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 ATP-ubiquitin-dependent proteolytic pathway (ubiquitin pathway) is believed to be involved in the formation of various neuronal inclusion bodies including Lewy bodies (LBs), a pathological hallmark of Parkinson disease and diffuse Lewy body disease (DLBD). Since multicatalytic proteinase (MCP) is involved in the ubiquitin pathway, an investigation of whether MCP is involved in neuronal inclusion bodies would provide a clue to the mechanism underlying the formation of neuronal inclusion bodies as well as to the pathogenesis of degenerative neurological disorders. In this study, we investigated detailed immunolocalization of MCP in LBs in DLBD brains using light and electron microscopy. We raised three different monoclonal antibodies against purified human MCP. Each of them recognized different sets of MCP subunits on Western blotting. Immunohistochemically, anti-MCP antibodies recognized all ubiquitin-positive cortical LBs in situ as well as those isolated from frozen DLBD cortices, suggesting that MCP is present in LBs as a whole molecule exhibiting protease activity. In electron microscopy, MCP immunoreactivity (MCP-IR) was exclusively localized on a characteristic oval structure with an approximate diameter of 100 nm. This structure was distributed throughout the LBs and was devoid of ubiquitin immunoreactivity. Treatment of isolated LBs with 2% SDS, but not with 0.5% Triton X-100, removed this structure from LBs in which fibrous materials predominated. Ubiquitin immunoreactivity was also decreased in isolated LBs treated with 2% SDS, suggesting that the fibrous structures in LBs were not ubiquitinated in situ. Thus, it is suggested that LBs are subjected to a proteolytic process in which MCP plays a role via processing of specific components of LBs.
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PMID:Multicatalytic proteinase is associated with characteristic oval structures in cortical Lewy bodies: an immunocytochemical study with light and electron microscopy. 802 94

The insulin degrading enzyme (IDE) is the first recognized member of a new class of metalloproteinases. Studies on the purification and the properties of this enzyme have led to divergent results and conclusions from different laboratories. The present manuscript suggests that many of the divergent results may be due to the interaction of this enzyme with other proteins as part of a proteolytic complex. IDE co-isolates with the multicatalytic proteinase (MCP) during a wide variety of purification approaches including affinity chromatography and conventional purification approaches. Ion exchange chromatography will partially or completely separate IDE and MCP. The SDS-PAGE protein bands at various purification steps suggest the presence of a cytosolic proteolytic complex containing IDE, MCP and other unidentified components and raise the possibility of a functional interaction among these proteins.
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PMID:Identification and isolation of a cytosolic proteolytic complex containing insulin degrading enzyme and the multicatalytic proteinase. 804 17

Subunit 6 (S6), an integral component of the 26S protease from human erythrocytes, has been studied by SDS-PAGE, peptide mapping and sequence analysis. S6 was cleaved with CNBr and three internal peptides were sequenced. A comparison with known proteins in Genbank revealed that all three S6 peptides match the predicted sequence of TBP7, Tat-binding protein 7. Based on peptide matches covering more than 10% of the TBP7 sequence, and the fact that the migration of S6 on SDS-PAGE is consistent with the estimated molecular mass for TBP7, we conclude that subunit 6 of the 26S protease is TBP7.
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PMID:Tat-binding protein 7 is a subunit of the 26S protease. 806 May 31

PA700 is a 700,000-dalton multisubunit protein that activates multiple proteolytic activities of the 20 S proteasome by a mechanism dependent upon ATP hydrolysis (Ma, C.-P., Vu, J.H., Proske, R.J., Slaughter, C.A., and DeMartino, G.N. (1994) J. Biol. Chem. 269, 3539-3547). In order to determine the identities of and structural relationships among the subunits of PA700, individual PA700 subunits were isolated by a combination of reverse phase high performance liquid chromatography (HPLC) and SDS-polyacrylamide gel electrophoresis. Seven of the 16 subunits of PA700 so isolated were subjected to solid phase protease digestion followed by reverse phase HPLC. Selected peptides from each protein were sequenced by automated Edman degradation. Comparison of the resulting amino acid sequences with those in current data bases indicated that three of the subunits represented novel proteins, whereas four subunits were homologous to previously describe proteins. Three subunits of the latter group were, in turn, homologous to one another and are members of a large family of proteins containing a consensus sequence for ATP binding. Purified PA700 demonstrated ATPase activity. Treatment of PA700 with alkylating agents, such as N-ethylmaleimide, inhibited with similar kinetics both proteasome activation and ATPase activity, suggesting that these two activities are functionally linked. Thus, PA700 is composed of multiple members of a protein family that may function in the ATP-dependent regulation of proteasome activity.
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PMID:PA700, an ATP-dependent activator of the 20 S proteasome, is an ATPase containing multiple members of a nucleotide-binding protein family. 806 4

A previously undescribed high molecular mass protein (HMP) from human erythrocyte membranes was solubilized by Triton X-100 and purified on a calmodulin-agarose column in the presence of Ca2+. It was shown to have a native molecular mass of 522-560 kDa, comprised of a single subunit of a molecular mass of 28 kDa (p28). The protein is associated with the lipid bilayer rather than with the cytoskeletal component of the membrane. The purified HMP showed peptidase-hydrolyzing activity toward substrates containing hydrophobic amino acids at the P1 position of the P2-P1 cleavage site. The activity was inhibited by serine proteinase inhibitors (leupeptin, phenylmethansulfonyl fluoride) and chymotrypsin inhibitors in particular (chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone). The enzyme exhibited maximal activity at slightly alkaline pH (7.5-8.5) and at 37 degrees C and was stimulated over a narrow range of SDS concentrations (maximal at 0.05%). HMP was found to cross-react in Western blots with an antibody raised against the rabbit multicatalytic proteinase. The single subunit of HMP therefore contains both the catalytic activity and a sequence necessary for its association into a multimeric complex. The properties of the human erythrocyte membrane HMP described indicate that it is a novel peptidase related to the ubiquitous multicatalytic proteinase.
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PMID:Characterization of a novel high molecular mass protein with peptidase activity purified from the human erythrocyte membrane by calmodulin affinity chromatography. 814 98

The 240-kDa proteasome inhibitor has been reported to be an ATP-stabilized component (CF-2) of the 26 S proteasome complex. We now report that this inhibitory factor is indistinguishable from delta-aminolevulinic acid dehydratase (ALAD), the second enzyme in the pathway of heme synthesis, based upon the following observations: 1) common sequence of the first 14 N-terminal amino acids; 2) identical migration on native and SDS-polyacrylamide gel electrophoresis; 3) identical isoelectric points of pH 7.1; 4) cross-reactivity of specific polyclonal antibodies; 5) similar dehydratase and proteasome inhibitor specific activities in both proteins; and 6) the presence of both activities in recombinant ALAD. The dual role of this protein as CF-2 in the ATP/ubiquitin-dependent pathway and in heme synthesis may be an example of "gene sharing" and explains the unexpected abundance of ALAD noted in earlier studies.
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PMID:240-kDa proteasome inhibitor (CF-2) is identical to delta-aminolevulinic acid dehydratase. 817 43

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

PA28, a protein activator of the 20 S proteasome, was previously identified in soluble extracts of bovine red blood cells (Ma, C.-P., Slaughter, C. A., and DeMartino, G. N. (1992) J. Biol. Chem. 267, 10515-10523). To determine whether this regulatory protein is as widely distributed as the proteasome, PA28 content and activity were examined in various eukaryotic tissues by immunoblot analysis and by functional assays of tissue extracts. PA28 protein was present in all sources examined. PA28 activity, however, was not detected in many of these sources, including those with the highest level of PA28 protein. To determine the biochemical basis of this result, PA28 was purified from extracts of rat liver, which had high levels of PA28 protein but no PA28 activity. The resulting purified PA28 had no detectable activity but had native and subunit molecular weights indistinguishable from the active PA28 of bovine red blood cells. Using the inactivation of purified PA28 as an assay, a protein that inactivated PA28 without altering its apparent molecular weight on SDS-polyacrylamide gel electrophoresis was identified, purified, and characterized from bovine liver. It had biochemical and catalytic characteristics similar to those of lysosomal carboxypeptidase B. When leupeptin, an inhibitor of lysosomal carboxypeptidase B, was included in the buffers used for the preparation of PA28, PA28 activity was detected in tissues which otherwise failed to demonstrate this activity. A similar result was obtained when extracts were prepared in a manner that minimized disruption of lysosomes. Other carboxypeptidases such as carboxypeptidase Y and pancreatic carboxypeptidase B also inactivated PA28 without altering its apparent molecular weight. Active PA28 binds to the proteasome to form a protease-activator complex that can be isolated after velocity sedimentation centrifugation through glycerol density gradients. Carboxypeptidase-inactivated PA28 failed to form such a complex, suggesting that the carboxyl terminus of PA28 is required for binding to the proteasome. These results indicate the importance of the carboxyl terminus of PA28 for proteasome activation.
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PMID:PA28, an activator of the 20 S proteasome, is inactivated by proteolytic modification at its carboxyl terminus. 822 60

The effect of phospholipids on the trypsin-like, chymotrypsin-like and peptidylglutamyl-peptide-hydrolysing activities of the so-called latent form of the rat liver multicatalytic proteinase was studied, assaying them with the following substrates: N-Cbz-ARR-4MNA (N-Cbz, N-benzyloxycarbonyl; 4MNA, 4-methoxy-beta-naphthylamide), N-Suc-LLVY-MCA (N-Suc, N-succinyl; MCA, methylcoumarin) and N-Cbz-LLE-beta-NA (beta-NA, beta-naphthylamide) respectively (amino acids are shown as their one-letter symbol). For the most part neither lysophospholipids nor phospholipids at 20 micrograms/ml have any effect on the activity of the enzyme (assayed at 50 microM peptide), except for phosphatidylserine, which activates 2-fold the hydrolysis of N-Suc-LLVY-MCA, and phosphatidylinositol, which inhibits by 20% the hydrolysis of N-Cbz-LLE-beta-NA. By contrast, cardiolipin (diphosphatidylglycerol) is a strong activator of the hydrolysis of N-Suc-LLVY-MCA (60-fold) and N-Cbz-LLE-beta-NA (30-fold), with half-maximal activation at concentrations of 0.15 micrograms/ml and 1.5 micrograms/ml respectively. The activation of N-Suc-LLVY-MCA hydrolysis is due to an increase of the affinity of the enzyme for the peptide and to an increase in the Vmax. (30-fold). The activation of N-Cbz-LLE-beta-NA hydrolysis is explained by suppressing the co-operativity for this substrate, producing hyperbolic kinetics with a Km of 60 microM and a 15-fold increase in the Vmax. of the enzyme. This activation by cardiolipin was completely suppressed by micromolar concentrations of fluophenazine, a drug known to inhibit other phospholipid-regulated process. Cardiolipin activation and the known activation by SDS are additive, either at suboptimal or optimal concentrations of both activators. Cardiolipin also activates the in vitro degradation of some proteins from metabolically labelled total cellular extracts by the latent multicatalytic proteinase. These results clearly show that cardiolipin is a natural positive modulator of the peptidase and proteolytic activities of the multicatalytic proteinase, probably acting through a binding site different from that of SDS.
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PMID:Kinetic mechanism of activation by cardiolipin (diphosphatidylglycerol) of the rat liver multicatalytic proteinase. 825 Aug 60

An endogenous activator of 20S proteasome was purified from human platelets and its effect on three peptidase activities of proteasome was studied. This activator had a molecular weight of 170 kDa, and was composed of 32 kDa polypeptides as determined by SDS-PAGE. It was highly labile upon heat treatment (56 degrees C, 20 s) and proteinase (pronase CB) digestion. Suc-LLVY-MCA degrading activity of the platelet proteasome showed positive cooperativity between two or more catalytic sites because the coefficient was 1.54 when analyzed by use of the Hill plot. The endogenous activator increased Vmax and caused a loss of cooperativity. The plot of reaction velocity as a function of activator concentration yielded a saturation curve, implying the binding of the activator to proteasome. Boc-LTR-MCA degrading activity followed Michaelis-Menten kinetics. The activator enhanced the activity by increasing Vmax and decreasing Km. In contrast, CBz-LLE-2NA degrading activity could not be analyzed according to any kinetic scheme reported so far. The activator stimulated this activity at lower substrate concentrations (below 200 microM), while it inhibited the activity at higher substrate concentrations (400-800 microM). It is concluded from these findings that the endogenous protein activator may regulate the intracellular proteasome activity by functioning as a positive allosteric effector.
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PMID:Purification and characterization of endogenous protein activator of human platelet proteasome. 828 19


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