunochemically compared between human and rat neutrophils. By subcellular fractionation and immunoprecipitation with discriminative antibodies specific for each enzyme, cathepsin D was shown to be present in the granular content fraction of both human and rat neutrophils and to account for about 35% of the total Hb-hydrolyzing activity. Cathepsin E was observed mainly in the cytoplasmic fraction of rat neutrophils from peripheral blood and peritoneal exudates and accounted for about 65% of the total activity, but it was not detected in human blood neutrophils. Immunoelectron microscopy on rat neutrophils revealed that cathepsin D was exclusively confined to lysosomes, whereas cathepsin E was localized mainly in the cytoplasmic matrix and often in the perinuclear spaces and the rough endoplasmic reticulum. The non-cathepsin D activity in human neutrophils, which represented about 65% of the total activity, appeared to be due to a serine proteinase, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride and was not inhibited by agents specific for aspartic-, cysteine-, or metallo proteinases. The enzyme(s) responsible for this activity was largely associated with the granular membranes, and a half of it could be described as an integral membrane protein on the basis of phase separation with Triton X-114 at 35 degrees C. The levels of these Hb-hydrolases in gingival crevicular fluid from human chronic inflammatory periodontitis patients were examined in order to clarify their participation in the periodontal tissue breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of hemoglobin-hydrolyzing acidic proteinases in human and rat neutrophils. 208 32


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