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)

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.
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PMID:Cathepsin D is membrane-associated in macrophage endosomes. 336 Aug 12

Cleavage of parathyroid hormone (PTH) by isolated Kupffer cells from rat liver was examined. Iodinated PTH labeled at position 43 was converted into two radioactive fragments which were shown by Edman degradation to have residues 35 and 38 as their NH2 termini. Cleavage at these positions is characteristic of cathepsin D. Amino-terminal fragments were detected by bioassay of fractions obtained by high performance liquid chromatography. These fragments eluted in positions characteristic of the 1-34 and 1-37 peptides also previously shown to be produced by purified cathepsin D. The putative 1-37 fragment was rapidly converted to 1-34 upon digestion with cathepsin D, whereas the putative 1-34 fragment was not further digested by this enzyme, behavior previously shown to be characteristic of 1-37 and 1-34 bovine PTH. Fragmentation of PTH as measured by generation of fragments soluble in trichloroacetic acid was inhibited by methylamine, monensin, and ammonium chloride. In addition, monensin significantly inhibited production of both carboxyl- and amino-terminal fragments. Finally, active PTH fragments were also produced by elicited peritoneal macrophages. It is concluded that Kupffer cells, and other macrophages, can produce active fragments of PTH which appear in the medium. These fragments may be generated by cathepsin D within the cells.
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PMID:Production of biologically active fragments of parathyroid hormone by isolated Kupffer cells. 377 58

Cleavage of parathyroid hormone by cathepsin D was studied. Four primary products were detected and separated by high performance liquid chromatography. Two of the fragments are fluorescent and therefore contain residue 23 (tryptophan). These fragments are NH2-terminal in origin. The other two cross-react with antisera directed against COOH-terminal portions of the hormone; they are the complementary COOH-terminal fragments. Microsequencing and amino acid analysis showed that the two COOH-terminal fragments are 35-84 and 38-84 bovine parathyroid hormone. By CNBr cleavage and amino acid analysis, the two NH2-terminal fragments were shown to be the complementary 1-37 and 1-34 fragments. The 1-37 fragment is transitory and is rapidly hydrolyzed to 1-34, so that only relatively small amounts are detected at any one time. However, 34-84 was not converted to 38-84, although cleavage at other sites in the COOH-terminal fragments was observed with more exhaustive digestion. The 1-34 fragment appears to be the final product of the action of cathepsin D on parathyroid hormone. Both enzymatically produced NH2-terminal fragments were fully active in the renal membrane adenylyl cyclase assay system.
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PMID:Characterization of parathyroid hormone fragments produced by cathepsin D. 396 81

Proteolytic fragments of fibronectin were used to identify regions of the molecule that support neurite extension and to investigate further the differential behavior of central and peripheral nervous system neurons in response to fibronectin (Rogers, S. L., P. C. Letourneau, S. L. Palm, J. B. McCarthy, and L. T. Furcht (1983) Dev. Biol. 98: 212-220). Fibronectin fragments with differing biological activities were produced by proteolytic digestion with trypsin and cathepsin D and sequential affinity chromatography on gelatin-agarose and heparin-Sepharose. The resulting fragments (described by Smith, D. E., D. F. Mosher, R. B. Johnson, and L. T. Furcht (1982) J. Biol. Chem. 257: 5831-5838; Smith, D. E., and L. T. Furcht (1982) J. Biol. Chem. 257: 6518-6523 included an NH2-terminal 27,000-dalton peptide that weakly binds heparin, a 46,000-dalton gelatin-binding fragment, a series of fragments (80,000 to 125,000 daltons) from the center of the molecule containing previously described cell-binding activity, two major peptides of Mr = 33,000 and 66,000 that bind heparin strongly and are thought to originate from the A and B chains, respectively, of plasma fibronectin, and a 31,000-dalton COOH-terminal peptide containing a free sulfhydryl from the A chain of the molecule. Tissue culture dishes were treated with these proteolytic preparations, and dissociated embryonic chick peripheral (PNS) and central nervous system (CNS) cells were cultured on each experimental substratum in serum-free medium. The fibronectin fragments were evaluated for ability to promote cell attachment, neurite initiation, and maintenance of neurite growth. The 27,000-, 46,000-, and 31,000-dalton preparations did not promote cell attachment or neurite extension. Both PNS and CNS neurons attached to and extended stable neurites upon the COOH-terminal heparin-binding preparation containing the 33,000- and 66,000-dalton peptides. A differential response of the neurons to the 80,000- to 125,000-dalton "cell-binding" peptides was observed: whereas PNS neurons maintained neuritic growth on this preparation for at least 48 hr, CNS neurons extended neurites during the first 24 hr of culture but, by 48 hr, withdrew these neurites and became increasingly clumped. On the basis of (1) the observed neuronal responses to the heparin binding and "cell binding" regions, and (2) the different ligand-binding properties of these regions, we propose that cell attachment and neurite extension can be mediated and/or modulated by two separate regions of fibronectin and that cellular response to the intact molecule may involve multivalent interactions.
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PMID:Neuron-specific interactions with two neurite-promoting fragments of fibronectin. 397 71

Various biosynthetic forms of porcine spleen cathepsin D (Erickson, A. H. and Blobel, G. (1979) J. Biol. Chem. 254, 11771-11774), isolated by immunoprecipitation of in vivo- and in vitro-synthesized products, have been characterized by partial NH2-terminal sequence analysis. Two short lived and functionally distinct NH2-terminal sequence extensions, a "pre" sequence and a "pro" sequence, have been detected. Both sequence extensions are present in preprocathepsin D which is the primary translation product immunoprecipitated after translation of porcine spleen mRNA in a wheat germ cell-free system. Preprocathepsin D is not glycosylated and has an approximate Mr = 43,000. Its 20-residue pre sequence resembles the signal sequences of presecretory proteins in abundance of Leu residues (7 out of 20 residues). Addition of dog pancreatic microsomal vesicles to the translation system resulted in the cleavage of the pre sequence and yielded segregated and glycosylated procathepsin D (Mr = 46,000) that was indistinguishable from its in vivo-synthesized counterpart detected after pulse-labeling of cultured porcine kidney cells. Some of this in vivo-synthesized procathepsin D was secreted and persisted as such in the culture medium. The remainder was converted within a period of 15 min to 2 h to single chain cathepsin D (Mr = 44,000) by removal of a pro sequence which was estimated to be 44 residues. Its partial sequence showed considerable sequence homology to the 44-residue activation peptide of pepsinogen. It is possible, therefore, that the prosequence of procathepsin D serves as an activation peptide that keeps the enzyme inactive during intracellular transport to the lysosome. The enzymatically active single chain form of cathepsin D undergoes further cleavage into a light and a heavy chain (Mr = 15,000 and 30,000, respectively) over a period of 2-24 h after synthesis. The oligosaccharide moieties of procathepsin D and of the single chain and heavy chain forms of cathepsin D are cleaved by endoglycosidase H. Treatment of cells with tunicamycin arrests the biosynthetic pathway of cathepsin D at procathepsin D. The nonglycosylated procathepsin D is not proteolytically processed and its secretion is greatly inhibited.
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PMID:Biosynthesis of a lysosomal enzyme. Partial structure of two transient and functionally distinct NH2-terminal sequences in cathepsin D. 611 13

During pulse-chase experiments in cultured porcine kidney cells, an early 75-kilodalton (kDa) form of beta-glucuronidase is converted to a late 72-kDa form. The relative molecular weight difference between the two forms is maintained on removal of high-mannose carbohydrate with endoglycosidase H. Both forms have the same partial NH2-terminal sequence, and both migrate as single polypeptide chains following reduction, alkylation, and electrophoresis under denaturing conditions. On treatment with carboxypeptidase Y, the early form released [35S]Met faster than the late form. Thus, the late form of beta-glucuronidase is generated by COOH-terminal proteolytic processing of the early form. During similar experiments, the mass of the 30-kDa heavy chain of porcine cathepsin D decreased by about 1 kDa. The heavy chain of the two-chain enzyme is derived from the COOH terminus of a 44-kDa single-chain enzyme. On treatment with carboxypeptidase Y, the early single-chain enzyme released COOH-terminal [35S]Met and [3H]Lys faster than the later 29-kDa heavy chain. Like beta-glucuronidase, cathepsin D evidently undergoes COOH-terminal proteolytic processing during biosynthesis.
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PMID:Carboxyl-terminal proteolytic processing during biosynthesis of the lysosomal enzymes beta-glucuronidase and cathepsin D. 636 Feb 5

The complete amino acid sequence of the light chain of cathepsin D from porcine spleen has been determined. The light chain consists of a single polypeptide chain with 97 amino acid residues. The sequence is: (formula; see text) The molecular weight of the light chain was calculated from this sequence to be 10,548 (without carbohydrates). A single disulfide bond links two half-cystine residues between positions 46 and 53. A cysteine residue is located at position 27. The light chain sequence is extensively homologous to the NH2-terminal sequence of other aspartyl proteases. It shows a 59% identity with the sequence of mouse submaxillary gland renin and a 49% identity with that of porcine pepsin. A single glycosylation site is located at residue 70 of the cathepsin D light chain. This site corresponds to position 67 of pepsin by homology. The active site aspartyl residue, corresponding to Asp-32 of pepsin, is located at residue 33 in the cathepsin D light chain.
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PMID:Amino acid sequence of porcine spleen cathepsin D light chain. 640 81

Preferential labeling of COOH-terminal sequences in newly synthesized fibronectin was achieved by short term incorporation of radiolabeled amino acids in the presence of pactamycin, an inhibitor of polypeptide chain initiation. The labeled fibronectin was then cleaved with cathepsin D under conditions that yield a large (137,000-dalton) fragment that lacks collagen-binding properties, and a smaller (72,000-dalton) fragment that retains the ability of fibronectin to bind to collagen. Determination of the relative specific radioactivities of the two fragments leads us to conclude that the collagen-binding domain in fibronectin is located in the NH2-terminal third of the polypeptide chain and not in a COOH-terminal region as previously indicated by other structural studies.
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PMID:Location of a collagen-binding domain in fibronectin. 644 47

Purified preparations of cathepsin D, BANA-hydrolase activity and dipeptidil aminopeptidase I from chicken liver, show a cooperative effect in the protein hydrolysis (acid-denatured haemoglobin and bovine serum albumin and native bovine serum albumin) at pH 5.0. The nature of the protein substrates determines their sensitivity to enzymatic digestion. The action of cathepsin D on proteins, in contrast which the BANA-hydrolase activity, releases polypeptides with high molecular weight, with scant--NH2 groups which can be valued by the ninhydrin method. These peptide fragments can then be further degraded by the protease BANA-hydrolase and the dipeptidil aminopeptidase I which is not active towards intact proteins.
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PMID:[Acid proteases from chicken liver. Cooperative hydrolysis of proteins (author's transl)]. 699 59

Ammonia is a natural lysosomotropic compound. Concentrations of ammonium acetate > 2 mM impaired the phagocytic activity of BV-2 cells, an immortalized microglial cell line, as was determined by the uptake of fluorescent latex microspheres of different sizes. In contrast, an increase in the uptake of fluorescent dextran was observed with the elevation in ammonium acetate concentrations. This indicates that ammonia affects phagocytotic and pinocytotic activities of BV-2 cells differently. Interferon-gamma- and polyinosinic-polycytidylic acid-stimulated secretion of IL1 alpha as well as LPS-stimulated secretion of IL6 decreased with an elevation in ammonium acetate concentrations. The constitutive secretion of IL1 alpha was not significantly affected by ammonium acetate. However, an increase in LPS-stimulated IL1 alpha secretion was observed at 10 mM and 20 mM ammonium acetate. High concentrations of ammonia affected the activity of lysosomal enzymes of the BV-2 cells. Acid phosphatase and alpha-glucosidase activities increased with the increase in ammonium acetate up to 20 mM. The activity of cathepsin D was increased at 5 mM, but decreased at higher ammonia concentrations. The effects of ammonia on microglial functions are discussed with respect to pathogenetic mechanisms of dementia of the Alzheimer type.
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PMID:Effect of ammonia on endocytosis, cytokine production and lysosomal enzyme activity of a microglial cell line. 782 5


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