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:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An artificially inserted extra peptide (21 amino acid peptide) between the B. subtilis alpha-amylase signal peptide and the mature thermostable alpha-amylase was completely cleaved by B. subtilis alkaline protease in vitro. The cleavage to form a mature enzyme was observed between pH 7.5 and 10, but not between pH 6.0 and 6.5, although a similar protease activity toward Azocall was observed between pH 6.0 and 7.5. To analyze the effects of pH on the cleavage, CD spectra at pH 6, 8, and 11 of the NH2-terminally extended thermostable alpha-amylase were analyzed and the results were compared with those of the mature form of the alpha-amylase. It is suggested that the cleavage of the NH2-terminally extended peptide is controlled by the secondary and tertiary structure of the precursor enzyme. Similar cleavage of different NH2-terminally extended peptides by the alkaline protease was also found in other hybrid thermostable alpha-amylases obtained.
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PMID:Maturation of an NH2-terminally extended thermostable alpha-amylase in Bacillus subtilis: a possible mechanism examined by in vitro experiments. 136 50

In vivo most extracellular iron is bound to transferrin or lactoferrin in such a way as to be unable to catalyze the formation of hydroxyl radical from superoxide (.O2-) and hydrogen peroxide (H2O2). At sites of Pseudomonas aeruginosa infection bacterial and neutrophil products could possibly modify transferrin and/or lactoferrin forming catalytic iron complexes. To examine this possibility, diferrictransferrin and diferriclactoferrin which had been incubated with pseudomonas elastase, pseudomonas alkaline protease, human neutrophil elastase, trypsin, or the myeloperoxidase product HOCl were added to a hypoxanthine/xanthine oxidase .O2-/H2O2 generating system. Hydroxyl radical formation was only detected with pseudomonas elastase treated diferrictransferrin and, to a much lesser extent, diferriclactoferrin. This effect was enhanced by the combination of pseudomonas elastase with other proteases, most prominently neutrophil elastase. Addition of pseudomonas elastase-treated diferrictransferrin to stimulated neutrophils also resulted in hydroxyl radical generation. Incubation of pseudomonas elastase with transferrin which had been selectively iron loaded at either the NH2- or COOH-terminal binding site yielded iron chelates with similar efficacy for hydroxyl radical catalysis. Pseudomonas elastase and HOCl treatment also decreased the ability of apotransferrin to inhibit hydroxyl radical formation by a Fe-NTA supplemented hypoxanthine/xanthine oxidase system. However, apotransferrin could be protected from the effects of HOCl if bicarbonate anion was present during the incubation. Apolactoferrin inhibition of hydroxyl radical generation was unaffected by any of the four proteases or HOCl. Alteration of transferrin by enzymes and oxidants present at sites of pseudomonas and other bacterial infections may increase the potential for local hydroxyl radical generation thereby contributing to tissue injury.
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PMID:Pseudomonas and neutrophil products modify transferrin and lactoferrin to create conditions that favor hydroxyl radical formation. 165 25

The present study was undertaken to examine and compare the direct effect of two Pseudomonas enzymes, elastase and alkaline protease, on the serum hemolytic complement as a whole, and on the two recognition molecules of complement, C1q and C3 in particular. The results of our study show that incubation of serum with 0-50 micrograms/ml elastase or protease (60 min, 37 degrees C) resulted in a dose-dependent depletion of hemolytic complement with the protease being 3-4 times more efficient than elastase. Incubation of highly purified C3 (20 hr, 37 degrees C) with protease (2% w/w) resulted in the conversion of the 190-kDa molecule to a 120-kDa fragment. When analyzed by SDS-PAGE under reducing conditions, the 120-kDa piece yielded three distinct bands: an intact 75-kDa beta-chain and two alpha-chain pieces of approximately 41- and 26-kDa. NH2-terminal end sequence analysis localized the 26-kDa fragment within the cysteine-rich 41-kDa, COOH-terminal piece. This in turn suggests that the 70-kDa fragment which is not accounted for on SDS-PAGE is derived from the NH2-terminal end of the alpha-chain molecule which is completely degraded into small fragments. While the degradation pattern obtained with elastase is similar to that of protease, the latter enzyme was found to be more efficient. Exposure of C1q (0-5 hr, 37 degrees C) to protease or elastase on the other hand appears to reveal preferential sensitivity of the 28-kDa A-chain and 24-kDa C-chain, of the C1q molecule, with the protease being more potent than the elastase. Since both C1q and physiologic fragments of C3 (C3b, iC3b, and C3dg) are important opsonins of varying efficiencies, degradation of these molecules by Pseudomonas enzymes may, in part, facilitate the survival and proliferation of the organism in plasma. Furthermore, degradation of the key recognition molecules of complement, C1q and C3, would enhance the virulence of this organism by aborting complement-mediated bacterial killing. In addition the results imply that during Pseudomonas bacteremia, PaAP may be a much more destructive enzyme than PaE with regards to C3 and C1q but combined, the synergistic effect may overwhelm not only the proteins of the complement system, but other proteins of the humoral immune defense system as well.
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PMID:Effect of Pseudomonas aeruginosa elastase and alkaline protease on serum complement and isolated components C1q and C3. 173 Jan 52

Pseudomonas aeruginosa elastase is a zinc metalloproteinase which is released during P. aeruginosa infections. Pseudomonas keratitis, which occurs following contact lens-induced corneal trauma, can lead to rapid, liquefactive necrosis of the cornea. This destruction has been attributed to the release of both host-derived enzymes and the bacterial products P. aeruginosa elastase, alkaline protease, exotoxin A, and lipopolysaccharide endotoxin. A synthetic metalloproteinase inhibitor, HSCH2 (DL)CH[CH2CH(CH3)2]CO-Phe-Ala-NH2, which we previously showed to be a potent inhibitor of corneal collagenase and alkali-induced corneal ulceration, was tested as a potential inhibitor of P. aeruginosa elastase. Inhibition constants (Kis) for the resolved diastereomers were determined with the chromogenic substrate furylacryloyl-glycyl-L-leucyl-L-alanine. One isomer had a Ki of 0.3 microM, while the other had a Ki of 0.4 microM. The more potent diastereomer was evaluated in vivo in experimentally induced Pseudomonas keratitis in rabbits. Following inoculation of one cornea of each rabbit, topical treatment with a 1 mM solution of the inhibitor significantly delayed the onset of corneal melting and perforation, as compared with the results for the control and gentamicin-treated groups. This protective effect suggests that the inhibitor may have a therapeutic application by delaying the progression of corneal destruction in Pseudomonas keratitis.
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PMID:Inhibition of Pseudomonas aeruginosa elastase and Pseudomonas keratitis using a thiol-based peptide. 212 41

To analyze the processing of extracellular enzymes of Bacillus subtilis, an NH2-terminally extended hybrid alpha-amylase [pTUBE638-alpha-amylase (E24)] was purified from the periplasm of E. coli(pTUBE638) as the substrate for the in vitro processing reaction, in which a 21-amino-acid extra-peptide was added at the NH2-terminus of the mature thermostable alpha-amylase. The extended peptide in pTUBE638-alpha-amylase (E24) was completely processed by the extracellular alkaline protease of B. subtilis alone at pH 7.5 to 10.0. The processing was inhibited by 2 mM PMSF. In contrast, the neutral protease did not process the extended peptide. The processing activity of the purified alkaline protease was fully active in 100 mM phosphate and glycine-NaCl-NaOH buffer while it was partially active in 100 mM Tris-HCl or MOPS buffer. The optimum pH of the activity ranged from 8.0 to 9.0, although the optimum pH of the alkaline protease activity toward casein and Azocoll was 10.5. The NH2-terminal amino acid sequences of the enzymes processed in vitro coincided with those of the mature extracellular thermostable alpha-amylases in the culture medium of B. subtilis (pTUBE638). The appearance of the processing activity of alkaline protease was correlated with the changes of the pH in the culture medium.
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PMID:Processing of an NH2-terminally extended thermostable alpha-amylase by Bacillus subtilis alkaline protease. 212 90

Rat liver proteasome (multicatalytic proteinase complex) is a 20S-ring shaped particle having a molecular mass of 750 kDa, and is composed of at least 13 non-identical components ranging from 21 to 31 kDa in size. We found here that the NH2-terminal residues of all the known 13 components, except for C5, are not reactive to phenylisothiocyanate. Among them, components C2, C3 and C8 are blocked in their NH2-termini with N alpha-acetyl-Met, N alpha-acetyl-Ala, and N alpha-acetyl-Ser, respectively. The NH2-terminal portions of C2, C3, and C8 exhibit sequence similarity to one another, but that of the non-blocked component C5 differs from those of C2, C3, and C8.
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PMID:The NH2-terminal residues of rat liver proteasome (multicatalytic proteinase complex) subunits, C2, C3 and C8, are N alpha-acetylated. 233 42

A multicatalytic proteinase complex present in the skin secretion of Xenopus laevis was purified and its enzymatic activity towards natural and synthetic peptides was investigated. We identified three activities: i) a C-terminal deamidation enzyme activity which exhibited selectivity for the Asp-Phe-NH2 and Phe-Leu-NH2 motifs of cerulein, minigastrin Leu-enkephalinamide, (des-Tyr1)Leu-enkephalinamide and diaminobenzylthiocyanate-DVDERDVRGFASFLNH2 (DABTC-DR8kermit); ii) an endopeptidase activity that cleaves peptide bonds on the carboxyl side of hydrophobic amino acid residues such as Tyr-Gly of LHRH, Ile-Ala of PGLa and Leu-Ala of buccalin; iii) an enzyme activity that cleaves peptide bonds at the dibasic sites of peptides of the dynorphin family. The molecular weight determined by Sephacryl S-400 molecular sieve filtration indicated an M(r) about 600 kDa. The activities characterized here exhibit an optimal pH of about 7.4. The activities of the multicatalytic complex were differentially inhibited by the classical inhibitors of proteases.
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PMID:Isolation and properties of a multicatalytic proteinase complex from Xenopus laevis skin secretion. 755 6

Human cells express cell surface complement regulatory molecules that inhibit the activity of the C3/C5 convertases (DAF, MCP, CR1) or inhibit the membrane attack complex (CD59). A single molecule that inhibits both the convertase activity and formation of the membrane attack complex has never been characterized. To this end, we have developed two reciprocal chimeric complement inhibitors (CD, NH2-CD59-DAF-GPI; and DC, NH2-DAF-CD59-GPI) that contain the functional domains of decay accelerating factor (DAF; CD55) and CD59. Cell surface expression of the CD and DC chimeric proteins was detected with DAF- and CD59-specific antisera. Cell surface C3d deposition was inhibited on cells expressing the chimeric molecules, thereby indicating that the DAF moiety was functional in both molecules. Conversely, Ab-blocking experiments demonstrated that only the DC molecule retained CD59 function. Therefore, the DC molecule represents a novel potent chimeric bifunctional complement inhibitor that retains the functional domains of two distinct complement regulatory molecules.
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PMID:A novel bifunctional chimeric complement inhibitor that regulates C3 convertase and formation of the membrane attack complex. 759 66

Six basic proteins of 26 to 38 kDa with isoelectric points (pI) > or = 8.5 were abundant in proteins separated by two-dimensional SDS-PAGE from adult rat peritoneal mast cells (MC). One was identified previously as rat mast cell proteinase (RMCP) 1, a chymase of 26 to 28 kDa, pI > 9.0. Microsequence analyses showed that two polypeptides of about 29 and 30 kDa had NH2 terminal amino acid sequences homologous to mouse MC proteinase 5 (MCP-5), whereas the amino terminals of the 33, 35, and 36 kDa proteins were homologous to MC carboxypeptidase A (MC-CPA). Rabbit Abs produced against synthetic peptides of the identified NH2 terminal sequences were used in immunoblot studies. At least three proteins reacted with Abs to MC-CPA, whereas Abs to MCP-5 detected three adjacent polypeptides, rather than just the two identified by using microsequence analysis. Removal of oligosaccharide side chains using peptide:N-glycosidase F reduced the heterogeneity of each set of three polypeptides (MCP-5 and MC-CPA) to a band of each protein of a lower M(r). The serine proteinase inhibitor [3H]diisopropylfluorophosphate ([3H]DFP) bound to a proteinase of 30 to 35 kDa, which is probably MC tryptase (pI < or = 6.0). Immunoblot analysis of proteins from intestinal mucosal mast cells showed RMCP-2, but not RMCP-1, MCP-5, or MC-CPA. This is the first report of MCP-5 in the rat and of clearly distinguishable glycosylated forms of MC CPA. These proteinases appear to be restricted in their distribution to selected MC populations, but little is known about their functions.
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PMID:Proteinases of rat mast cells. Peritoneal but not intestinal mucosal mast cells express mast cell proteinase 5 and carboxypeptidase A. 759 1

Thermostable alkaline protease from an alkaliphilic thermophile Bacillus sp. B18' was purified by using DEAE- and CM-Toyopearl 650M column chromatographies. Molecular weights of the enzyme determined by SDS-PAGE and gel filtration were 30,000 and 28,000, respectively. The optimum pH and temperature toward the hydrolysis of casein were pH 12-13 and 85 degrees C, both of which are higher than those of a mesophilic alkaline protease from an alkaliphile, Bacillus sp. B21-2. The enzyme was stable at pH 5.0-12.0 and about 60% of the initial enzymatic activity was retained after a 60 min incubation period at pH 10.0 and 70 degrees C. Thermostability of the enzyme was enhanced by Ca2+. The enzyme activity was inhibited by DFP, suggesting that the enzyme is a serine protease. The NH2-terminal amino acid is Gln, which is that of many subtilisin-type proteases. The 20 residues of the NH2-terminal amino acid sequence have a comparative high homology with those of other alkaline proteases from alkaliphiles (40-50%), especially thermostable alkaline protease from Bacillus sp. No. AH-101 (95%) and Thermoactinomyces sp. HS682 (95%).
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PMID:Purification and properties of the highly thermostable alkaline protease from an alkaliphilic and thermophilic Bacillus sp. 776 36


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