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

A new set of allergens from Dermatophagoides pteronyssinus and D. farinae (provisionally named DP5 and DF5, respectively) was isolated from the whole culture of mites. The apparent molecular weights of both allergens were shown to be 25,000 on SDS-PAGE under a reducing condition and 27,000 on Sephadex G-75 gel filtration chromatography. Both DP5 and DF5, as well as Der f III, possessed proteolytic activity. The results of substrate specificity and susceptibility to various protease inhibitors of DP5 and DF5 strongly suggested that they belonged to the chymotrypsin-like serine protease family. In sera from 88 mite-allergic patients, specific IgE antibodies to DP5 and/or DF5 were detected in only 41% of the sera by radio-allergosorbent test, while 90% and 93% had specific IgE antibodies to Der p I and/or Der f I and Der p II and/or Der f II, respectively.
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PMID:Allergens from Dermatophagoides mites with chymotryptic activity. 768 8

By comparison of the cell surface proteins derived from the outer membrane and fibrils from 14 Prevotella intermedia and 19 Prevotella nigrescens strains using SDS and analysed by SDS-PAGE, it was possible to distinguish the two species. A polypeptide of approx. 21 kDa distinguished P. intermedia strains, whereas two polypeptides of approx. 18 and 22 kDa could be used to identify P. nigrescens strains. Four other human oral black pigmented bacterial species (Porphyromonas gingivalis, Prevotella denticola, Prevotella loescheii and Prevotella melaninogenica) did not have the 18-, 21- or 22-kDa polypeptides shown by P. intermedia or P. nigrescens. The cell-associated proteolytic activity of eight strains of P. intermedia, 14 strains of P. nigrescens and one strain of P. gingivalis (W50) was assessed using four chromogenic substrates. The hydrolysis of the substrate GPPNA (indicative of dipeptidyl peptidase IV-like activity) and SAAPPNA (elastase-like activity) by P. intermedia strains varied from 32 to 114 units and 0.5 to 12.6 units of activity respectively, where one unit was defined as the amount of protease enzyme catalysing the formation of 1 nmol of p-nitroaniline under experimental conditions. 37.5% (3 of 8) of P. intermedia strains hydrolysed SAAPPNA (chymotrypsin-like enzyme activity) with activities of between 7 and 12 units. The hydrolysis of GPPNA and SAAAPNA by P. nigrescens strains was 32-149 and 3-16 units, respectively. 57% (8 of 14) of P. nigrescens strains hydrolysed SAAPPPNA with activities ranging from 3 to 8 units. None of the P. intermedia or P. nigrescens strains examined were found to have trypsin-like enzyme activity (BAPNA hydrolysis). The GPPNA and SAAAPNA hydrolytic activity associated with the proteases from Porphyromonas gingivalis W50 was at least twice that of P. intermedia and P. nigrescens strains. The similar peptidase activities of P. intermedia and P. nigrescens against chromogenic substrates cannot be used to differentiate the species, but SDS-PAGE of cell surface protein extracts allowed unambiguous speciation between P. intermedia and P. nigrescens. This simple technique of cell surface protein analysis can be performed in most laboratories and offers a convenient way by which to differentiate the two species.
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PMID:An investigation into the use of SDS-PAGE of cell surface extracts and proteolytic activity to differentiate Prevotella nigrescens and Prevotella intermedia. 886 94

A wide range of intra- and extracellular microbial proteases has been studied and characterized. These enzymes are mostly extracellular and in some cases they may resemble 'classical' serine proteases. As part of a programme in which the lipase and protease activities of the fungus Geotrichum candidum are being studied, an intracellular protease with an apparent chymotrypsin-like specificity was detected. The serine protease was isolated from biomass using ion-exchange and exclusion chromatography. Kinetic characterization was done using a series of synthetic substrates and inhibitors. Aprotinin-sepharose affinity chromatography was used to isolate a fraction for molecular size determination on SDS-PAGE. The purified protease, which could hydrolyse haemoglobin as protein substrate, was obtained with a 30-fold purification and a yield of 44%, but it was very unstable and rapidly lost activity. The enzyme which bound to the affinity column had a single subunit mass of 278 kDa. Kinetic analysis showed a similarity with trypsin and chymotrypsin, but tending more towards chymotrypsin in that a bulky aromatic group, e.g. phenylalanine in the P1 position, was preferred. The optimum pH was in the region of 7-8.25. Inhibition patterns indicated that the enzyme was a serine protease with no metal dependence, although it was stabilized by magnesium ions. The enzyme seems to share some properties with other intra- and extracellular microbial serine proteases. The exact function of the enzymatic activity is still unclear, but it is suggested that it may be involved with intracellular protein turnover.
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PMID:Geotrichum candidum P-5 produces an intracellular serine protease resembling chymotrypsin. 893 Jan 36

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for more advanced squamous cell tumors. Molecular cloning of the SCCA genomic region revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. Analysis of the primary amino acid sequences shows that both genes are members of the high molecular weight serpin superfamily of serine proteinase inhibitors. Although SCCA1 and SCCA2 are nearly identical in primary structure, the reactive site loop of each inhibitor suggests that they may differ in their specificity for target proteinases. SCCA1 has been shown to be effective against papain-like cysteine proteinases. The purpose of this study was to determine whether SCCA2 inhibited a different family of proteolytic enzymes. Using recombinant DNA techniques, we prepared a fusion protein of glutathione S-transferase and full-length SCCA2 . The recombinant SCCA2 was most effective against two chymotrypsin-like proteinases from inflammatory cells, but was ineffective against papain-like cysteine proteinases. Serpin-like inhibition was observed for both human neutrophil cathepsin G and human mast cell chymase. The second order rate constants for these associations were on the order of approximately 1 x 10(5) M-1 s-1 and approximately 3 x 10(4) M-1 s-1 for cathepsin G and mast cell chymase, respectively. Moreover, SCCA2 formed SDS-stable complexes with these proteinases at a stoichiometry of near 1:1. These data showed that SCCA2 is a novel inhibitor of two physiologically important chymotrypsin-like serine proteinases.
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PMID:Squamous cell carcinoma antigen 2 is a novel serpin that inhibits the chymotrypsin-like proteinases cathepsin G and mast cell chymase. 899 71

Soybean Bowman-Birk protease inhibitor (BBI) is an inhibitor of serine proteases with two functional inhibitory domains of different specificities: one is specific for chymotrypsin-like proteases, the other for trypsin-like proteases. Chymase and tryptase are serine proteases which are stored in mast cell granules and released upon degranulation. This work investigated the inhibition of human chymase and tryptase by BBI. Active-site titration of human skin chymase by BBI demonstrated that BBI was a highly effective inhibitor of human chymase. Virtually stoichiometric inhibition of chymase by BBI was observed at 10 nM chymase. Kinetic studies of the inhibition reaction yielded an association rate constant of 4.0 x 10(5) M(-1) s(-1) and a dissociation rate constant of 1.7 x 10(-5) s(-1). From these two constants we estimate a K(i) of 50 pM. Chymase/BBI complexes did not dissociate in SDS-PAGE analyses under nonreducing conditions, consistent with the formation of a very tight complex with little tendency to dissociate. In contrast to chymase, human tryptase was not inhibited by BBI. These studies demonstrate that BBI is a good inhibitor of human chymase, exhibiting reaction properties better than physiological inhibitors described to date.
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PMID:Soybean Bowman-Birk protease inhibitor is a highly effective inhibitor of human mast cell chymase. 924 90

Macrophage infiltration into inflammatory sites is generally preceded by neutrophils. This suggests neutrophils may be the source of chemotactic factors for monocytes. To identify these putative monocyte attractants, we have systematically prepared neutrophil granules, lysed them, and sequentially purified the released proteins by several reverse phase chromatography procedures. Assays for monocyte chemotactic activity of the chromatography fractions yielded a major peak of activity associated with a protein of 30 kD, according to SDS-PAGE analysis. NH2-terminal sequence of the protein revealed this to be identical to cathepsin G. The monocyte chemotactic activity of human cathepsin G was dose dependent with optimal concentration at 0.5-1 microg/ml. Cathepsin G is chemotactic rather than chemokinetic for monocytes, as demonstrated by checkerboard analysis. Cathepsin G-induced monocyte chemotaxis is partially pertussis toxin sensitive implying the involvement of a G protein-coupled receptor. Enzymatic activity of cathepsin G is associated with its monocyte chemotactic activity, since DFP- or PMSF-inactivated cathepsin G no longer induced monocyte migration. The chemotactic activity of cathepsin G can also be completely blocked by alpha1 antichymotrypsin, a specific inhibitor of chymotrypsin-like proteinases present in human plasma. In addition, cathepsin G is also a potent chemoattractant for neutrophils and a chemokinetic stimulant for T cells. In the course of pursuing these in vitro studies, we established that the T cell chemoattractant, azurocidin/CAP37 from human neutrophil granules, at doses of 0.05 to 5 microg/ml, was chemotactic for monocytes and neutrophils. As predicted from the in vitro chemotactic activity, subcutaneous injection of cathepsin G into BALB/c mice led to infiltration of both mononuclear cells and neutrophils. Thus, the transition of inflammatory exudate from neutrophil to mononuclear cells can be mediated, at least in part, by extracellular release of neutrophil granule proteins such as cathepsin G and azurocidin/CAP37.
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PMID:Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils. 927 89

20 and 26 S proteasomes were isolated from rat liver. The procedure developed for the 26 S proteasome resulted in greatly improved yields compared with previously published methods. A comparison of the kinetic properties of 20 and 26 S proteasomes showed significant differences in the kinetic characteristics with certain substrates and differences in the effects of a protein substrate on peptidase activity. Observed differences in the kinetics of peptidylglutamyl peptide hydrolase activity suggest that the 26 S complex cannot undergo the conformational changes of 20 S proteasomes at high concentrations of the substrate benzyloxycarbonyl (Z) -Leu-Leu-Glu-beta-naphthylamide. Various inhibitors that differentially affect the trypsin-like and chymotrypsin-like activities have been identified. Ala-Ala-Phe-chloromethyl (CH2Cl) inhibits chymotrypsin-like activity assayed with succinyl (Suc) -Leu-Leu-Val-Tyr-AMC, but surprisingly not hydrolysis of Ala-Ala-Phe-7-amido4-methylcoumarin (AMC). Tyr-Gly-Arg-CH2Cl inhibits Suc-Leu-Leu-Val-Tyr-AMC hydrolysis as well as trypsin-like activity measured with t-butoxycarbonyl (Boc) -Leu-Ser-Thr-Arg-AMC, while Z-Phe-Gly-Tyr-diazomethyl (CHN2) was found to inhibit only the two chymotrypsin-like activities. Radiolabeled forms of peptidyl chloromethane and peptidyl diazomethane inhibitors, [3H]acetyl-Ala-Ala-Phe-CH2Cl, [3H]acetyl- and radioiodinated Tyr-Gly-Arg-CH2Cl, and Z-Phe-Gly-Tyr-(125I-CHN2), have been used to identify catalytic components associated with each of the three peptidase activities. In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3, 4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by two-dimensional polyacrylamide gel electrophoresis followed by autoradiography/fluorography and immunoblotting with subunit-specific antibodies. In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in different relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities. The results strongly suggest a relationship between trypsin-like activity and chymotrypsin-like activity. They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase activities.
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PMID:Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities. 931 91

Prostate specific antigen (uPSA) was purified to homogeneity from human urine using SuperQ-Toyopearl, Sulfate-Cellulofine, Phenyl-Toyopearl, CM-Sepharose, anti-urokinase IgG Sepharose and Sephadex G-100. The purified uPSA gave a major band at 32.9 kDa on SDS-PAGE under the reduced condition. However, it shows multiple bands on native PAGE. Substrate specificity of purified uPSA is identical with that of PSA from human seminal plasma and uPSA shows the kallikrein and chymotrypsin-like activities. On the analysis of N-terminal amino acid, two amino acid residues at N-terminal position of uPSA were detected and other amino acid sequence of uPSA was identical with that of sPSA. In addition, we isolated the multiple components of uPSA using anion-exchange chromatography. They were almost the same in amino acid composition and N-terminal amino acid sequences and showed differences in lectin-blotting pattern.
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PMID:Purification and characterization of prostate specific antigen from human urine. 936 70

Proteolytic degradation of plasma vitellogenins during purification procedure has been noted in several teleost fishes. We have characterized here a trypsin-like serine protease in the plasma of the tilapia, Oreochromis niloticus, which degrades vitellogenins. The molecular mass of the protease was estimated as 230 kDa by gel filtration and as 170 kDa both by nondenaturing and by SDS-polyacrylamide gel electrophoresis. The protease efficiently hydrolyzed the synthetic peptide substrates for trypsin-like proteases but not the substrates for chymotrypsin-like proteases nor aminopeptidases. Hydrolysis of the peptide substrates was strongly inhibited by leupeptin, aprotinin and N-tosyl-L-lysine chloromethyl ketone and to certain extent by chymostatin, 3,4-dichloroisocoumarin, phenylmethanesulfonyl fluoride, and soybean trypsin inhibitor. Leupeptin and aprotinin also inhibited the degradation of a vitellogenin in the plasma. Although the physiological functions of the 170 kDa protease in vivo have not been elucidated, the results on exzymatic properties of this protease will be useful for the isolation and characterization of vitellogenin not only in tilapia but also in other organisms.
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PMID:Degradation of vitellogenins by 170 kDa trypsin-like protease in the plasma of the tilapia, Oreochromis niloticus. 941 96

An anionic chymotrypsin-like enzyme was isolated from a crude extract of camel pancreas by a three-step procedure consisting of anion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The purified enzyme was homogeneous on native and SDS gel electrophoresis and on gel isoelectric focusing. Its molecular mass was estimated as 28.5 kDa and its isoelectric point was found to be 4.4. The enzyme differed markedly from bovine chymotrypsin A in its substrate specificity, showing considerably lower values of the specificity constant for its action on tyrosine, tryptophan, and phenylalanine esters. Its pH optimum was found to be 7.8. It showed lower kininase activity and was more susceptible to inhibition by a number of inhibitors than the bovine cationic chymotrypsin. On the other hand, the camel enzyme showed a much greater hydrolytic activity than the bovine enzyme toward a leucine ester. In terms of its size, charge, and substrate specificity the camel enzyme was very similar to anionic chymotrypsins that have been isolated from other species and thus appears to be a camel anionic chymotrypsin.
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PMID:Purification and partial characterization of camel anionic chymotrypsin. 943 49


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