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)

The effect of chemical compounds like sodium dodecyl sulfate (SDS), fatty acid esters of glycerol, carnitine and coenzyme A, phospholipids, histones, polylysines as well as homobifunctional chemical cross-linkers on the various proteolytic activities of mammalian proteasomes have been tested. Most of the reagents enhance these activities, and some, e.g. fatty acid CoA esters, histones and the chemical cross-linkers, exert dual effects, i.e. activation and inhibition at the same time, depending on the activity measured. With optimally activating concentrations of SDS, no structural changes in proteasomes can be detected by electron microscopy. Formation of micelles at supra-optimal detergent concentrations may be a reason for irreversible denaturation of the proteasome.
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PMID:In vitro activation of the 20S proteasome. 769 25

The proteasome plays a central role in ubiquitin-dependent and -independent proteolysis in eukaryotic cells. The hawkmoth proteasome was purified from larval body wall and characterized with respect to substrate specificity, sensitivity to protease inhibitors, and cross-reactivity with monoclonal antibodies (mAbs) raised against human placenta proteasome. Leupeptin selectively inhibited the trypsin-like activity (T-L) and N-ethylmaleimide inhibited both T-L and chymotrypsin-like activities, whereas 0.02% sodium dodecyl sulfate stimulated the peptidylglutamyl peptide hydrolase, branched-chain amino acid preferring, and caseinolytic activities 20-, 18-, and 3.8-fold, respectively. All four peptidase activities were inhibited by 3,4-dichloroisocoumarin. One-dimensional immunoblot analysis showed that the level and subunit composition of the proteasome varied between tissues. The relative levels of proteasome were high in intersegmental muscle and ovary, lower in Malpighian tubule, male accessory gland, and ventral nerve cord, and lowest in flight muscle and fat body. The tissues differed in the relative amount of a 41-kDa doublet; a 22-kDa subunit was present only in the male accessory gland. Two-dimensional polyacrylamide gel electrophoresis showed that the hawkmoth proteasome contained at least 26 subunits, compared with 28 subunits in lobster. Immunological analysis using four subunit-specific mAbs identified the putative homologs of the human zeta, C2, C3, and C8 alpha-type subunits in the hawkmoth and lobster enzymes. Two of the four mAbs reacted with three or more of the hawkmoth subunits and three of the mAbs reacted with two or more of the lobster subunits. In addition, two other mAbs that recognize epitopes shared by a number of alpha-type subunits indicated that at least 15 (lobster) or 16 (hawkmoth) subunits were alpha-type. These results suggest that much of the subunit complexity of the arthropod proteasomes is a consequence of extensive post-translational modifications.
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PMID:The multicatalytic proteinase (proteasome) of the hawkmoth, Manduca sexta: catalytic properties and immunological comparison with the lobster enzyme complex. 772 56

We have identified 27- and 26-kDa polypeptides in sea urchin egg jelly, both of which cross-reacted with the antibody against 20 S proteasome (multicatalytic proteinase) isolated from sea urchin sperm. Separation of egg jelly fraction by gel filtration or sucrose density gradient centrifugation revealed that these polypeptides comigrated as a complex with a molecular size much smaller than that of proteasome: the apparent molecular mass and the sedimentation coefficient were 200 kDa and 10 S, respectively. This protease significantly hydrolyzed the fluorogenic synthetic substrates for trypsin-like protease but little hydrolyzed those for chymotrypsin-like protease. Trypsin-like activity of sperm proteasome was activated up to more than threefold by a low concentration of sodium dodecyl sulfate (SDS), whereas the egg jelly 10 S protease was inhibited by SDS. Two-dimensional immunoblot and peptide mapping revealed that the 26-kDa polypeptide is a degradative product of 27-kDa polypeptide and that the 10 S protease is composed of a proteasome-related single 27-kDa polypeptide and its modified forms. These results indicate the presence of a 10 S novel assembly of a proteasome subunit only with trypsin-like activity.
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PMID:Identification of a 10 S trypsin-like protease that cross-reacts with anti-proteasome antibody in sea urchin egg jelly. 777 82

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

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

The multicatalytic proteinase complex or proteasome is a high-molecular-mass multisubunit proteinase which is found in the nucleus and cytoplasm of eukaryotic cells. Electron microscopy of negatively stained rat liver proteinase preparations suggests that the particle has a hollow cylindrical shape (approximate width 11 nm and height 17 nm using methylamine tungstate as the negative stain) with a pseudo-helical arrangement of subunits rather than the directly stacked arrangement suggested previously. The side-on view has a 2-fold rotational symmetry, while end-on there appears to be six or seven subunits around the ring. This model is very different from that proposed by others for the proteinase from rat liver but resembles the structure of the simpler archaebacterial proteasome. The possibility of conformational changes associated with the addition of effectors of proteolytic activity has been investigated by sedimentation velocity analysis and dynamic light-scattering measurements. The results provide the first direct evidence for conformational changes associated with the observed positive co-operativity in one component of the peptidylglutamylpeptide hydrolase activity as well as with the stimulation of peptidylglutamylpeptide hydrolase activities by MnCl2. In the latter case, there appears to be a correlation between changes in the shape of the molecule and the effect on activity. KCl and low concentrations of SDS may also act by inducing conformational changes within the complex. Sedimentation-velocity measurements also provide evidence for the formation of intermediates during dissociation of the complex by urea, guanidinium chloride or sodium thiocyanate. Dissociation of the complex either by these agents or by treatment at low pH leads to inactivation of its proteolytic components. The results suggest that activation and inhibition of the various proteolytic activities may be mediated by measurable changes in size and shape of the molecules.
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PMID:The multicatalytic proteinase complex (proteasome): structure and conformational changes associated with changes in proteolytic activity. 831 14

A metalloprotease (MEP) secreted by Aspergillus fumigatus was isolated from an alkaline protease-deficient mutant after the fungus was cultivated in the presence of collagen as the sole nitrogen and carbon source. The enzyme was purified 50-fold from the culture supernatant after adsorption to hydroxylapatite and carboxy-methyl-Sephadex and after gel filtration. The molecular mass was determined to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was estimated at pH 5.5 by isoelectric focusing. Reducing agents and divalent cations strongly inhibited enzyme activity, whereas nonionic detergents had no effect. A. fumigatus MEP was totally inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. MEP is not able to cleave elastin and is thermosensitive. Sera from patients suffering from aspergilloma reacted with MEP in Western blotting (immunoblotting) analyses, suggesting that MEP promotes an antigenic response in these patients.
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PMID:Isolation and characterization of a secreted metalloprotease of Aspergillus fumigatus. 840 98

Initial studies on the specificity of the multicatalytic proteinase complex (MPC; EC 3.4.99.46) led to the identification of three distinct proteolytic components designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing, all sensitive to inactivation by 3,4-dichloroisocoumarin (DCI), a general serine proteinase inhibitor. The three components cleave the peptidyl-arylamide bonds in the model synthetic substrates, Z-(D)-Ala-Leu-Arg-2-naphthylamide, Z-Gly-Gly-Leu-p-nitroanilide, and Z-Leu-Leu-Glu-2-naphthylamide, respectively. We report here evidence for the presence in the MPC of two additional distinct components, neither of them capable of cleaving the three model substrates. One of these components cleaves the Leu-Gly and the Leu-Ala bonds in the substrates Cbz-Gly-Pro-Ala-Leu-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Leu-Ala-p-aminobenzoate, respectively, and is activated by treatment of the MPC with DCI, N-ethylmaleimide, Mg2+, Ca2+, and low concentrations of sodium dodecyl sulfate and fatty acids. This component is apparently identical with the previously identified DCI-resistant component of the MPC that cleaves preferentially bonds on the carboxyl side of branched chain amino acids in natural peptides including neurotensin and proinsulin [Cardozo, C., Vinitsky, A., Hidalgo, M. C., Michaud, C., & Orlowski, M. (1992) Biochemistry 31, 7373-7380]. It is probably also identical with the component proposed to be the main factor responsible for the caseinolytic activity [Pereira, M. E., Nguyen, T., Wagner, B. J., Margolis, J. W., Yu, B., & Wilk, S. (1992a) J. Biol. Chem. 267, 7949-7955]. The designation "branched chain amino acid preferring" (BrAAP) is proposed for this component. The second component cleaves peptide bonds between the small neutral amino acids Ala-Gly and Gly-Gly in the substrates Cbz-Gly-Pro-Ala-Ala-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Gly-Gly-p-aminobenzoate, respectively. This component is sensitive to inactivation by DCI, N-ethylmaleimide, and organic mercurials, but unlike the BrAAP it is significantly activated neither by Mg2+ or Ca2+ nor by fatty acids or sodium dodecyl sulfate. The designation "small neutral amino acid preferring" (SNAAP) is proposed for this component. Both components are sensitive to inhibition by the peptidyl-aldehydes N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO; calpain inhibitor I) and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO; calpain inhibitor II) but are resistant to inhibition by Z-LLF-CHO, a potent inhibitor of the chymotrypsin-like activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Evidence for the presence of five distinct proteolytic components in the pituitary multicatalytic proteinase complex. Properties of two components cleaving bonds on the carboxyl side of branched chain and small neutral amino acids. 843 36

The LMP7 gene maps to the major histocompatibility complex class II region. The derived protein sequence shares homology with N-terminal amino acid sequence from proteasome subunits (Glynne, R., Powis, S. H., Beck, S., Kelly, A., Kerr, L.-A. and Trowsdale, J., Nature 1991. 353: 357) and it has been suggested that LMP7 is involved in the degradation of endogenous antigens prior to their presentation through class I (Robertson, M., Nature 1991. 353: 300). We have isolated a second LMP7 transcript which has a different first exon to the published sequence. Both transcripts were expressed in cell lines from a number of tissues and both responded to interferon-gamma. An anti-LMP7 antiserum precipitated proteins similar in their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to those precipitated by an anti-proteasome serum. Western blot analysis of anti-proteasome precipitates demonstrated that the LMP7 protein is incorporated into the proteasome but has a molecular mass of 23 kDa, 7 kDa smaller than expected fro the derived protein sequence of either of the cDNA. A pulse-chase experiment indicated that post-translational cleavage of the LMP7 N terminus precedes the formation of the 23-kDa proteasome subunit. To our knowledge, LMP7 provides the first biochemical evidence for such processing of proteasome components.
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PMID:The major histocompatibility complex-encoded proteasome component LMP7: alternative first exons and post-translational processing. 845 75

A sperm protein of molecular mass 43 kDa (the spermatozoa membrane cofactor protein, smMCP) and a seminal plasma protein of 60 kDa (ssMCP) were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with four monoclonal antibodies (mAb) against membrane cofactor protein (MCP, CD46). These proteins served as factor I cofactors for the cleavage of methylamine-treated C3 (C3ma), the activity of which was blocked by M75, an MCP cofactor-activity-blocking mAb. Thus, these semen proteins are antigenic and functional homologous of MCP. On SDS-PAGE analysis these MCP migrated as single-band proteins which differed from the two-band forms of MCP expressed on other cells. smMCP was N-glycosylated but not O-glycosylated, while ssMCP was O-glycosylated: after deglycosylation of these proteins bands were detected at 38-40 kDa and 43 kDa on SDS-PAGE, respectively. These semen MCP are therefore, structurally different from the conventional MCP. ssMCP in both normal and "sterile" subject groups was determined by sandwich enzyme-linked immunosorbent assay. Seminal plasma in the two groups contained 250-700 ng/ml ssMCP. The difference between the two groups was marginal, although samples from normal subjects tended to show higher concentrations of ssMCP than samples from "sterile" subjects. No molecular difference was observed with ssMCP and smMCP in the two groups by SDS-PAGE/immunoblotting analysis. Immunohistochemical analysis suggested that MCP was positive in glandular epithelial cells and the lumen of the prostate, and in most intra-lumen cells of the testis. Using antibody M177, solubilized prostate and testis were analyzed by immunoblotting and compared with other cell MCP. The major band of MCP in the testis, but not in the prostate, was of 60 kDa, which aligned with ssMCP. No band of testis or prostate MCP, however, aligned with smMCP. ssMCP may be produced in the testis, while the origin of smMCP remains unknown. We hypothesize that ssMCP is important in the survival of spermatozoa, protecting them against local secretion of immunoglobulin and complement in the female genital tract, and that smMCP, which is expressed on acrosome-reacted spermatozoa, plays an essential role in the interaction of spermatozoa with oocytes.
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PMID:Membrane cofactor protein (MCP, CD46) in seminal plasma and on spermatozoa in normal and "sterile" subjects. 850 May 28


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