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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)

The multicatalytic endopeptidase complex (20S proteasome) is a latent high-molecular-mass multisubunit proteinase. In many investigations, SDS has been used as a proteasome activator at some fixed concentration that was apparently optimal. This study examined the effects of various divalent cations on the SDS-dependent peptidase and casein degradation activities of 20S proteasome purified from Xenopus laevis oocytes at a series of SDS concentrations and the correlation between these effects and the critical micelle concentration (CMC) of SDS. Surprisingly, it was found that divalent cations such as Mg2+ markedly shifted the SDS-dependent activation profiles to a lower concentration range. Ca2+, Mn2+, Co2+, and Zn2+ also markedly reduced the optimum SDS concentration in the Suc-Leu-Leu-Val-Tyr-MCA hydrolysis reaction: for example, 5 mM Co2+ reduced the optimum SDS concentration from 0.065 to 0.005%. However, in all cases examined the optimum concentrations were below the CMC. Cu2+, Hg2+, and Cd2+ strongly inhibited the SDS-dependent maximum activity without remarkably shifting the optimum SDS concentration. No correlation between the shift and the inhibition was recognized. Most interestingly, remarkable activation of casein degradation by SDS was observed only by addition of the divalent cations Mg2+, Ca2+, and Mn2+. These cations might be essential for casein degradation. The activation and inactivation ranges of SDS concentration varied with the species of substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reaction of 20S proteasome: shift of SDS-dependent activation profile by divalent cations. 749 Feb 55

High-alkaline protease (HAP) has been entrapped in Manugel DMB (an alginate gel) and assayed with two sizes and types of substrates: neutral protein casein and synthetic chromogenic tripeptide substrate, Z-Gly-Pro-Cit-PNA. Increasing the concentration of calcium chloride used for capsule formation decreased the measured enzyme activity with both substrates. Capsules were found to be stable in water for long periods of time, but they dissolved in both phosphate and carbonate-bicarbonate buffers. The pH vs activity profiles of encapsulated enzyme showed pH optima between 10 and 11 with both substrates. The calcium alginate matrix surrounding the enzyme was quite effective in stabilizing the enzyme at 20-25 degrees C and even more so at 4 degrees C. Enzyme stability at 50 degrees C was quite impressive, some enzyme activity being evident even after remaining for 1 wk at this temperature in water. Increasing concentrations of sodium dodecyl sulfate (SDS) were also found to inhibit the protease progressively, whereas a polyhexamethylene biguanidium chloride (PHMBH+Cl-) and SDS:PHMBH+Cl- combination showed the opposite effect. Optical microscopy, especially polarized light microscopy, provided a sensitive physical means of ascertaining some of the structural properties (sphericity, disorganization or organization, distinct layer enveloping the capsules, intensity of the maltese cross) of the capsules with and without enzyme before and after different chemical treatments and the presence or absence of the substrate.
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PMID:High-alkaline protease from Bacillus PB92 entrapped in calcium alginate gel. Physicochemical and microscopic studies. 749 34

A protein kinase phosphorylating the 45-kDa proteasome subunit was co-purified with the 26 S proteasome from the porcine heart. This kinase appears to be associated with the 26 S proteasome, since the kinase activity was co-eluted with the 26 S proteasome on Superose 6 FPLC and immunoprecipitated with anti-20 S proteasome antibody. This kinase also phosphorylated the casein. Furthermore, the phosphorylated casein was more efficiently hydrolyzed by the 26 S proteasome than the dephosphorylated casein without ATP. Inhibition patterns of kinase inhibitors against the 45 kDa subunit and casein were well in accord with the inhibition pattern against the ATP-dependent proteolysis of the 26 S proteasome, suggesting that the phosphorylation of casein by a protein kinase associated with the 26 S proteasome is linked to the ATP-dependent proteolysis of the 26 S proteasome.
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PMID:Phosphorylation of proteasome substrate by a protein kinase associated with the 26 S proteasome is linked to the ATP-dependent proteolysis of the 26 S proteasome. 763 64

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

Two high molecular mass proteinases, multicatalytic proteinase (MCP) and a new high molecular mass proteinase (HMP) with only chymotrypsin-like activity (Khan et al. (1994) J. Biol. Chem. 269, 10016-10021) from human erythrocyte membranes, have been compared. For this purpose, MCP was purified from human erythrocyte membranes in the active form towards synthetic peptide substrates; it also hydrolysed the protein substrates [14]methyl casein and [14C]oxidised insulin beta chain at 37 degrees C. MCP from plasma membranes exhibited hollow cylindrical structures also typical of cytosolic forms. Radiolabelled diisopropyl fluorophosphate, [3H]DFP, a serine proteinase inhibitor, labelled a band of Mr 23 000 in membrane MCP. By contrast, no labelling was obtained with HMP. Chymotrypsin-like activity of HMP was also found to be insensitive to DFP. On the other hand, DFP inhibited chymotrypsin-like and peptidylglutamyl peptide hydrolysing activities of membrane MCP, with no effect on its trypsin-like activity. The inhibition of MCP by DFP was concentration-dependent. These studies showed that MCP and HMP represent two distinct kinds of proteinases with chymotrypsin-like activities and can be distinguished by the serine proteinase inhibitor DFP.
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PMID:Membrane-bound high molecular mass proteinases from human erythrocytes. 781 93

The multicatalytic proteinase (MCP or proteasome) is a large proteolytic complex that contains at least five catalytic components: the trypsin-like, chymotrypsin-like, peptidylglutamyl-peptide hydrolase (PGPH), branched-chain-amino-acid-preferring (BrAAP) and small-neutral-amino-acid-preferring activities. We have shown that brief heating of the lobster muscle proteasome activates a proteolytic activity that degrades casein and myofibrillar proteins and is distinct from the trypsin-like, chymotrypsin-like and PGPH components. Here we identify the BrAAP activity as a catalytic component involved in the initial degradation of myofibrillar proteins in vitro. This conclusion is based on the following. (1) The BrAAP component was activated by heat-treatment, whereas the other four peptidase activities were not. (2) The BrAAP and proteolytic activities showed similar sensitivities to cations and protease inhibitors: both were inhibited by 3,4-dichloroisocoumarin, chymostatin, N-ethylmaleimide and Mg2+, but were not affected by leupeptin, phenylmethanesulphonyl fluoride or Li+. (3) The BrAAP activity was inhibited most strongly by casein substrates and troponin; conversely, the troponin-degrading activity was inhibited by the BrAAP substrate. Another significant finding was that incubation of the heat-activated MCP in the presence of chymostatin resulted in the limited cleavage of troponin-T2 (45 kDa) to two fragments of 41 and 42 kDa; this cleavage was completely suppressed by leupeptin. These results suggest that under certain conditions the trypsin-like component can cleave endogenous protein.
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PMID:Branched-chain-amino-acid-preferring peptidase activity of the lobster multicatalytic proteinase (proteasome) and the degradation of myofibrillar proteins. 786 22

The subunit patterns of the proteasomes, that were purified from muscle, liver and brain, were found to be significantly different from one another. Furthermore, the proteasomes from adult and embryonic tissues of the same types also differed from each other in their subunit patterns. In addition, the specific activities of the purified proteasomes for peptide-cleavage, but not for casein-hydrolysis, appeared to be varied among the enzymes isolated from the different tissues. Thus, expression of a large number of proteasome subunits appears to be tissue-specific and under developmental control, although its relation with the multicatalytic activities of the proteasomes remains unclear.
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PMID:Tissue-specific expression of the subunits of chick 20S proteasomes. 803 22

We studied 5 strains of Pseudomonas fluorescens, its ability to produce proteolytic enzymes and the antigenic relatedness between P. fluorescens and P. aeruginosa proteases. Cells were grown in tryptic soy broth plus 2% skim milk powder, at 4 C during 5 days. All the proteases acted on gelatin, casein, and showed limited activity on congo redelastin. By zymograms in polyacrylamide gel (PAA), one enzyme responsible of whole enzymatic activity was shown. The extracellular protease of the strain P. fluorescens ATCC 17400 was purified by ammonium sulfate precipitation (60% saturation) and chromatography on DEAE cellulose with ionic strength gradient, and Sephadex G 100. A 181 fold increase in specific activity with a recovery of 21% was obtained. PAA-sodium dodecyl sulfate revealed a single band with a molecular weight of approximately 45,700 +/- 1,000 Daltons. P. fluorescens antiprotease rabbit serum showed by immunodiffusion (ID) and countercurrent immunoelectrophoresis (CIEF) identity pattern of reaction with the homology strains studied. Rabbit sera antielastase and anti-alkaline protease of P. aeruginosa did not exhibit by ID, CIEF and immunoblotting immunological reactivity with antigen (protease) from P. fluorescens; by enzyme linked immunosorbent assay (ELISA), P. aeruginosa antielastase rabbit serum showed a weak response with P. fluorescens protease. These preliminary observations showed analogy in enzymatic functions, such as specificity, between the enzymes produced by phylogenetically related species, but the immunological studies showed very little interspecific homology.
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PMID:[Proteases from Pseudomonas: immunologic comparison]. 814 Mar 33

A ubiquitin (Ub)/ATP-dependent proteolytic complex (26S proteasome) purified from rabbit skeletal muscle was dissociated into two subcomplexes, a 20S proteasome and a regulatory subunit complex, by preparative non-denaturing polyacrylamide gel electrophoresis (PAGE). The isolated regulatory subunit complex preparation gave a single broad band on analytical non-denaturing PAGE, and several bands ranging between 33 and 110 kDa on SDS-PAGE. This complex was found to consist of about 20 subunits on the basis of two-dimensional PAGE, the pattern of which appeared identical or very similar to that of the 33-110 kDa 26S proteasome subunits. The apparent molecular mass of the complex was estimated to be 1100 kDa by Ferguson plot analysis and also by Superose 6 gel filtration. Unlike the 26S proteasome, neither ATPase activity nor protease activities toward Suc-Leu-Leu-Val-Tyr-MCA, Boc-Phe-Ser-Arg-MCA, Z-Leu-Leu-Glu-beta NA, [14C]-casein, [125I]-lysozyme and Ub-[125I]-lysozyme were significantly detectable in the regulatory subunit complex. This complex was found to be capable of associating with itself in MgATP-dependent manner. These results suggest that a regulatory subunit complex dissociated from the 26S proteasome comprises all the higher molecular mass subunits of the 26S proteasome, and has no detectable ATPase and protease activities, although the homo-oligomerization occurs in an ATP-dependent fashion.
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PMID:Regulatory subunit complex dissociated from 26S proteasome: isolation and characterization. 854 9

The effects of age and food restriction on proteasome function in rat liver supernatant (100,000 x g) were investigated. The cellular level of the proteasome has been quantitated by using Western blot analysis. The level of the proteasome was not affected by either age or food restriction. The three best-characterized proteasomal peptidase activities, chymotrypsin-like (ChT-L), trypsin-like (T-L), and peptidylglutamyl peptide hydrolyzing (PGPH) activities, were measured in the presence and absence of the proteasomal activator, sodium dodecyl sulfate (SDS). Basal ChT-L, T-L, and PGPH activities were not markedly affected by either age or food restriction. SDS-stimulated ChT-L and T-L activities increased approximately 15% and approximately 30%, respectively, between 7 and 26 months of age, and the increase of both activities was prevented by food restriction. In marked contrast, the SDS-stimulated PGPH activity decreased approximately 40% with age. Food restriction, while not preventing the age-related decline, maintained higher levels of SDS-stimulated PGPH activity at all ages. The proteolytic activity of the proteasome toward casein was not altered by either age or food restriction. In conclusion, the cellular level of the proteasome as well as the caseinolytic activity of the proteasome appear to be unaffected by either age or food restriction. It appears unlikely that the proteasome activity changes are related to the reported age-associated decline of protein degradation. Simultaneously, proteasomal peptidase activities appear to be differentially regulated by both age and food restriction. It suggests more subtle age-related changes in proteasome function, which could include an effect on proteasomal subunit composition.
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PMID:Alteration of rat liver 20S proteasome activities by age and food restriction. 880 79


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