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)

Protease activity was detected in membranes of human bovine erythrocytes prepared by the conventional procedures which include washing and removal of the "buffy layer". The enzyme was extracted by 0.75 M KCNS or (NH4)2SO4 and was activated by 0.4 to 0.5 M of the same salts. Colored, particulate hide powder-azure, membrane fractions and soluble proteins such as hemoglobin, casein or albumin were susceptible to hydrolysis by the membraneous protease. Partial purification of the enzyme was accomplished through disc-gel electrophoresis on polyacrylamide in the presence of 0.25% positively charged detergents like cetyltrimethylammonium bromide. An alkaline protease (pH 7.4) with properties similar to those of the erythrocyte enzyme was found in leucocytes. The similarity between the properties of the leucocytic and erythrocytic proteases and the correlation of the activity in erythrocyte membranes with content of white cells in these preparations, suggest that enzymatic activities in the contaminating leucocytes are responsible for the activity of membraneous proteases in erythrocytes.
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PMID:Membrane-bound enzymes. III. Protease activity in leucocytes in relation to erythrocyte membranes. 0 92

The properties of the homogeneous exoprotease preparation from Bacillus subtilis varamyloliquefaciens 759 possessing the coagulase activity were studied. The enzyme is an alkaline protease, has the isopoint at pH 7.8, and not only clots blood plasmo but also hydrolyses such protein substrates as casein, hemoglobin, fibrinogen and fibrin. The enzyme is relatively stable at pH 6.0--9.0. Bivalent metal ions have virtually no effect on the enzyme activity though some of them stabilize it. The inhibitors PCMB and EDTA do not affect the activity of the enzyme whereas diisopropylfluorophosphate completely inactivates it. Fibrinogen is clotted by the enzyme only in the presence of blood plasma factors.
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PMID:[Exoprotease properties of Bacillus subtilis var. amyloliquefaciens capable of coagulating blood plasma]. 3 Aug 84

Studies were carried out to characterize further the cytoplasmic ATP- and ubiquitin-independent proteolytic system in red blood cells that degrades hemoglobin damaged by exposure to oxidants (Fagan, J. M., Waxman, L., and Goldberg, A. L. (1986) J. Biol. Chem. 261, 5705-5713). Several proteases were ruled out as having a major role in the degradation of oxidant-treated hemoglobin (Ox-Hb). Acid hydrolases are not active in this process since the degradation of Ox-Hb has a pH optimum between 6 and 8. The calpains are also not involved since inhibitors of cysteine proteases (leupeptin and trans-epoxysuccinyl-L-leucylamido-(3-methyl)butane) did not diminish the increased proteolysis in intact erythrocytes treated with oxidants or in lysates to which Ox-Hb was added. The degradation of Ox-Hb was unaffected by inhibitors of serine and aspartic proteases. Removal of the high M(r) multicatalytic proteinase by immunoprecipitation also did not significantly affect the degradation of Ox-Hb in erythrocyte lysates. The degradation of Ox-Hb was sensitive to metal chelators and sulfhydryl-modifying reagents but not to specific inhibitors of known metalloproteases. Insulin, which is rapidly degraded in lysates, completely blocked the degradation of Ox-Hb. Insulin- and Ox-Hb-hydrolyzing activity was also inhibited following immunoprecipitation of the 100-kDa metalloinsulinase. The metalloinsulinase, which is inhibited by sulfhydryl-modifying reagents and which requires divalent metals, may therefore participate in the degradation of hemoglobin damaged by oxidants in erythrocytes.
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PMID:The ATP-independent pathway in red blood cells that degrades oxidant-damaged hemoglobin. 142 49

Exposure of human red cells to oxidants such as phenylhydrazine, 2,4-dimethylphenylhydrazine and 4-hydrazinobenzoic acid stimulates the proteolysis of hemoglobin as evidenced by the increase in the rate of the free alanine and acid soluble amino groups released. An enzyme responsible for proteolytic degradation of oxidized hemoglobin, was purified from cytosolic fraction of erythrocytes by a DEAE-batch procedure followed by gel-filtration and ion-exchange chromatography. The final enzyme preparation produces a single band in non-denaturing polyacrylamide gel electrophoresis, and eight different bands of 23-32 kDa when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has a molecular mass of about 700 kDa as estimated by gel filtration. The enzyme, unable to hydrolyze native hemoglobin, cleaves phenylhydrazine-treated hemoglobin into small peptides without free amino acid release. In addition, the enzyme shows an endopeptidase activity towards synthetic peptides having a tyrosine or an arginine in the P1 position, whereas it does not hydrolyze shorter peptides and those with a proline in the P1 or P2 position. The proteolytic activity of the enzyme against oxidized hemoglobin is inhibited by chymostatin and p-chloromercuribenzoate, while it is stimulated by N-ethylmaleimide and epoxysuccinylleucylamido-(4-guanidino)butane (E-64). The peptidase activity assayed on succinyl-Leu-Leu-Val-Tyr-MCA is inhibited by chymostatin, hemin, N-ethylmaleimide and p-chloromercuribenzoate. The results obtained show that in human erythrocytes oxidized hemoglobin is cleaved into peptides by a high molecular mass proteinase identified as a member of the multicatalytic proteinase family. It is also suggested that the complete degradation of oxidized hemoglobin to free amino acids requires the involvement of a further proteolytic enzyme(s) which remain(s) to be identified.
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PMID:Purification of human erythrocyte proteolytic enzyme responsible for degradation of oxidant-damaged hemoglobin. Evidence for identifying as a member of the multicatalytic proteinase family. 217 87

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

Eukaryotic cells contain a 700-kDa proteolytic complex (the "proteasome" or multicatalytic endopeptidase complex), whose role in intracellular protein breakdown is unclear. It has been suggested that the proteasome functions in the rapid degradation of oxidant-damaged proteins and in the ATP-dependent proteolytic pathway. To test these possibilities, oxidant-damaged hemoglobin and albumin were produced by treating hemoglobin and albumin with phenylhydrazine, with hydroxyl radicals, or with both hydroxyl and superoxide radicals. After oxidant damage, these proteins were degraded more rapidly in erythrocyte extracts and also by the purified proteasome. However, complete removal of proteasomes from these extracts by immunoprecipitation (or inhibitors of its proteolytic activity) did not reduce the breakdown of oxidant-damaged hemoglobin and decreased degradation of hydroxyl- and superoxide-treated proteins by only 30-40%. Thus, erythrocytes must contain another proteolytic system for degradation of oxidant-damaged proteins. In contrast, immunoprecipitation of proteasomes with polyclonal or monoclonal antibodies prevented the ATP/ubiquitin-dependent degradation of lysozyme and also blocked the ATP-stimulated degradation of ubiquitin-conjugated lysozyme in reticulocyte and skeletal muscle extracts. These data indicate a critical role of the proteasome in the degradation of ubiquitin-conjugated proteins and suggest that the proteasome is associated with or is a component of the larger ubiquitin-conjugate-degrading enzyme complex.
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PMID:Involvement of the proteasome in various degradative processes in mammalian cells. 253 95

It would appear to us that the patients with rheumatoid arthritis (RA), in whom articular bone lesions were confined to the wrist and/or carpal joints in X-ray films, may follow a milder disease activity than do the patients with the hand and finger joint lesions. To clarify it, the present study was performed retrospectively. RA patients showing stage II or more lesions in the wrists and/or carpal joints but no lesions over stage II in any hand and finger joint (MCP, IP and PIP) radiologically after 5 years or more duration were regarded as the carpal type (C type). The clinical and laboratory data were compared between 44 patients of the C type RA and 44 patients of other type RA, matched in the sex, age and disease duration. Significant differences were observed in the following parameters between the 2 groups; the functional class, Lansbury's activity index, number of the affected joints, ESR, CRP and hemoglobin values, and ADL scores. That is, Hb values and ADL scores were higher, but the others were lower, in the C type RA group than in other type RA group. The positive percentage and titer of rheumatoid factor were not significantly different between the 2 groups. It was concluded that C type RA patients are milder in the activity and fewer in the number of affected joints than other type RA patients. Furthermore it was suggested that C type RA patients may have milder clinical course and better prognosis than other type RA patients.
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PMID:[Evaluation of disease activity by hand X-ray findings in rheumatoid arthritis]. 277 54

Exposure to various forms of mild oxidative stress significantly increased the intracellular degradation of both "short-lived" and "long-lived," metabolically radiolabeled, cell proteins in cultures of Clone 9 liver cells (normal liver epithelia). The oxidative stresses employed were bolus H2O2 addition; continuous H2O2 flux; the redox cycling quinones, menadione and paraquat; and the aldehydic products of lipid peroxidation, 4-hydroxynonenal, malonyldialdehyde, and hexenal. In general, exposure to more severe oxidative stress produced a concentration-dependent decline in intracellular proteolysis, in some cases to below baseline levels. Oxidatively modified "foreign" proteins (superoxide dismutase and hemoglobin) were also selectively degraded, in comparison with untreated foreign proteins, when added to lysates of Clone 9 liver cells. As with intracellular proteolysis, the degradation of foreign proteins added to cell lysates was greatly increased by mild oxidative modification, but depressed by more severe oxidative modification. The proteinase activity was recovered in > 300-kDa cell fractions, and inhibitor profiles and immunoprecipitation studies indicated that the multicatalytic proteinase complex, proteasome, was responsible for most of the selective degradation observed with mild oxidative stress; up to approximately 95% for intracellular proteolysis and 65-80% for degradation of foreign modified proteins. Seven days of daily treatment with an antisense oligodeoxynucleotide, directed against the initiation codon region of the proteasome C2 subunit gene, severely depressed the intracellular levels of several proteasome subunit polypeptides (by Western blot analysis), and also depressed the H2O2 induced increase in intracellular proteolysis by approximately 95%, without significantly affecting baseline proteolytic rates. Extensive studies revealed only small or no increases in the overall capacity of oxidatively stressed cells to degrade oxidatively modified protein substrates; a finding supported by both Western blot and Northern blot analyses which revealed no significant increase in the levels of proteasome subunit polypeptides or mRNA transcripts. We conclude that mild oxidative stress increases intracellular proteolysis by modifying cellular proteins, thus increasing their proteolytic susceptibility. In contrast, severe oxidative stress diminishes intracellular proteolysis, probably by generating severely damaged cell proteins that cannot be easily degraded (e.g. cross-linked/aggregated proteins), and by damaging proteolytic enzymes. We further conclude that the multicatalytic proteinase complex proteasome is responsible for most of the recognition and selective degradation of oxidatively modified proteins in Clone 9 liver cells.
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PMID:Proteolysis in cultured liver epithelial cells during oxidative stress. Role of the multicatalytic proteinase complex, proteasome. 783 68

The physiologically relevant stress of a flux of H2O2 increased hemoglobin (Hb) degradation in red blood cells (RBC) and increased the proteolytic susceptibility of Hb in vitro. After exposure to low H2O2 flux rates (6-32 microM/min) Hb exhibited increased exposure of hydrophobic (Trp, Met) and basic (Lys) amino acid R groups, increased hydrophobicity, and increased proteolytic susceptibility during subsequent incubation with RBC extracts, a partially purified preparation called Fraction II (which retains all of the proteolytic activities of RBC extracts), or the purified 670-kDa RBC multicatalytic proteinase complex proteasome. Hydrophobicity was measured by butyl-Sepharose hydrophobic interaction chromatography, by the free energy of transfer from water to ethanol, and by heat denaturation assays. Proteolytic susceptibility was measured by release of free alanine, by fluorescamine-reactive free amino groups, and by release of acid-soluble radioactivity from radiolabeled Hb. Low H2O2 flux rates also caused significant charge changes in Hb (isoelectric focusing gels) and extensive noncovalent aggregation (presumably due to increased hydrophobic interactions) but only limited covalent cross-linking (comparison of sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE). Exposure to higher H2O2 flux rates (56-120 microM/min) caused progressive oxidative destruction of exposed hydrophobic amino acids, decreased hydrophobicity as judged by butyl-Sepharose chromatography and heat denaturation assays, increased hydrophilicity as judged by measurements of the free energy of transfer (delta G') from water to ethanol, and decreased proteolytic susceptibility during incubation with RBC extracts, Fraction II, or purified proteasome. High H2O2 flux rates also caused further charge changes and the extensive formation of covalently cross-linked Hb molecules. Linear regression analyses revealed correlations of 0.8-0.99 for the relationship between Hb hydrophobicity and proteolytic susceptibility for both Fraction II and proteasome. Inhibitor studies and SDS activation experiments indicate that proteasome is responsible for most of the Hb degradation during exposure of RBC to H2O2. Previous work yielded essentially identical conclusions for Hb exposed to hydroxyl radicals (R. E. Pacifici, Y. Kono, and K. J. A. Davies, J. Biol. Chem. 268, 15405-15411, 1993). Thus, nonspecific oxidation by .OH and site-specific (metal-catalyzed) oxidation by H2O2 both yield a more hydrophobic Hb molecule with increased proteolytic susceptibility. We propose that increased exposure of hydrophobic, and perhaps basic, amino acids is the general common cause for degradation of oxidized proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Exposure of hydrophobic moieties promotes the selective degradation of hydrogen peroxide-modified hemoglobin by the multicatalytic proteinase complex, proteasome. 820 95

Red blood cells (RBC) and many other cell types exhibit increased rates of proteolysis during exposure to oxygen radicals and other activated oxygen species (oxidative stress). One of the major RBC proteins modified and proteolytically degraded during oxidative stress is hemoglobin (Hb). We now show that Hb undergoes a partial unfolding (or denaturation) during exposure to hydroxyl radicals (.OH), with an increase in hydrophobicity (hydrophobic interaction chromatography). At low .OH/Hb molar ratios, oxidatively modified Hb exhibits increased proteolytic susceptibility during incubation with RBC lysates, cell-free extracts, Fraction II, a 40-80% (NH4)2SO4 fraction, and purified proteasome (the 670-kDa RBC multicatalytic proteinase complex that we have previously called macroxyproteinase. At higher .OH/Hb molar ratios covalent cross-linking between Hb tetramers, and decreased proteolytic susceptibility are observed. The selective degradation of .OH-modified Hb is an ATP- and ubiquitin-independent process (in fact ATP is slightly inhibitory), and antibody precipitation studies, as well as inhibitor studies, indicate that proteasome is responsible for at least 60-70% of the activity in RBC. We propose that the mechanism of oxidation-induced proteolysis involves exposure of hydrophobic amino acid R groups during the partial Hb unfolding (or partial denaturation) that occurs at relatively low .OH/Hb molar ratios. Peptide bonds flanked by hydrophobic residues are preferred substrates for the proteasome complex, which degrades .OH-modified Hb in a processive process involving apparent serine-protease, sulfhydryl-protease, and metallo-peptidase activities. Highly denatured and covalently cross-linked Hb molecules, produced at high .OH/Hb molar ratios, are poorly degraded in RBC lysates and at all stages of proteasome purification. These cross-linked Hb tetramers have molecular sizes of 120-180 kDa and are presumably too large to fit in the proteasome active site(s). Recognition of exposed hydrophobic amino acid R groups provides a simple, energy-independent, and universal explanation for the proteasome-dependent proteolysis that accompanies oxidative stress.
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PMID:Hydrophobicity as the signal for selective degradation of hydroxyl radical-modified hemoglobin by the multicatalytic proteinase complex, proteasome. 839 40


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