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)

Proteins conjugated to ubiquitin are degraded by a 26S (1500-kDa) proteolytic complex that, in reticulocyte extracts, can be formed by the association of three factors: CF-1, CF-2, and CF-3. One of these factors, CF-3, has been shown to be the proteasome, a 650-kDa multicatalytic protease complex. We have purified a 250-kDa inhibitor of the proteasome and shown that it corresponds to CF-2. In the presence or absence of ATP, this factor inhibited hydrolysis by the proteasome of both fluorogenic tetrapeptides and protein substrates. When the inhibitor, proteasome, and CF-1 were incubated together in the presence of ATP and Mg2+, degradation of ubiquitin-125I-lysozyme occurred. Both the inhibitory activity and the ability to reconstitute ubiquitin-125I-lysozyme degradation were very labile at 42 degrees C, but both activities were stabilized by ATP or a nonhydrolyzable ATP analog. SDS/PAGE indicated that the 250-kDa inhibitor fraction contained a major subunit of 40 kDa (plus some minor bands). The 125I-labeled inhibitor and purified proteasome formed a complex. When CF-1, ATP, and Mg2+ were also present, the 125I-labeled inhibitor along with the proteasome formed a complex of 1500 kDa. The inhibitor (CF-2) thus appears to be an ATP-binding component that regulates proteolysis within the 1500-kDa complex.
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PMID:An ATP-stabilized inhibitor of the proteasome is a component of the 1500-kDa ubiquitin conjugate-degrading complex. 131 79

We have purified proteasomes to apparent homogeneity from the archaebacterium Thermoplasma acidophilum. This proteinase has a molecular mass of about 650 kDa and an isoelectric point of 5.6. The proteasome hydrolyses peptide substrates containing an aromatic residue adjacent to the reporter group, as well as [14C]methylated casein optimally at pH 8.5 and 90 degrees C. The enzyme activity is enhanced severalfold by Mg2+ and Ca2+ at 25-500 mM. This increase in activity results primarily from a change in Km. The serine-proteinase inhibitors diisopropylfluorophosphate and 3,4-dichloroisocoumarin irreversibly inhibit the enzyme, obviously by modification of both the alpha and beta subunits in the proteasome. The inhibition of proteasomal activity by the peptidylchloromethanes, Cbz-Leu-Leu-CH2Cl and Cbz-Ala-Ala-Phe-CH2Cl (Cbz, benzyloxycarbonyl), is reversible and predominantly of a competitive type. The enzyme is not activated by any of the compounds that typically stimulate the activities of the eukaryotic proteasome.
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PMID:Biochemical properties of the proteasome from Thermoplasma acidophilum. 139 84

The effect of magnesium ions on the catalytic activities of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. Mg2+ markedly stimulated the breakdown of dephosphorylated beta-casein (caseinolytic activity) and the hydrolysis of Cbz-Leu-Leu-Glu-2-naphthylamide (peptidylglutamyl peptide bond hydrolyzing activity) by a 1700-fold purified preparation of MPC. Cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide (trypsin-like activity) was strongly inhibited and cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide (chymotrypsin-like activity) was weakly inhibited. Similar results were produced when enzymatic activities in the absence of Mg2+ were measured at 52 degrees C rather than at 37 degrees C. Trace protein impurities were removed by phenyl-Sepharose chromatography. This additional chromatographic step, while not changing the specific activities of hydrolysis of the three synthetic chromogenic substrates, led to a marked activation of the breakdown of dephosphorylated beta-casein. Mg2+ was not able to further stimulate the caseinolytic activities of either the phenyl-Sepharose-treated preparation or the preparation measured at 52 degrees C. Mg2+ therefore converts a "repressed" form of MPC to an "activated" form, possibly by promoting dissociation of a protein inhibitor, and may serve as a physiological regulator of this enzyme complex.
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PMID:Enzymatic changes of the bovine pituitary multicatalytic proteinase complex, induced by magnesium ions. 155 Mar 35

We have investigated the proteolytic degradation of [14C]methylcasein and 125I-labeled bovine serum albumin at pH 7.8 and 37 degrees C by lysates of rabbit reticulocytes purified from rabbit blood by two different procedures. (I) Lysates obtained from reticulocytes after removal of plasma and buffy coat as well as after washing of cells, degraded casein and albumin, and released from the two substrates 1.3%/h and 0.4%/h, respectively, of acid-soluble radioactivity. The activity towards both substrates was stimulated about 4-fold by ATP/Mg2+. Chromatography of whole blood on a column of cellulose prior to washing and lysis of cells had profound but differential effects on these activities in that stimulation of casein-degradation by ATP/Mg2+ was almost completely lost, whereas degradation of albumin, albeit at a low rate, was measurable in the presence of ATP/Mg2+ only. (II) Degradation of casein by these lysates is largely inhibited by a monospecific antibody against rabbit multicatalytic proteinase, whereas digestion of albumin is not affected by the antibody, either in the presence or absence of ATP/Mg2+. The latter activity is partially inhibited by a specific antibody against rabbit alpha 1-macroglobulin. (III) The immunoreactive amount of multicatalytic proteinase is about 1.2 micrograms per mg of lysate protein and almost identical in the two lysates. In contrast, the immunologically detectable levels of alpha 1-macroglobulin vary and are much lower in reticulocyte-lysates following chromatography on cellulose than in lysates from washed reticulocytes. (IV) Caseinolytic activity of multicatalytic proteinase, purified from rabbit reticulocyte lysate, is not activated by ATP/Mg2+ and the enzyme is proteolytically inactive towards albumin. On the other hand, a complex consisting of the proteinase inhibitor alpha 1-macroglobulin and the cysteine proteinase, cathepsin B, does degrade both substrates at pH 7.8, in an ATP/Mg2+-activated fashion. From these results it is concluded that the multicatalytic proteinase is an ATP-independent enzyme and a cellular constituent of rabbit reticulocytes whereas the activity stimulated by ATP/Mg2+ appears to be associated, at least in part, with a cysteine proteinase complexed to alpha 1-macroglobulin.
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PMID:High-molecular-mass proteinases in rabbit reticulocytes: the multicatalytic proteinase is an ATP-independent enzyme and ATP-activated proteolysis is in part associated with a cysteine proteinase complexed to alpha 1-macroglobulin. 247 Apr 11

The proteasome (the multicatalytic endoproteinase complex) in mammalian tissues hydrolyzes proteins and several types of peptides. When this structure was isolated rapidly from rabbit skeletal muscle in the presence of glycerol, its various peptidase and protease activities showed a large reversible activation by physiological concentrations of ATP (Ka = 0.3-0.5 mM). Hydrolysis of succinyl-Leu-Leu-Val-Tyr-(4-methylcoumaryl-7-amide) was stimulated up to 12-fold by ATP, whereas degradation of casein and bovine serum albumin increased 4- to 7-fold. Neither ADP nor AMP had any effect. CTP, GTP, UTP, and the nonhydrolyzable analogs adenosine 5'-[beta,gamma-imino]triphosphate (AMPP[NH]P) and adenosine 5'-[alpha,beta-methylene]triphosphate (AMP[CH2]PP) increased peptide hydrolysis as well as ATP did. However, only ATP stimulated casein breakdown and only in the presence of Mg2+. Thus, nucleotide binding allows activation of the peptidase functions, but ATP hydrolysis seems necessary for enhanced degradation of proteins. The ATP effect on proteolysis was reversible and did not require ubiquitin. Sensitivity to ATP was labile, and with storage at 4 degrees C the enzyme became fully active in the absence of ATP or Mg2+. The ATP-activated form closely resembles the proteasome complex described previously, which did not show ATP dependence: both have molecular masses of 650 kDa, contain the same 8-10 subunits, and are precipitated by the same antibodies. A similar ATP-activated form was found in rabbit liver but not in rabbit reticulocytes. The proteasome seems to represent a ubiquitin-independent, ATP-stimulated proteolytic activity within nucleated mammalian cells.
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PMID:Skeletal muscle proteasome can degrade proteins in an ATP-dependent process that does not require ubiquitin. 253 33

Previously, we isolated an ATP-dependent proteolytic pathway in muscle, liver, and reticulocytes that requires ubiquitin and the enzymes which conjugate ubiquitin to proteins. We report here that skeletal muscle contains another soluble alkaline energy-dependent (but ubiquitin-independent) proteolytic activity. The cleavage of non-ubiquitinated protein substrates by the partially purified protease requires ATP hydrolysis since ATP in the absence of Mg2+, nonhydrolyzable ATP analogs, and pyrophosphate all fail to stimulate proteolysis. Proteolytic activity is also stimulated by UTP, CTP, and GTP, although not as effectively as by ATP (Km(ATP) = 0.027 mM). The enzyme is inactivated by the serine protease inhibitors diisopropyl fluorophosphate and 3,4-dichloroisocoumarin, but not by specific inhibitors of aspartic, thiol, or metalloproteases. It is maximally active at pH 8 and has a molecular weight of approximately 600,000. This new activity differs from the 720-kDa multicatalytic proteinase, but resembles the soluble ATP-dependent proteolytic system that we previously isolated from murine erythroleukemia cells.
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PMID:A novel ATP-requiring protease from skeletal muscle that hydrolyzes non-ubiquitinated proteins. 255 95

Soluble extracts of cultured cells (BHK 21/C13) degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated this proteolytic activity up to 10-fold. The ATP effect was dependent on Mg2+ and was not elicited by nonhydrolyzable analogs of ATP. After the extract was fractionated on DEAE-cellulose, ATP-stimulated protease activity was in the fraction that bound to the resin and eluted in buffer containing 0.4 M NaCl. This activity had characteristics that were indistinguishable from those of the unfractionated extract but the degree of ATP stimulation was two- to three-fold lower. Although no protease activity was detected in the unbound fraction, reconstitution of this material with the bound fraction enhanced the ATP stimulation up to twofold. The component responsible for the enhancement of the ATP stimulation had properties similar to ubiquitin and purified ubiquitin enhanced the ATP-stimulated protease activity in the fractionated extract. Substrates whose amino groups were almost completely blocked by various chemical modifications were still degraded in an ATP-stimulated fashion, but the degradation of these substrates was not affected by ubiquitin. The protease activity isolated by ion-exchange chromatography was fractionated further by gel filtration chromatography on Sephacryl S-300. ATP-stimulated protease activity eluted with an apparent molecular weight of 750,000. Protease activity was enhanced up to eightfold by Mg2+-ATP but was not increased further by ubiquitin. An activity that hydrolyzed the synthetic peptide Z-Val-Leu-Arg-MNA coeluted with ATP-stimulated protease activity, but peptide hydrolysis was not affected by ATP. These and other catalytic and biochemical characteristics suggested that the protease might be related to the high-molecular-weight protease, macropain, recently purified by us from human erythrocytes (M. J. McGuire and G. N. DeMartino Biochim. Biophys. Acta (1986) 873, 279-289). Antibodies raised against macropain specifically reacted with proteins characteristic of macropain in the column fractions containing ATP-stimulated protease activity. These antibodies also specifically immunoprecipitated 70-100% of the ATP-stimulated protease activity as well as Z-Val-Leu-Arg-MNA hydrolyzing activity. Thus BHK cell extracts appear to contain both ubiquitin-mediated and ubiquitin-independent pathways for the ATP-stimulated degradation of proteins. Furthermore, at least one of these pathways appears to involve a high-molecular-weight, ATP-stimulated protease related to macropain.
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PMID:ATP-stimulated proteolysis in soluble extracts of BHK 21/C13 cells. Evidence for multiple pathways and a role for an enzyme related to the high-molecular-weight protease, macropain. 283 71

Two forms of a neutral protease that catalyzed the hydrolysis of succinyl-Leu-Leu-Val-Tyr-MCA were isolated from porcine skeletal muscle cytosol by fractionation on DEAE-cellulose, hydroxyapatite and Sephadex G-100. The native enzyme had a molecular mass of above 1000 kDa. Peak A, which was eluted from hydroxyapatite at 50 mM phosphate, was activated 37-fold by the detergent, SDS, while peak B which was eluted at 150 mM phosphate, was activated only 2-fold. After dialysis against water, the B form showed restored ability to be activated by SDS (9.6-fold with 0.04% SDS). The activated peak B was extremely sensitive to divalent and monovalent cations such as Ca2+, Mg2+, Na+, K+ and NH+4 as well as protease inhibitors such as leupeptin, chymostatin and DFP. These results suggest that these proteases are generally latent in the cells and may be regulated by changes in the concentrations of cations in the cytosol. We call this new type of protease, ingensin.
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PMID:Isolation of two forms of the high-molecular-mass serine protease, ingensin, from porcine skeletal muscle. 389 52

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

The 26 S proteasome complex is thought to catalyse the breakdown of ubiquitinated proteins within eukaryotic cells. In addition it has been found that the complex also degrades short-lived proteins such as ornithine decarboxylase in a ubiquitin-independent manner. Both proteolytic processes are paralleled by the hydrolysis of ATP. Here we show that ATP also affects the hydrolytic activity towards fluorigenic peptide substrates by the 26 S proteasome complex from rat skeletal muscle tissue. Low concentrations of ATP (about 25 microM) optimally activate the so-called chymotryptic and tryptic activity by increasing the rate of peptide hydrolysis but not peptidylglutamylpeptide hydrolysis. Activation of the enzyme by ATP is transient but this effect can be enhanced and prolonged by including in the assay an ATP-regenerating system, indicating that ATP is hydrolysed by the 26 S proteasome complex. Although ATP cannot be substituted for by adenosine 5'-[beta,gamma-methylene]triphosphate or AMP, hydrolysis of the phosphoanhydride bond of ATP seems not to be necessary for the activation process of the proteasome complex, a conclusion drawn from the findings that ATP analogues such as adenosine 5'-[beta,gamma-imido]triphosphate, adenosine 5'-O-[gamma-thio]triphosphate, adenosine 5'-O-[beta-thio]-diphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate give the same effect as ATP, and vanadate does not prevent ATP activation. These effects are independent of the presence of Mg2+. Thus, ATP and other nucleotides may act as allosteric activators of peptide-hydrolysing activities of the 26 S proteasome complex as has also been found with the lon protease from Escherichia coli.
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PMID:Studies on the activation by ATP of the 26 S proteasome complex from rat skeletal muscle. 761 56


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