Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thyroglobulin (Tg) is the major secretory product of thyroid epithelial cells and is stored in the lumen of thyroid follicles at high concentrations. Thyroid hormone liberation is assumed to occur separately from this storage compartment within lysosomes. However, for the transfer of Tg to lysosomes, mechanisms to solubilize the luminal content must precede its endocytosis, because part of the luminal Tg occurs in a covalently cross-linked form. Here, by immunoprecipitation and immunoblotting we show that the majority of procathepsin B or L and a fraction of mature cathepsin B are released from porcine thyrocytes in vitro. Released cathepsins were detectable on the cell surface of the thyrocytes by immunocytochemistry and amounted to 27% of the total cathepsin B. Cytochemical studies revealed the proteolytic activity of cathepsin B at neutral pH on the cell surface of thyrocytes. Therefore, the possibility of extracellular proteolysis by cathepsins was investigated by incubating plasma membrane preparations, conditioned media, or lysosomes with Tg. The liberation of thyroid hormones was quantitated by RIA, and the degradation of Tg was determined by SDS-PAGE. Extracellular and plasma membrane-associated proteases rapidly mediated up to 54% of the total T4 liberation by limited proteolysis of Tg at neutral pH under conditions where cysteine proteases were reactivated. We propose that released and proteolytically active cysteine protease i.e. cathepsins B and L, provide thyrocytes with a pathway of limited extracellular proteolysis of Tg before endocytosis.
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PMID:Evidence for extracellularly acting cathepsins mediating thyroid hormone liberation in thyroid epithelial cells. 861 37

Plasma and ascitic fluid of rats bearing the Yoshida ascites hepatoma AH-130 were shown to contain high levels of proteolytic enzymes belonging to different classes active at neutral and acidic pH. Relative to those measured in control rat plasma, in tumor-bearing animals, the activity levels of lysosomal cathepsins B and L, in their latent, acidic-activatable form, were approximately 5-fold higher in plasma and 9-fold higher in ascitic fluid, and cathepsin D activity was about 5-fold higher in both plasma and ascitic fluid. Plasma and ascitic fluid of tumor-bearing rats also contained novel neutral and acidic gelatinolytic activities. The latter, as revealed by zymographic analysis conducted at pH 6.0, in the presence of dithiothreitol and in the absence of divalent metal ions, was sensitive to iodoacetamide inhibition but not to EDTA, showed a molecular mass of approximately 90 kD on SDS-PAGE, and was lost upon limited proteolysis with pepsin. Therefore, this enzyme is not identifiable as cathepsin B or L or their related latent forms and may represent a novel, so far undescribed, gelatinase. Its presence exclusively in the body fluids of AH-130-bearing rats suggests its possible use as a tumor marker.
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PMID:High levels of proteolytic enzymes in the ascitic fluid and plasma of rats bearing the Yoshida AH-130 hepatoma. 862 Dec 67

The ciliate Paramecium tetraurelia secretes large amounts of a cysteine protease into the growth medium, presumably for extracellular food digestion. Two endoprotease isozymes (30 and 33 kDa on SDS/PAGE, respectively), both present in cell homogenates and in spent growth medium, were purified to homogeneity. Peptide sequence analysis revealed that these isozymes share identities at the amino acid level but are probably differently processed. Enzymatic characterization of the isolated proteases and sequencing of the cloned cDNA demonstrated that the enzymes belong to the cathepsin-L protease subfamily. Although the identity with mammalian and other protozoan L cathepsins was only around 30%, all important signature sequences for cathepsin L in the preproregion as well as in the catalyst of the enzyme were fully retained. The cDNA of this cysteine protease codes for a preproregion of 108 amino acids. The putative proregion of 86 amino acids which contained the characteristic conserved ERFNIN motif, was fused with a His6 tag, expressed in Escherichia coli, and purified. Both cathepsin L isozymes of Paramecium tetraurelia were inhibited by their cognate propeptide in the nanomolar concentration range. All other cysteine proteases tested (papain and mammalian cathepsin B, G and H) were unaffected by the propeptide up to 10 microM.
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PMID:Cathepsin L is an intracellular and extracellular protease in Paramecium tetraurelia. Purification, cloning, sequencing and specific inhibition by its expressed propeptide. 866 38

Cathepsin B was isolated from buffalo liver by salt fractionation, ion-exchange resin treatment, gel filtration and repeated ion-exchange chromatography using a linear salt gradient. The enzyme showed activity, against denatured hemoglobin (or ovalbumin), alpha-N-benzoyl-DL-arginine p-nitroanilide (BAPNA), and alpha-benzoyl-DL-arginine-naphthylamine (BANA). It inactivated buffalo muscle aldolase with a half life period of 21 min. The pH-activity profiles obtained for the digestion of hemoglobin (or ovalbumin) and aldolase inactivation by the enzyme were found to be different. The enzyme (mol wt 27,800 by SDS-PAGE) eluted in gel filtration with a molecular weight of 27,000 and a Stokes radius of 2.31 nm. The results showed buffalo cathepsin B to be a single-chain molecule. The N- and C-terminal amino acids of the enzyme were found to be leucine and aspartic acid, respectively. It contained 0.7% concanavalin A reactive neutral carbohydrate. The amino acid composition of buffalo cathepsin B was found to be similar to that of human liver cathepsin B. The optical properties of the buffalo enzyme were found consistent with its aromatic amino acid content. The isoionic pH of the enzyme was found to be 5.70 and the intrinsic viscosity was 3.48 ml/g whence the frictional ratio, f/f0 was computed to be 1.10 suggesting that the native enzyme conformation is compact and is globular in solution.
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PMID:Isolation, purification and properties of cathepsin B from buffalo liver. 893 19

The affect of methylprednisolone (MP), an anti-inflammatory drug upon purified calpain and the Ca2+-mediated degradation of endogenous proteins of spinal cord homogenate in vitro has been examined. Activity of calpain purified from rabbit muscle was greatly inhibited in a dose-dependent fashion by MP. A 50% inhibition was obtained with 3.2 mM MP concentration and the activity was inhibited further (80%) at 8.1 mM. More potent inhibition of the purified enzyme (70-80%) was produced by dexamethasone (3.9 mM) and prednisolone (4.1 mM). Calpain-mediated degradation of myelin basic protein (MBP) was also inhibited by MP as was cathepsin B-mediated MBP breakdown. The effect of MP and other steroids upon calcium-mediated degradation of spinal cord homogenate was also evaluated. SDS-PAGE analysis revealed significant inhibition of neurofilament protein breakdown by MP and other corticosteroids. This inhibitory effect was much less than that exerted by the calpain inhibitors calpeptin and/or E64-d. These results indicate that MP acts as a proteinase (calpain) inhibitor and define a new mechanism for its actions.
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PMID:A new mechanism of methylprednisolone and other corticosteroids action demonstrated in vitro: inhibition of a proteinase (calpain) prevents myelin and cytoskeletal protein degradation. 906 63

To improve the level of purity of cathepsin B, we have modified the published procedure [Agarwal, S.K. and Khan, M.Y. (1987) Biochem. Int. 15,785-792] by incorporating CM-Sephadex ion exchange chromatography and chromatofocusing. The enzyme thus isolated could be resolved into one 26 kDa major and a minor 27 kDa protein bands on SDS-PAGE. The two components, however, could not be separated by gel filtration and they eluted, in a single peak corresponding to a molecular mass of 28.1 kDa. Among the various substrates tested, Z-Phe-Arg-MCA with a Km of 0.058 mM and hemoglobin with a Km of 1.449 microM were the most preferred synthetic and protein substrates respectively. It was found to be a glycoprotein with an acidic pI of 4.8. The enzyme was activated by various thiol-reducing reagents and inhibited by cysteine proteinase inhibitors, divalent cations, lysyl group modifiers, anti-inflammatory drug and denaturing agents. The hydrodynamic behaviour of cathepsin B suggested a compact and globular conformation. Immunodiffusion studies with anti-goat cathepsin B indicated a tissue/ species dependence.
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PMID:Catalytic and physico-chemical characteristics of goat spleen cathepsin B. 930 39

Characterization of cathepsin B from buffalo kidney and goat spleen showed the presence of isozymes in case of the goat spleen (GSCB-I and GSCB-II) whereas cathepsin B from buffalo kidney exhibited only one form (BKCB). The molecular weights determined by SDS-PAGE for GSCB-I, GSCB-II, and BKCB were 25.7, 26.6 and 25.5 kDa respectively. The kinetic parameters (Km and Vmax) of GSCB-I showed close similarities with BKCB against alpha-N-benzoyl-DL-arginine-2-napthylamide whereas GSCB-II was closer to the buffalo enzyme with regards to its activity against Z-Arg-Arg-MCA and Z-Phe-Arg-MCA. All the three enzymes had similar sensitivities towards urea, antipain and leupeptin. However, clear differences were observed in the inhibition patterns of the enzyme with iodoacetic acid and iodoacetamide. Differences in the kinetic, immunogenic and some catalytic properties of GSCB-I and II, which had similarities with regard to most of their physico-chemical properties, were considered to be due to the existence of two isozyme forms in goat spleen cathepsin B preparations. Absence of such a multiplicity in forms of the enzyme from buffalo kidney was accordingly attributed to the absence of cathepsin B isozymes in this species. These observations taken together therefore, indicate a probable species/tissue dependence of cathepsin B.
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PMID:On the tissue/species dependence of cathepsin B isozymes. 945 Jun 49

Proteolytic activity present in the excreted/secreted (ES) material of newly excysted juvenile (NEJ) Fasciola hepatica was biochemically analyzed. By gelatin substrate SDS-PAGE, only one region of activity was observed in the NEJ ES material at a molecular mass of 29 kDa. Both the secreted cathepsin L from adult fluke and the 29-kDa proteolytic activity of NEJ ES show a common pH optimum of 7.5, a cysteine protease inhibition profile, and preference for the N-benzyloxycarbonyl (Z)-Phe-Arg-NHMec fluorogenic substrate over Z-Arg-Arg-NHMec and Z-Arg-NHMec. In vitro analysis revealed that the NEJ protease activity digested sheep immunoglobulin heavy chain and bovine serum albumin but not bovine hemoglobin. Amino-terminal protein sequence analysis of the 29-kDa NEJ protease band revealed two sequences with homology to the cathepsin B family of proteases. Using degenerate oligonucleotides designed from the N-terminal sequence, reverse transcriptase polymerase chain reaction with NEJ RNA amplified a cDNA sequence encoding the first 236 amino acids of mature cathepsin B. Using this cDNA fragment an overlapping cDNA was isolated from a LambadaZAP cDNA library constructed with poly(A)+ RNA from immature 5-week-old liver fluke. Together with the N-terminal sequence, these cDNAs predict a mature cathepsin B sequence of 254 amino acids which shows 48-51% sequence identity to mammalian and Schistosoma mansoni cathepsin B. We conclude that, in contrast to the major proteases released by adult fluke, the major secreted protease of NEJ of F. hepatica is of the cathepsin B class.
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PMID:Fasciola hepatica: characterization and cloning of the major cathepsin B protease secreted by newly excysted juvenile liver fluke. 953 62

A novel proteolytic activity was identified in epimastigote, amastigote and trypomastigote forms of Trypanosoma cruzi using the fluorogenic substrate N-Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Epimastigotes showed enzyme activity to be 2-fold higher than amastigotes and trypomastigotes. The protease that displays this activity was purified from epimastigote forms by a four step chromatographic procedure: Diethylaminoethyl-Sephacel, Phenyl-Sepharose, Phenyl-Superose, and Concanavalin A Sepharose columns. The purified enzyme is a glycoprotein that migrates as a 30 kDa protein in 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), under reducing conditions. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. The inhibition pattern of the purified 30 kDa protease showed that it belongs to the cysteine-protease class. In addition to the synthetic substrate, the purified protease hydrolysed bovine serum albumin (BSA) and human type I collagen. The N-terminal amino acid sequence of the protease shows similarity to the mammalian cathepsin B protease.
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PMID:Characterisation of a Trypanosoma cruzi acidic 30 kDa cysteine protease. 956 19

A simple purification scheme was developed for isolation and purification of cathepsin B from buffalo kidney. The use of CM-Sephadex and chromatofocusing helped in better and simultaneous separation of cathepsin B, H and L. As judged by PAGE and SDS-PAGE studies, the enzyme was found to be pure on the basis of charge and had a molecular mass of 25.5 kDa. The amino acid composition, number of free sulfhydryl groups and other major physico-chemical properties of the purified enzyme were similar to the properties reported for cathepsin B from other sources/tissues. However, the NH2-terminal amino acid residue of the enzyme was found to be Ala as against Leu reported from other tissues/species. The total carbohydrate content was also found to be significantly lower (3.6%) as compared to 7.0-7.6% reported for the enzyme from other sources. Thiol reducing compounds activated the enzyme whereas thiol blocking compounds inhibited it. The buffalo kidney enzyme hydrolyzed Z-Phe-Arg-MCA (Vmax/K(m) = 17.1) as the most efficient substrate followed by Z-Arg-Arg-MCA, BANA and BAPNA. Among the protein substrates, goat hemoglobin (Vmax/K(m) = 874) was found to be the most preferred. Rabbit muscle aldolase, usually considered to be a good substrate for cathepsin B, proved to be a poor substrate for this enzyme; only 25-30% inactivation of aldolase was observed. Antibodies raised against the enzyme recognised only cathepsin B and did not have any cross reactivity with cathepsin H or L from the same or different sources. These differences in the properties of the buffalo kidney enzyme vis-a-vis the same enzyme from other tissue/species have been attributed to specialized function of cathepsin B in diversified tissues.
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PMID:Purification and tissue/species dependence of the specificity of buffalo kidney cathepsin B. 959 26


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