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: UMLS:C0272170 (SDS)
50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An oral parasite Trichomonas tenax ATCC 30207 synthesizes and secretes various proteinases. By gelatin-SDS-PAGE, we found five proteinases bands (30, 37, 46, 51 and 60 kDa) in cell lysate and four bands (37, 45, 52 and 60 kDa) in culture filtrate. The proteinases hydrolyzed acid soluble type I collagen as well as gelatin. The enzymes were suggested to possess typical characteristics of cysteine proteinases based on the patterns of inhibition and activation by various factors. Based on relative efficiencies of synthetic substrates, most of them were most likely cathepsin B-like enzymes.
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PMID:Characterization of the cathepsin B-like proteinases of Trichomonas tenax ATCC 30207. 1115 32

Although papain-like enzymes are strongly inhibited by their natural tight-binding inhibitors of the cystatin superfamily, cathepsins B and L may still retain some residual proteolytic activity toward Z-Phe-Arg-AMC in the presence of an excess of kininogen. This activity is abolished by adding E-64 or chicken cystatin. Cathepsins B and L show a single band of gelatinolytic activity when subjected to gelatin-SDS-PAGE. Adding high Mr kininogen, low Mr kininogen, T-kininogen, or chicken cystatin to cathepsin L results in additional intense bands of enzyme activity corresponding to the protease-inhibitor complexes. Cathepsin B does not produce these additional bands. This gelatinolytic activity was inhibited by E-64, but not by EDTA, PMSF or Pefabloc. Cathepsin L also specifically generated kinins from high and low molecular weight kininogens in vitro, but cathepsin B did not. T-kininogen did not release any immunoreactive kinins when complexed with cathepsin L, as previously observed using tissue kallikreins. The ability of cathepsin L to generate vasoactive peptides raises the question of the physiological significance of this mechanism during inflammation.
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PMID:Cathepsin L, but not cathepsin B, is a potential kininogenase. 1151 35

The luminal fluid of estrogen or DES-stimulated uterus of immature rats contains 10-12 isoforms of peroxidase between pI 4.5-6.0. N-terminal amino acid sequencing of diaminobenzidine-peroxidase bands eluted from IEF and SDS-PAGE gels showed the presence of cathepsin B and the complement family of proteins as the major comigrants. Sequential treatment of uterine fluid by cation, anion, and size exclusion chromatography resulted in a five-fold purification of peroxidase having a specific activity of 273 units/mg. Mass spectrometric studies of bands isolated from SDS-PAGE gels from the size-exclusion purified peroxidase fraction showed the presence of complement C3 along with novel previously uncharacterized proteins. Two dimensional electrophoresis followed by N-terminal amino acid sequencing confirmed the presence of cathepsin B isoforms and isoforms of a novel protein at approximately 87 kDa. Identification by mass spectrometry from the database for this novel protein was inconclusive but could most likely be a candidate for estrogen-induced peroxidase. Results conclusively prove that cathepsin B and complement C3 are major proteins in the estrogen-induced peroxidase fraction of uterine fluid.
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PMID:Cathepsin B and complement C3 are major comigrants in the estrogen-induced peroxidase fraction of rat uterine fluid. 1184 90

Papain-like cysteine endopeptidases have been recognized as potential targets for chemotherapy and serodiagnostic reagents in infections with the human parasitic helminth Schistosoma. A novel cathepsin B endopeptidase from adult S. mansoni has been isolated and characterized. The enzyme is termed SmCB2 to distinguish it from the first recorded schistosome cathepsin B, SmCB1, also known as Sm31. A rapid and convenient protocol involving anion exchange and affinity chromatography is described for the isolation of SmCB1 and SmCB2 from the same parasite starting material. SmCB2 has been functionally expressed in and purified from Pichia pastoris. Both native and recombinant SmCB2 migrate similarly (33 kDa) by SDS-PAGE. Both display strict acidic pH activity profiles and similar K(m) and k(cat) for dipeptidyl amidomethylcoumarin substrates. We conclude that the recombinant enzyme is properly folded. The S(2) subsite specificity of recombinant SmCB2 exhibits the preferences Phe>Leu>Val>>Arg. By immunoblotting with anti-SmCB2 IgG, a 33 kDa protein was identified in soluble extracts of male schistosomes. By immunohistochemistry, SmCB2 was localized in the tegumental tubercles and parenchyma of males with less product being visualized in the parenchyma of females. The enzyme may be lysosomal and function at the host parasite-interface.
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PMID:SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni. 1198 62

The excretory-secretory product (ESP) derived from Cyathostominea in vitro was assessed, in terms of subunit composition, and proteolytic activity using as substrates azocasein and two synthetic fluorogenic peptides. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved 13 subunits, and the presence of the protein cysteine proteinase activator dithiothreitol (DTT) revealed 21 subunits. DTT also enhanced azocaseinolysis, and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NHMec) and carbobenzoxy-arginyl-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-NHMec). At the optimum pH of 5.5, hydrolysis of Z-Phe-Arg-NHMec was three-fold greater than that of Z-Arg-Arg-NHMec suggesting that the proteolytic specificities of the ESP are more like those of papain or cathepsin L, rather than cathepsin B. In SDS-PAGE gelatin gels, DTT was a requirement for proteolysis by the ESP. Optimum resolution was at pH=5.5, resolving six bands ranging from 114-20kDa. Cysteine proteinase inhibitors abolished all gelatinolytic activity at the pH values tested. Such data indicate the presence of cysteine-class proteinases in the ESP of Cyathostominea.
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PMID:A preliminary analysis of proteolytic activity of excretory-secretory products from Cyathostominea. 1207 15

This study demonstrates that endopin 2 is a unique secretory vesicle serpin that displays cross-class inhibition of cysteine and serine proteases, indicated by effective inhibition of papain and elastase, respectively. Homology of the reactive site loop (RSL) domain of endopin 2, notably at P1-P1' residues, with other serpins that inhibit cysteine and serine proteases predicted that endopin 2 may inhibit similar proteases. Recombinant N-His-tagged endopin 2 inhibited papain and elastase with second-order rate constants (k(ass)) of 1.4 x 10(6) and 1.7 x 10(5) M(-1) s(-1), respectively. Endopin 2 formed SDS-stable complexes with papain and elastase, a characteristic property of serpins. Interactions of the RSL domain of endopin 2 with papain and elastase were indicated by cleavage of endopin 2 near the predicted P1-P1' residues by these proteases. Endopin 2 did not inhibit the cysteine protease cathepsin B, or the serine proteases chymotrypsin, trypsin, plasmin, and furin. Endopin 2 in neuroendocrine chromaffin cells was colocalized with the secretory vesicle component (Met)enkephalin by confocal immunonfluorescence microscopy, and was present in isolated secretory vesicles (chromaffin granules) from chromaffin cells as a glycoprotein of 72-73 kDa. Moreover, regulated secretion of endopin 2 from chromaffin cells was induced by nicotine and KCl depolarization. Overall, these results demonstrate that the serpin endopin 2 possesses dual specificity for inhibiting both papain-like cysteine and elastase-like serine proteases. These findings demonstrate that endopin 2 inhibitory functions may occur in the regulated secretory pathway.
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PMID:The novel serpin endopin 2 demonstrates cross-class inhibition of papain and elastase: localization of endopin 2 to regulated secretory vesicles of neuroendocrine chromaffin cells. 1217 26

An enzyme purified from the ovaries of Helicoverpa armigera, as an active form with molecular mass of 30 kDa on SDS-PAGE, was identified as a cysteine proteinase because it could be inhibited by E-64, a specific inhibitor of cysteine proteinase, and required reducing conditions for activity. This enzyme was further identified as a cathepsin B-like cysteine proteinase by partial amino acid sequencing. A cDNA encoding this proteinase was cloned from H. armigera, using degenerate primers and RACE techniques. Results of Northern blots indicated that the mRNA encoding the proteinase was transcribed in the ovaries, the fat bodies of female and male adults, pupae and in the larvae. No mRNA was detected from the larval epidermis or from the midgut. Hence, transcription of the cathepsin B-like cysteine proteinase from H. armigera was tissue-specific, but not gender- or developmental stage-specific. However, proteolytic activities were only detected from ovaries, and adult female and male fat bodies. No activity was observed from pupal and larval fat bodies, from the larval epidermis or from the midgut. Only one form of mRNA of approximately 1100 bases was detected, and in situ hybridization showed that the transcripts were distributed in the adult female fat bodies, follicular cells and the oocytes. Since the proteinase expressed in ovaries was able to degrade vitellin in vitro, it may be involved in the degradation of vitellin during embryonic development.
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PMID:Molecular cloning and characterization of the cathepsin B-like proteinase from the cotton boll worm, Helicoverpa armigera. 1242 14

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.
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PMID:Novel secretory vesicle serpins, endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities. 1243 89

Two cysteine proteinase inhibitors, CPI-L and CPI-H, were purified from rabbit skeletal muscle by means of successive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. The molecular mass of CPI-L was 13 kDa on gel permeation chromatography and SDS-PAGE under reducing conditions and was 15 kDa on SDS-PAGE under non-reducing conditions. The molecular mass of CPI-H was 23 kDa on gel permeation chromatography and it was converted to 13 kDa by SH-reducing agent. Although CPI-H showed single protein band with 13 kDa on SDS-PAGE under reducing conditions, it showed four protein bands with 21, 20, 15 and 13 kDa on SDS-PAGE under non-reducing conditions. Therefore, CPI-H was suggested to have a complicated subunit structure for which S-S bonds and some non-covalent bonds would be responsible. CPI-L and CPI-H were stable in the range of pH 3.0-9.5 and up to 80 degrees C. CPI-L and CPI-H were suggested to inhibit cathepsins B, H and L by a non-competitive mechanism. The inhibition constants (Ki) of CPI-L and CPI-H showed that both CPIs have much higher affinity against cathepsins H and L than against cathepsin B.
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PMID:Purification and properties of cysteine proteinase inhibitors from rabbit skeletal muscle. 1452 56

Cathepsin B (EC 3.4.22.1) has been highly purified (14,225 fold) from bovine kidney by a rapid procedure that included the preparation of an enriched lysosomal extract, a selective fractionation with ammonium sulphate, size-exclusion chromatography, two cation-exchange chromatographies, and anion-exchange chromatography on diethylaminoethyl-Sephacel. After the last purification step, two hydrolytic peaks against Z-Phe-Arg-AMC were separated from each other, a minor peak corresponding to the cathepsin B single-chain form and a major one representing the double-chain form of cathepsin B. The single-chain form showed a molecular mass of 31 kDa on sodium dodecyl sulphate - polyacrylamide gel electrphoresis (PAGE) under reducing conditions, whereas the heavy chain of the double-chain form appeared as a doublet with molecular masses of 23.4 and 25 kDa, respectively. The identity of the different cathepsin B isoforms and the quality of the final enzyme preparation were confirmed by using two types of antibodies, one against a synthetic peptide sequence and one against purified cathepsin B. The proteomic study confirmed the identity of the different SDS-PAGE protein bands as cathepsin B isoforms and allowed the comparison and study of some structural differences between them at the level of their primary structures.
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PMID:Purification of bovine cathepsin B: proteomic characterization of the different forms and production of specific antibodies. 1456 4


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