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)

Six cathepsin D isozymes have been purified from porcine spleen using a large scale purification procedure. Five isozymes, I to V, have an identical molecular weight of 50,000 and are similar in specific activity. Isozymes I to IV contained two polypeptide chains each. The light and heavy chains have Mr = 15,000 and 35,000, respectively. Isozyme V is a single polypeptide. The molecular weight of the sixth isozyme is about 100,000 and it has only 5% of the specific activity of the other isozymes. On Ouchterlony immunodiffusion, an antiserum formed precipitin lines against the urea-denatured isozyme with Mr = 100,000. This immunoreactivity showed immunoidentity with those formed against other isozymes. The NH2-terminal sequence of light chains was identical for the isozymes. This sequence is homologous to the NH2-terminal sequence of other acid proteases, especially near the region of the active center aspartate-32. The NH2-terminal sequence of the single chain, isozyme V, Is apparently the same as the light chain sequence. The NH2-terminal sequence analysis of the heavy chain from isozyme I produced two sets of related sequences, suggesting the prescene of structural microheterogeneity. The carbohydrate analysis of the isozymes, the light chain, and the heavy chain revealed the presence of possibly four attachment sites, with one in the light chain and three in the heavy chain. Each carbohydrate unit contains 2 residues of mannose and 1 residue of glucosamine. The results suggest that the high molecular weight cathepsin D (Mr = 100,000) is the probable precursor of the single chain (Mr = 50,000), which in turn produces the two-chain isozymes. These are likely in vivo processes.
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PMID:Cathepsin D isozymes from porcine spleens. Large scale purification and polypeptide chain arrangements. 11 68

Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin--Sepharose 4B and concanavalin-A--Sepharose 4B, and chromatography on Sephadex G-100 and DEAE-Sephacel. The purified major enzyme (85% of the cathepsin D activity after DEAE-Sephacel chromatography), termed cathepsin D-I, represented about a 1000-fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15%), termed cathepsin D-II, represented about a 900-fold purification and about a 3% recovery. Both enzymes showed four (pI: 4.2, 4.9, 6.1 and 6.5) and three (pI: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corresponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D-I contained 6.6% carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8:2:1:1:5 with a trace of sialic acid. The properties of purified enzymes were also compared.
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PMID:Cathepsin D of rat spleen. Affinity purification and properties of two types of cathepsin D. 44 74

A content of neutral sugars and N-acetyl-glucosamine in homogeneous cathepsin D preparations from a variety of vertebrate organs was determined. A more detailed study of the carbohydrate component was carried out with chicken liver cathepsin D preparation. It was shown that carbohydrates constitute 20% of the molecule of this cathepsin and contain glucosamine (11.6%) and mannose (10%). Removal of the major portion of the carbohydrates by treatment with mixture of glycosidases did not lead to any significant decrease of biological activity. This finding suggests that the carbohydrate component is not essential for the biological activity of the enzyme. Analysis of distribution of carbohydrates in the peptides of the trypsin hydrolyzate of cathepsin D allows conclusion that the enzyme molecule has several carbohydrate chains attached to different sites of the molecule.
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PMID:[Study of the carbohydrate component of cathepsin D]. 59 21

Two-dimensional electrophoretograms of extracts of [3H]glucosamine-labeled human renal cancer cells demonstrated a series of components (Mr 48,000 and 30,000) that are only poorly expressed in similarly labeled normal kidney epithelial cell cultures [S. Ogata, R. Ueda, and K. O. Lloyd (1981) Proc. Natl. Acad Sci. USA 78, 770-774]. These characteristics are also exhibited by [3H]Man-labeled samples and by concanavalin A-binding glycoproteins from [35S]Met-labeled cells. It is now shown that these species are the precursor chain (Mr 48,000) and native heavy chain (Mr 30,000) forms of the lysosomal enzyme, cathepsin D. These results were obtained by precipitation with a specific anti-cathepsin D serum and by binding of the components to pepstatin-Sepharose. Cathepsin D heavy chain is heterogeneous, having three major species with pI's of 5.7, 5.3, and 4.9; all forms are glycosylated with high mannose-type chains [approximate size: Man5(GlcNAc)2] and are partially phosphorylated. Despite these indications of dissimilarities in cathepsin D levels, the actual levels of total acid protease activity were not significantly higher in renal cancer cells than in normal kidney epithelial cells.
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PMID:Characteristic [3H]glucosamine-labeled glycoproteins in two-dimensional electrophoretograms of human renal cancer cells: identification as cathepsin D. 388 15

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.
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PMID:[Purification and properties of cathepsin D from the mammary glands of lactating rabbits]. 400 22

1. Rat kidney lysosomal glycoproteins, prelabelled in the N-acetylneuraminic acid and polypeptide portions with N-acetyl[(3)H]mannosamine and [(14)C]lysine, or with N-acetyl-[(14)C]glucosamine, were incubated under various conditions. Autolytic cleavage of labelled N-acetylneuraminic acid and peptide was maximum at pH5.0. 2. N-Acetylneuraminic acid was released more rapidly than peptide during incubation at 37 degrees or 4 degrees C at pH5. p-Nitrophenyloxamic acid, an inhibitor of bacterial neuraminidase (Edmond et al., 1966), inhibited the cleavage of N-acetylneuraminic acid and peptide, and also inhibited cathepsin D activity. 3. Galactono-, mannono-, and glucono-lactone, inhibitors of the corresponding glycosidases, blocked the autolytic cleavage of N-acetyl[(14)C]glucosamine and protein without inhibiting beta-N-acetylhexosaminidase or cathepsin D activity. These findings suggest that the carbohydrate side chains protect the polypeptide portion of the lysosomal glycoproteins against proteolytic attack by lysosomal cathepsins. 4. In electrofocusing experiments, autolysis was minimized by adding 0.1% p-nitrophenyloxamic acid to the media used for extraction and electrofocusing, and by maintaining an alkaline pH (pH8.8-9) during extraction and dialysis. Arylsulphatase occurred in two forms with pI values of 4.4 and 6.4-6.7, and beta-glucuronidase in two forms with pI values of 4.4 and 6.1. When [(14)C]lysine and N-acetyl[(3)H]mannosamine were given to rats 1.5 and 1 h before killing, (14)C and (3)H were largely restricted to highly acidic glycoprotein species with pI values of 2.1-5.1. 5. When a lysosomal extract was adjusted to pH5 and incubated at 20 degrees C for 16h and then at 37 degrees C for 1 h before electrofocusing, 32 and 58% of the labelled peptide and N-acetylneuraminic acid was cleaved and the pI values of the labelled glycoproteins were markedly increased. About 80% of the acidic form of arylsulphatase and beta-glucuronidase was recovered with the basic form, and the pI of the basic form of both enzymes rose to 7.0. Similar, though less marked changes, were observed when a lysosomal extract was kept at pH5 for 2h at 4 degrees C before electrofocusing. 6. When an acidic lysosomal fraction (pI4.2-4.6) was incubated at pH5 for 2.5h and refocused, 80% of the arylsulphatase now occurred in two forms with pI values of 5 and 6.4. When a basic lysosomal fraction (pI5.8-6.4) was similarly incubated, the pI of arylsulphatase increased from 6.4 to 7.2. The relative increase in pI of arylsulphatases was accompanied by a proportional loss of N-acetylneuraminic acid from the glycoprotein associated with these forms. 7. These experiments show that lysosomal glycoproteins and two representative hydrolases, when exposed to a mildly acidic pH, readily undergo autolytic degradation and their pI values increase. These observations may have a bearing on the origin of the molecular heterogeneity of the lysosomal enzymes.
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PMID:Autolysis of glycoproteins in rat kidney lysosomes in vitro. Effects on the isoelectric focusing behaviour of glycoproteins, arylsulphatase and beta-glucuronidase. 445 20

Cathepsin D from porcine spleen contained mannose (3.3%), glucosamine (1.4%), and mannose 6-phosphate (0.08%). Essentially all of the oligosaccharides of cathepsin D could be released by endo-beta-N-acetylglucosaminidase H, pointing to oligomannoside types of structures. Three neutral oligosaccharide fractions, containing 5, 6, and 7 mannose residues, respectively, were isolated by gel permeation chromatography on Bio-Gel P-2. Studies using exoglycosidase digestions and 500-MHz 1H NMR spectroscopy revealed that their structures are [Man alpha 1 leads to 2]0 or 1 Man alpha 1 leads to 6[Man alpha 1 leads to 3]Man alpha 1 leads to 6[Man alpha 1 leads to 2)0 or 1 Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4 GlcNAc. These structures are identical to what have recently been proposed by Takahashi et al. for the major oligosaccharide units of cathepsin D from the same source (T. Takahashi, P. G. Schimidt, and J. Tang (1983) J. Biol. Chem. 258, 2819-2930), except for the occurrence of two isomeric oligosaccharides containing six mannoses. Only a part (3.4%) of the oligosaccharides were acidic, containing phosphates in monoester linkage. The phosphorylated oligosaccharides also consisted of oligomannoside-type chains which were analogous to, but more heterogeneous in size than the neutral oligosaccharides. Cathepsin D was bound to a mannose- and N-acetylglucosamine-specific lectin (mannan-binding protein) isolated from rabbit liver with the Ki value of 5.4 X 10(-6) M.
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PMID:Oligosaccharides on cathepsin D from porcine spleen. 642 53

It has been shown that low concentrations of E. coli lipopolysaccharides (LPS) greatly and selectively stimulate phagocytosis and related functions in mouse bone marrow-derived macrophages. Culture in the presence of 50 ng/ml LPS induced on average a 10-fold enhancement of phagocytosis of IgG-coated sheep erythrocytes. Activation was in two stages--a small increase observed during the first 8 to 12 hr, and the major increase noted between 16 and 24 hr. Phagocytic activity remained at the maximal level for 24 hr and then declined progressively. Stimulation by LPS was dose-dependent; significant effects could be observed at 0.8 ng/ml and the maximum was reached at 10 ng/ml. LPS-treated cells also showed a markedly increased tendency to form colonies. All these effects could be prevented by the addition of 100 ng/ml polymyxin B together with LPS, indicating that the active principle is lipid A. The LPS-dependent increase in phagocytic activity is probably mediated by increased Fc receptor capacity because both parameters were influenced in parallel by the stimulus. Phagocytosis-related events, such as enhanced hexose monophosphate shunt activity, H2O2 formation, and nitroblue tetrazolium reduction were also stimulated by LPS. By contrast, pinocytosis was unaffected. Measurements of cell-associated enzyme activities showed that lactate dehydrogenase, acid phosphatase, and cathepsin D were significantly increased. Beta-glucuronidase, beta-galactosidase, alkaline phosphodiesterase, and aminopeptidase were unchanged and NAD nucleosidase was markedly decreased after LPS treatment. 5'-Nucleotidase and glucosamine uptake were undetectable both in control and LPS-stimulated cells. LPS treatment induced a significant increase in cell-associated protein, but did not result in cell proliferation or increased cell loss as shown by the DNA content that remained constant. LPS-induced changes were dependent on de novo protein synthesis; cycloheximide prevented enhancement of phagocytosis, Fc receptor capacity, and colony formation.
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PMID:Stimulation of phagocytosis in bone marrow-derived mouse macrophages by bacterial lipopolysaccharide: correlation with biochemical and functional parameters. 673 51

The purpose of study was to explore the possible functions of Bcl-xL in the glucosamine sulfate-induced apoptosis of chronic myeloid leukemia K562 cells. Light microscopy and Wright-Giemsa staining were used to investigate the morphologic evidences for apoptosis of K562 cells induced by glucosamine sulfate (GS); immunofluorescence was used to observe the translocation of cathepsin D and cytochrome C during the apoptosis; Western blot was performed to detect the expression of Bcl-xL, Bid, Bax in K562 cells treated by GS. The results showed that many vacuoles were observed in the cytoplasma of the K562 cells treated by GS; fluorescent signals of cathepsin D and cytochrome were fransformed from granules to disperse form by using immunofluorescence; the expression of Bcl-xL was found down-regulated in K562 cells treated by GS, but not in the cells pre-treated with pepstatin A; the significant changes were not detected in expression of Bax and Bid protein before or after apoptosis. It is concluded that Bcl-xL protein may mediate relationship between cathepsin D and mitochondia pathway, Cathepsin D may play an important role in the GS inducing apoptosis of K562 cells through downregulation of Bcl-xL expression.
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PMID:[Role of Bcl-xL in the cathepsin D-associated apoptosis of K562 cells]. 1597 24

Chemical composition of extracellular matrix (ECM) plays a pivotal role in cellular and tissue development, regeneration, and differentiation. It also plays a key role in pathogenesis of hepatocellular carcinoma (HCC). This study explored premalignant changes in the liver tissue content of collagen (as hydroxyproline, HP), total glycosaminoglycans (TGAGs), free glucosamine (FGA), total sialic acid (TSA), lysosomal membrane integrity variations (calculated as total and free cathepsin D activities), and liver histology. Serum alfa-fetoprotein (AFP) level was used as an early marker for HCC in two groups of Wistar rats. One group of rats served as control and was provided normal saline orally. The other group was provided trichloroacetic acid (TCA) as 0.5 g/kg/day for five consecutive days by oral gavage. Animals were killed before tumor development. The treatment revealed dysplastic changes in addition to microsteatosis (fatty changes). Both sinusoids and the portal vein among dysplastic cells were dilated and congested. These dysplastic foci are believed to be premalignant and may be precancerous lesions. The following things were observed: a highly significant increase in serum AFP (as a key marker for HCC), a significant decrease in HP and TSA, a significant increase in FGA, nonsignificant decrease in TGAGs, significant up-regulation of free cathepsin D, nonsignificant decrease in total cathepsin D activities, and destabilization of lysosomal membrane integrity. Down-regulation of HP, TSA, and TGAGs seems to be a prerequisite for cancer development. This might be stimulated by up-regulation of free cathepsin D activity. Perhaps tissue fibrosis is not a condition for developing HCC because collagen was significantly depressed. Up-regulated FGA could be assumed to be a defense mechanism against TCA-induced proteolysis of membrane proteins because it is frequently reported to be of value in cancer chemotherapy. Studied ECM perturbations can be assumed as preliminary changes during chemical hepatocarcinogenesis at the tissue level. Prospective studies on blood levels of cathepsins, TGAGs, and individual ECM variables such as TSA, FGA, and Hp in patients at risk for HCC, performed in parallel with assessments of AFP, may provide a cost-effective way to find new links between tissue changes and circulation that would permit early prediction of disease. It may also provide a way to monitor HCC and compensate for the missed peak AFP values.
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PMID:Premalignant variations in extracellular matrix composition in chemically induced hepatocellular carcinoma in rats. 1968 84


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