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)

Four natural protease inhibitors have been partially purified by heat treatment, ion-exchange chromatography pand gel filtration from Neurospora crassa. The inhibitory activity has been estimated by measuring the inhibition of proteolysis of casein as well as by the protection of Neurospora tryptophan synthase from proteolytic inactivation. The inhibitors are all oligopeptides and possess molecular weights in the range 5000-24 000 and appear to be very specific to Neurospora proteases. They may be classified into two types. The first are specific to Neurospora alkaline protease and the second to acidic protease. None of them exhibited any effect on other proteases including trypsin, chymotrypsin, papain, pepsin, thermolysin, subtilisin and proteinase K. The possible physiological role of these inhibitors is discussed.
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PMID:Isolation of specific protease inhibitors from Neurospora crassa. 13 53

Two metalloendopeptidases, designated as Streptomyces griseus metalloendopeptidases I and II (SGMPI and SGMPII), were isolated from a commercial Pronase P by a method including affinity chromatography on carbobenzoxy-L-alaninyl-triethylenetetraminyl-Sepharose (Z-Ala-T-Sepharose). The two enzymes differed from each other in behavior on ion-exchange chromatography but showed the same amino-terminal sequence at least up to the 20th residue. Their molecular weights were both estimated to be 37,000 by SDS-polyacrylamide gel electrophoresis. Elemental and amino acid composition analyses indicated that both of them contained about 1 g atom of zinc and one cystine residue per mol of protein. Cleavage specificities of the two enzymes toward synthetic peptide-substrates were very similar to those observed with thermolysin. EDTA, o-phenanthroline, and phosphoramidon strongly inhibited these enzymes, while typical serine-protease inhibitors and cysteine-protease inhibitors had no effect. The findings clearly indicate that SGMPI and SGMPII can be classified into the family of zinc-endopeptidases. It was unexpectedly found, however, that these metalloendopeptidases were strongly inhibited by protein serine-protease inhibitors produced by Streptomycetes, such as Streptomyces subtilisin inhibitor (SSI), alkaline protease inhibitor-2c' (API-2c'), and plasminostreptin (PS).
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PMID:Purification and characterization of Streptomyces griseus metalloendopeptidases I and II. 176 59

The amino acid sequence of the neutral zinc protease from Bacillus mesentericus strain 76 (MCP 76) has been determined by using peptides derived from digests with trypsin, chymotrypsin, and cyanogen bromide and from cleavage with o-iodosobenzoic acid. The peptides were purified by means of gel filtration and reversed-phase high-performance liquid chromatography and analyzed by automatic sequencing. The protein contains 300 amino acid residues. It proved to be identical with the neutral protease deduced from the DNA precursor sequence of Bacillus subtilis. The residues for zinc and substrate binding are conserved, whereas the number of calcium binding sites is reduced compared to thermolysin. A classification of the neutral zinc protease is discussed.
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PMID:Primary structure of a zinc protease from Bacillus mesentericus strain 76. 230 86

The proteolytic specificity of the neutral zinc proteinase from Bacillus mesentericus strain 76 (MCP 76)/Bacillus subtilis was determined by using the alpha-chain of walrus hemoglobin as substrate. The resulting peptides were fractionated by gel filtration and than isolated by reversed-phase HPLC. The peptides were identified on the basis of their amino-acid compositions and aligned with the known sequence of the walrus alpha-chain. The proteolytic specificity of MCP 76, deduced from the experimental cleavage pattern is compared to that of thermolysin. The amino-acid residues in positions P1 and P'1 on both sides of the scissible bond are considered as most important for the cleavage. MCP 76 prefers leucine, valine, phenylalanine and threonine in position P'1 as well as lysine, threonine, leucine and alanine in position P1 and thus differs from thermolysin which shows no preference for threonine in P'1 and accepts numerous amino-acid residues of different type in P1.
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PMID:Proteolytic specificity of the neutral zinc proteinase from Bacillus mesentericus strain 76 determined by digestion of an alpha-globin chain. 251 21

Pseudomonas toxin is produced as a proenzyme which is cytotoxic for cells in culture but must be activated to express full enzymatic activity. The ability of purified pseudomonas alkaline protease and elastase or of culture filtrates of two strains of Pseudomonas aeruginosa to modify the activity of pseudomonas toxin was examined. Two parameters of toxin activity were followed: enzymatic activity, i.e., the adenosine diphosphate (ADP) ribosylation of elongation factor 2, and biological activity, i.e., inhibition of protein synthesis in cultured mouse fibroblasts. Biological activity of toxin depends upon an intact toxin molecule, whereas enzyme activity requires only a functional A region. Incubation with purified pseudomonas proteolytic enzymes did not alter either enzymatic or biological activity. The toxin is not refractory to the action of all proteolytic enzymes, since thermolysin rapidly destroyed the toxin molecule. Treatment of toxin with culture filtrates of P. aeruginosa reduced ADP ribosylation activity, but increased the ability of toxin to inhibit protein synthesis in cell monolayers. Incubation of culture filtrates with one of the protease inhibitors alpha-2-macroglobulin or phosphoramidon did not alter the effect of the filtrates on biological activity. Alpha-2-macroglobulin, however, caused a fourfold stimulation of ADP ribosylation activity of the toxin. We conclude that pseudomonas alkaline protease and elastase are not responsible for the modifications in toxin activity induced by culture filtrates of P. aeruginosa; the factors responsible have not yet been identified, but are not inactivated by phosphoramidon or alpha-2-macroglobulin.
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PMID:Resistance of exotoxin A to purified Pseudomonas proteolytic enzymes. 615 7

Tetanus and botulinum neurotoxins constitute a new group of Zn-endopeptidases which has been recently actively investigated with the purpose of correlating their biochemical properties to their neurobiocytosis inhibitory capacity. Crystallographic data show that Zn-endopeptidases are characterized by an active site with a Zn atom coordinated to two histidines and glutamate-bound water molecule. The two histidines and glutamate resides belong to the HEXXH motif which is characteristic of most Zn-endopeptidases. A forth metal ligand is a glutamate in thermolysin-like proteinases, but it is an histidine in the astacin family of proteinases and in alkaline protease. Astacin and alkaline protease possess a tyrosine as fifth Zn ligand, whose position in the case of alkaline protease could not be determined by X-ray crystallography. Not much is known about the atom arrangement around the active site in tetanus neurotoxin. In this work X-ray absorption spectroscopy has been used to obtain information on the Zn coordination mode in tetanus neurotoxin. The near-edge and extended fine-structure absorption spectra of this toxin are compared with those of astacin, alkaline protease and thermolysin. The present data and sequence information suggest a new pattern of Zn coordination in tetanus neurotoxin with one water molecule and three aromatic residues as metal ligands. These residues are the two histidines of the characteristic motif and a tyrosine which is tentatively identified with Tyr242, on the basis of sequence comparison and mutagenesis experiments. The mean distances of the Zn from the nearest coordinated atoms is reported. Our results indicate that alkaline protease, like astacin, also possesses a tyrosine as a fifth ligand.
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PMID:X-ray absorption spectroscopy study of zinc coordination in tetanus neurotoxin, astacin, alkaline protease and thermolysin. 865 8

Pseudomonas aeruginosa is a zinc metalloprotease which may be involved in many infection processes, especially in the lung. In order to evaluate the production of the enzyme in culture supernatants, we developed an assay using peptide derivatives; the conductimetric method was used for monitoring the enzymatic activities. Tetrapeptide derivatives were enzymatically synthesized by coupling Z-Ala2 and X-AlaR using either thermolysin or P. aeruginosa elastase itself. In these substrates, X could be phenylalanine, tyrosine, or leucine and C-protection was performed by either an amide (NH2) or a methyl (OMe) group. Z-Ala2-Phe-AlaNH2 was found to be the best substrate, giving a catalytic ratio kcat/KM of 8600 mM-1.s-1. The evaluation of the alkaline protease activity with this substrate showed that the catalytic ratio is 1000-fold lower. The sensitivity of the conductimetric method was also demonstrated with as little as 1 nM elastase (0.13 microgram), being easily and accurately detected (SD, 3.8% for 10 measurements). Furthermore, the enzymatic activity was measured in a culture supernatant from a clinical strain.
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PMID:Synthetic peptide substrates for a conductimetric assay of Pseudomonas aeruginosa elastase. 866 May 69

Two monoclonal antibodies (MAbs) to a 36-kDa extracellular metalloprotease (PSCP) from Burkholderia (Pseudomonas) cepacia were found to react with thermolysin, Pseudomonas aeruginosa elastase, alkaline protease (Apr) and LasA, Serratia marcescens protease (SMP), Aeromonas hydrophila protease (AhP), and both the lethal factor (LF) and protective antigen (PA) of Bacillus anthracis on immunoblots. The MAbs were capable of neutralising the proteolytic activity of thermolysin, P. aeruginosa elastase and PSCP but not that of Apr, SMP, and AhP. These results suggest that these MAbs may be able to differentiate between the thermolysin and serralysin family of metalloproteases on the basis of their neutralisation capability and could, therefore, be useful tools in the characterisation of new bacterial proteases.
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PMID:Differentiation of thermolysins and serralysins by monoclonal antibodies. 881 Sep 50

Monoclonal antibodies (MAbs) to a Burkholderia (Pseudomonas) cepacia 36-kDa protease (PSCP) which neutralize PSCP and Pseudomonas aeruginosa elastase but not P. aeruginosa alkaline protease have been isolated (C. Kooi et al., Infect. Immun. 62:2811-2817, 1994). These MAbs, designated 36-6-6 and 36-6-8, react with N-chlorosuccinimide cleavage products of P. aeruginosa elastase, consistent with the recognition of a 13.9-kDa fragment which contains the active site. Overlapping 9-mer peptides that span this region were synthesized. Neutralizing MAbs to PSCP reacted strongly with two peptides (341HGFTEQNSG349 and 395RYM DQPSRD403). Peptide 341HGFTEQNSG349 overlaps the motif 337HEXXH341, which has been found in many zinc-dependent endopeptidases. Peptide 395RYMDQPSRD403 lies between E361, which binds a zinc atom, and H420, which acts as a proton donor at the active site. Polyclonal rabbit sera raised against these peptides reacted with elastase on Western immunoblots and by enzyme-linked immunosorbent assay. With hide powder azure as the substrate, antisera to either HGFTEQNG and RYMDQPSRD completely neutralized the activities of elastase, thermolysin, Vibrio cholerae hemagglutinin/protease, and PSCP but had no effect on P. aeruginosa alkaline protease or the Serratia marcescens major protease. These results suggest that the MAbs recognize two different epitopes on P. aeruginosa elastase and that antibodies raised against synthetic peptides corresponding to either of these epitopes neutralize proteolytic activity.
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PMID:Identification of neutralizing epitopes on Pseudomonas aeruginosa elastase and effects of cross-reactions on other thermolysin-like proteases. 900 99

The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif in Rpn11 and Csn5 underlies isopeptidase activities intrinsic to the proteasome and signalosome, respectively. We show here that the archaebacterial protein AfJAMM possesses the key features of a zinc metalloprotease, yet with a distinct fold. The histidine and aspartic acid of the conserved EX(n)HS/THX(7)SXXD motif coordinate a zinc, whereas the glutamic acid hydrogen-bonds an aqua ligand. By analogy to the active site of thermolysin, we predict that the glutamic acid serves as an acid-base catalyst and the second serine stabilizes a tetrahedral intermediate. Mutagenesis of Csn5 confirms these residues are required for Nedd8 isopeptidase activity. The active site-like architecture specified by the JAMM motif motivates structure-based approaches to the study of JAMM domain proteins and the development of therapeutic proteasome and signalosome inhibitors.
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PMID:JAMM: a metalloprotease-like zinc site in the proteasome and signalosome. 1473 82


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