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)

Procathepsin L, the precursor to a powerful lysosomal cysteine proteinase, has been purified to apparent homogeneity from guinea pig spermatozoa, a novel and previously unrecognized source of this catalytically active zymogen. In the range of pH 5.0, procathepsin L (39,000 M(r)) readily self-processed yielding a mature, single-chain proteinase (29,000 M(r)) and an intact propeptide (10,000 M(r)) by what appeared kinetically to be an intramolecular reaction mechanism. These characteristics resembled those reported for the "major excreted protein" (MEP) of malignantly transformed mouse fibroblasts-a protein that has been characterized as the precursor to the mouse analog of human cathepsin L (B. R. Troen, S. Gal, and M. M. Gottesman (1987) Biochem. J. 246, 731-735). Other characteristics shared by the guinea pig and mouse zymogens included proteolytic activity at pH 5.0, homologous N-terminal amino acid sequences, and immunological relatedness. It was thus concluded that acrosomal procathepsin L is the guinea pig analog of MEP. Acrosomal procathepsin L had a specific activity on benzyloxy-carbonyl-Phe-Arg-7-(4-methyl)coumarylamide (Z-Phe-Arg-NMec) of 30 mumol min-1 mg-1 enzyme at pH 3.2 and 37 degrees C. Relative to the assay substrate, rates on other fluorogenic substrates were 90% for Z-Phe-Cit-NMec, 63% for Z-Leu-Leu-Arg-NMec, 43% for D-Phe-Ser(Bzl)-Phe-Phe-Ala-Ala-p-aminobenzoate (a "specific" cathepsin D assay substrate), and 32% for Z-Val-Val-Arg-NMec. No action was detected on Z-Arg-Arg-NMec or Arg-NMec. Mature cathepsin L showed the same relative order of substrate specificity as its proenzyme form, but the absolute rates were about 5-fold greater. Additionally, the mature (single-chain) form of cathepsin L displayed Km and kcat values on Z-Phe-Arg-NMec that yielded an exceptionally high catalytic coefficient (11,600 s-1 mM-1) compared to values reported for two-chain forms of cathepsin L. Self-processing by acrosomal procathepsin L at pH 5.5 was totally inhibited by leupeptin, cystatin C, Ep-475, and Z-Phe-Phe-CHN2 at 1 microM levels. Gossypol (0.1 mM) gave 94% inhibition. Interestingly, dextran sulfate (100 micrograms ml-1) gave a 3.6-fold increase in the rate of self-processing seen at pH 5.5--a phenomenon of potential physiological relevance in view of the high-negative-charge density present within the hyaluronic acid-rich outer layer (cumulus oophorus) of the ovum.
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PMID:Purification and characterization of procathepsin L, a self-processing zymogen of guinea pig spermatozoa that acts on a cathepsin D assay substrate. 748 6

The structural domains of human apolipoprotein(a) [apo(a)] and its interaction with apolipoprotein B-100 (apo B-100) in the lipoprotein(a) [Lp(a)] particle were investigated by limited proteolysis with thermolysin and cathepsin D. We characterized the proteolytic products by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis, followed by immunoblotting using different antibodies. For apo B-100 in Lp(a), the digestion patterns were found to be identical to those previously described [Chen et al. (1989) J. Biol. Chem. 264, 14369-14375; Chen et al. (1991) J. Biol. Chem. 266, 12581-12587] for apo B-100 in LDL. Thus, we compared the digestion patterns of apo B-100 in Lp(a) resolved under reducing and nonreducing migrating conditions. Using an antibody specific for a synthetic peptide of apo B-100 (residues 4004-4021), we confirmed that apo B-100 was linked to apo(a) by its C-terminal end. Various Lp(a)s isolated from several donors, and containing different isoforms, were used to study the structural domains of apo(a). Using the same procedure as for apo B-100, several common features were found for the different isoforms. (1) Apo(a) can be cleaved into two structural domains: one was of constant size (170 kDa) and was linked to apo B-100. Using an antibody specifically directed against kringle V, we demonstrated that this fragment corresponded to the C-terminal part of apo(a). (2) The other domain, whose size varied according to the digested apo(a) isoform, was not linked to apo B-100.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structural domains of apolipoprotein(a) and its interaction with apolipoprotein B-100 in the lipoprotein(a) particle. 813 70

The p53 protein was identified in primary breast carcinomas by specific binding of PAb1801 and PAb240 antibodies. Using sodium dodecyl sulfate electrophoresis followed by immunoblotting on nitrocellulose membrane, the p53 protein was identified in 36 nuclear fractions obtained from 60 primary breast cancers; semiquantitation of p53 was performed by densitometric scanning. The total cathepsin D content, the estrogen and progesterone receptor concentration values and the axillary lymph node involvement were also assessed. Tumors expressing p53 had significantly higher levels of cathepsin D than those in which p53 was undetectable. p53 expression was strongly associated with low or negative estrogen receptor values; progesterone receptor concentrations were also significantly higher in p53-negative tumors than in those tumors with detectable p53 levels. Finally, a significant relationship between p53 expression and lymph node metastasis was observed. It was concluded that a positive association between p53 and cathepsin D values exists which is of prognostic interest in that both cathepsin D and p53 are associated with a high tumor grade and metastatic activity.
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PMID:p53 associated with cathepsin D in primary breast cancer. 851 12

Cell surface molecules on adherent cells that bind 125I-labeled fibronectin or its 70-kDa N-terminal fragment were identified by cross-linking with factor XIIIa and by photoaffinity labeling. Such cross-linking caused the 70-kDa fragment to become associated irreversibly to cell layers and was greater in cells treated with lysophosphatidic acid, an enhancer of fibronectin assembly and strong modulator of cell shape. Cross-linking of the 70-kDa fragment with factor XIIIa was to molecules that migrated in discontinuous sodium dodecyl sulfate-polyacrylamide gels at the top of the 3.3% stacking gel and near the top of the separating gel. Estimated sizes of these large apparent molecular mass molecules (LAMMs) were >>3 MDa and approximately 3 MDa. The label in 70-kDa fragment conjugated with 125I-sulfosuccinimidyl 2-(p-azidosalicylamido)-1, 3'-dithiopropionate was associated with >>3-MDa LAMMs without reduction and with approximately 3-MDa LAMMs after reduction and transfer of the cleavable label. The LAMMs were expressed on monolayer cells shortly after adherence, required both 1% Triton X-100 and 2 M urea for efficient extraction, and were susceptible to digestion with trypsin but not to cathepsin D digestion. Complexes of 125I-70-kDa fragment and LAMMs were also susceptible to limited acid digestion and Glu-C protease digestion but were not cleaved by chondroitin lyase or heparitinase. Neither the uncleaved complexes nor the cleavage products were immunoprecipitated with anti-fibronectin antibodies directed toward epitopes outside the 70-kDa region. Thus, cell surface molecules that are either very large or not dissociated in sodium dodecyl sulfate comprise the labile matrix assembly sites for fibronectin.
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PMID:Cross-linking of the NH2-terminal region of fibronectin to molecules of large apparent molecular mass. Characterization of fibronectin assembly sites induced by the treatment of fibroblasts with lysophosphatidic acid. 896 87

Fatty acid-binding protein (FABP) has been isolated from rat liver cytosol by two steps of gel-permeation chromatography on Sephadex G-75 and Sephacryl S-100 after ammonium sulfate precipitation. FABP fraction was eluted as two well-separated peaks, fractions A and B, by reversed-phase high-performance liquid chromatography (HPLC). The structural difference between the two fractions was investigated by lysyl endopeptidase digestion followed by reversed-phase HPLC of the digests, which identified a peptide corresponding to residues 58 through 78 as the modified peptide. Matrix-assisted laser-desorption-ionization mass spectrometry and other chemical analyses of the peptides established the modification in fraction A as cystein-thiolation at cysteine-69. This was confirmed by reduction and reoxidation of the peptide and the parent molecules. The modification did not affect binding of fluorescent derivatives of fatty acids. However, the modified species was more susceptible to proteolysis by bovine spleen cathepsin B and cathepsin D than the unmodified species. The presence of a relatively large amount of cysteine (but not of glutathione) mixed-disulfide form of FABP suggests some physiological role of this modification related to the redox status of the cell [Thomas, J.A., Poland, B., and Honzatko, R. (1995) Arch. Biochem. Biophys. 319, 1-9], and accounts, at least in part, for the extensive heterogeneity of liver FABP.
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PMID:Rat liver fatty acid-binding protein: identification of a molecular species having a mixed disulfide with cysteine at cysteine-69 and enhanced protease susceptibility. 898 55

We have determined the primary cleavage sites in the bone Gla protein (BGP; osteocalcin) for several of the proteases that could act on the protein during bone resorption and turnover, cathepsins B, D, L, H, and S. The time course of BGP digestion by each cathepsin was first determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We then incubated human and bovine BGP with each cathepsin for a sufficient time to reduce the level of intact protein by at least 20-fold, isolated the major cleavage peptides, and identified each by N-terminal sequence analysis and by amino acid analysis. Our results show that BGP has relatively few cathepsin-sensitive sites and that these sites are located at the N and C terminus of the 49-residue protein. Cathepsins B, L, H, and S readily cleave BGP at the G7-A8 bond; cathepsin L also cleaves at R43-R44; cathepsin B also cleaves at R44-F45; and cathepsin D cleaves only at A41-Y42. The immunoreactivity of the major peptides generated by cathepsin cleavage was evaluated using the original radioimmunoassay developed for the detection of BGP in human serum. The BGP 8-49 fragment cross-reacts identically with native BGP, while the 8-43 and the 1-44 fragments require 20- to 40-fold higher concentrations to achieve the same level of displacement as the native protein. The 1-41 and 8-41 fragments are unable to significantly displace the labeled native BGP tracer at any concentration tested. These results demonstrate the utility of peptides generated by cathepsin digestion in the mapping of the antigenic epitopes recognized by a given BGP immunoassay.
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PMID:Identification of peptide fragments generated by digestion of bovine and human osteocalcin with the lysosomal proteinases cathepsin B, D, L, H, and S. 907 88

Employing isoelectric focusing on immobilized pH gradients followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we have obtained a map of C. elegans proteins, from a mixed culture containing all developmental stages, presenting over 2000 spots within the window of isoelectric points (pI) 3.5-9 and a molecular mass of 10-200 kDa. Edman microsequencing yielded successful results in 12 out of 24 analyzed spots. All but one of the N-terminal sequences retrieved C. elegans sequences in cosmid and/or expressed sequence tag clones. Structurally related protein sequences found in data banks included enzymes in energy metabolism (cytochrome oxydase, ATP synthase, enolase), a fatty acid-binding protein, a translationally controlled tumor protein, an unknown C. elegans protein, an acidic ribosomal protein, a titin-like protein, a G-protein beta chain, cyclophilin, and cathepsin D. Experimental determination of N-termini allowed us to define sites of signal cleavage providing further information on the physiological role of the newly found C. elegans proteins. This report demonstrates the possibility of two-dimensional gel electrophoresis and Edman microsequencing in the elucidation of C. elegans proteome.
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PMID:Two-dimensional gel electrophoresis of Caenorhabditis elegans homogenates and identification of protein spots by microsequencing. 915 Sep 41

Age related changes in the activity of lysosomal enzymes have been studied in the cultured human retinal pigment epithelium cells collected from 26-85 year old donors. Among four such enzymes studied, activities of cathepsin D and beta-glucuronidase increased with the age of the donors while no notable change in activity of arylsulfatase B and alpha-mannosidase was observed. Kinetic parameters of beta-glucuronidase was measured in retinal pigment epithelium cells isolated from donors of different ages. Similar kinetic parameters for beta-glucuronidase at different ages suggest that the observed increase in the activity of the enzyme with age is not due to post-translational modification of the enzyme. Western blot analysis provides evidence for increased synthesis of beta-glucuronidase with aging. Relative proportions of glycosaminoglycans, the natural substrates of beta-glucuronidase and arylsulfatase B, in the retinal pigment epithelium altered with the age of the donors. A significant decrease of dermatan sulfate levels with aging correlates well with the observed increase in the level of beta-glucuronidase activity.
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PMID:Age-related increase in activity of specific lysosomal enzymes in the human retinal pigment epithelium. 926 91

Three protein inhibitors of proteolytic enzymes with molecular weights 21, 22, and 23 kD were isolated from potato tubers (Solanum tuberosum L.) by ammonium sulfate precipitation followed by gel and ion-exchange chromatography. The 21- and 22-kD proteins were shown to be serine proteinase inhibitors with different specificities. The 21-kD protein inhibits human leucocyte elastase and trypsin effectively, but it is less effective towards chymotrypsin. The 22-kD protein is an inhibitor of cysteine proteinases and suppresses the activities of papain, ficin, and bromelain with the same affinities. None of the isolated proteins inhibit subtilisin, pepsin, or cathepsin D. The 21-kD protein consists of two disulfide-linked polypeptide chains with molecular weights of 16.5 +/- 1 kD and 4.5 +/- 1 kD. The 22-kD and 23-kD proteins have a single polypeptide chain. The N-terminal 22-25 amino acid sequences of these three proteins were determined. These sequences have significant homology to other plant inhibitors from the Kunitz soybean inhibitor superfamily.
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PMID:Potato tuber protein proteinase inhibitors belonging to the Kunitz soybean inhibitor family. 948 70

The action of purified cathepsin D on hemoglobin was examined using micellar electrokinetic chromatographic separation of peptide products. Purified cathepsin D was incubated with hemoglobin in 40 mM Na-formate pH 3.1 at 37 degrees C for varying lengths of time. The reaction was stopped by the addition of the inhibitor pepstatin, and the peptide products were isolated from the reaction mixture by ultrafiltration with a 10,000 molecular weight cut-off (MWCO) microfuge type filter. Filtered samples were then separated in 100 mM Tris-Cl pH 8.5 containing sodium dodecyl sulfate (SDS) or Na-deoxycholate at micellar concentrations using a 50-microns (i.d.) fused-silica capillary. Detection was performed at 214 nm. It was found that Na-deoxycholate containing separations were superior in resolution and required less time. This technique was used to determine initial velocity (expressed as peak area per unit time) for nine peptides. Several peptides resulted after very short incubation times (< 10 min). This suggests that this approach may be useful for the determination of cathepsin D activity.
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PMID:Capillary electrophoretic analysis of cathepsin D action on hemoglobin. 948 58


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