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 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

The breakdown of beta-casein (caseinolytic activity) by the bovine pituitary multicatalytic proteinase complex (MPC) is initiated by a fourth active site different from the previously described chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide, where Cbz is benzyloxycarbonyl), trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide), and peptidylglutamyl peptide bond-hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) (Yu, B., Pereira, M. E., and Wilk, S. (1991) J. Biol. Chem. 266, 17396-17400). 3,4-Dichloroisocoumarin, a serine proteinase inhibitor, stimulated the caseinolytic activity of bovine pituitary or lens MPC, 3-18-fold under conditions under which the other three catalytic activities were inactivated. Addition of hydroxylamine to the modified enzyme did not reverse the effects of the inhibitor. A form of the proteinase exhibiting only 2-4% of control chymotrypsin-like, trypsin-like, and PGP activities degraded beta-casein with no accumulation of intermediate peptides. 3,4-Dichloroisocoumarin, by reacting with the chymotrypsin-like, trypsin-like, and/or PGP-active sites, may promote a conformational change of MPC, rendering the caseinolytic active site accessible to the substrate. Once bound to the active site, beta-casein is rapidly degraded either by the caseinolytic component itself or by a cooperative interaction with catalytic centers that are not affected by the serine proteinase inhibitor. These results imply that the caseinolytic component does not belong to the class of serine proteinases. Other proteins tested were not degraded by the 3,4-dichloroisocoumarin-treated enzyme, suggesting that the conformation of beta-casein may be more adequate for degradation by the caseinolytic component.
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PMID:3,4-dichloroisocoumarin-induced activation of the degradation of beta-casein by the bovine pituitary multicatalytic proteinase complex. 156 24

The effect of N-acetylimidazole, a mild acetylating reagent, on the catalytic activities and subunit structure of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. The trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide) and the peptidylglutamyl-peptide bond hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) of MPC were rapidly inactivated by N-acetylimidazole, whereas the chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide) was inactivated slowly. However, the hydrolysis of casein was markedly stimulated. Hydrolysis of casein by the acetylated enzyme generated a stable intermediate (21 kDa) which could be further degraded by native MPC. Treatment of acetylated MPC with hydroxylamine reversed the changes in trypsin-like and caseinolytic activities but did not restore the PGP activity. N-Acetylimidazole did not dissociate MPC but altered its migration on nondissociating gels presumably by acetylation of epsilon-amino groups of lysine residues. Hydroxylamine did not alter the gel electrophoretic appearance of the acetylated enzyme. These results indicate that acetylation of thiol or tyrosyl groups changes the trypsin-like and caseinolytic activities, and that amino group acetylation inhibits the PGP activity. Degradation of casein by MPC appears to be a sequential process with initial cleavage catalyzed by a component distinct from the chymotrypsin-like, trypsin-like, and PGP activities. The latter three components likely participate in the secondary proteolysis of the generated intermediates.
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PMID:Chemical modification of the bovine pituitary multicatalytic proteinase complex by N-acetylimidazole. Reversible activation of casein hydrolysis. 189 26

The activity of multicatalytic proteinase against synthetic substrates and the kinetics of its inhibition by a range of class-specific inhibitors have been investigated. The enzyme was found to have a broader pH activity profile than previously noted, being active against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin optimally at pH 4.5 and against benzyloxycarbonyl-Gly-Gly-Arg-7-amino-4-methylcoumarin optimally at pH 10.5. Neither activity was inhibited by the class-specific inhibitors 1,10-phenanthroline, EDTA, pepstatin, di-isopropyl fluorophosphate, peptidyl chloromethanes, peptidyl diazomethanes or L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido-(4-guanidin o)butane (E-64), indicating that the enzyme is not a typical metallo-, aspartic, serine or cysteine proteinase. Inhibition by HgCl2, iodoacetamide and N-ethylmaleimide suggests that free thiols are necessary for the enzyme to maintain activity, but that these thiols are not particularly reactive as is the case for cysteine proteinases of the papain superfamily. The peptidyl aldehydes chymostatin and leupeptin were found to be reversible inhibitors of multicatalytic proteinase. Chymostatin inhibited activity against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin at pH 4.5 (Ki 160 +/- 22 microM) whereas leupeptin (200 microM) was not inhibitory. Inhibition of activity against benzyloxycarbonyl-Gly-Gly-Arg-7-amino-4-methylcoumarin by these compounds was more complex, in that they behaved as slow tight-binding inhibitors. kon values were determined to be 12 +/- 2 M-1.s-1 and 1290 +/- 125 M-1.s-1 for chymostatin and leupeptin, respectively. The upper limit for Ki values for these two inhibitors was estimated as 5 +/- 1.5 microM and 25 +/- 5 nM, respectively. The different inhibition characteristics for each substrate were also apparent at an intermediate pH of 8.5, showing that the two activities are distinct. Dichloroisocoumarin, a mechanism-based inhibitor of serine proteinases, did inhibit activity against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin with a rate constant of 250 M-1.s-1, suggesting that multicatalytic proteinase is an atypical serine proteinase.
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PMID:Characterization of the active site of human multicatalytic proteinase. 230 79

In Drosophila melanogaster the population of proteasome particles consists of three distinct subclasses. By fractionation of a 40,000 x g supernatant of Drosophila homogenate on a DEAE-Sephacel column, proteasome particles which elute at salt concentrations of 200, 300, and 500 mM KAc can be separated. The proteasomes of all three subfractions sediment at 19 S in sucrose gradients and are shown by two-dimensional gel electrophoretic analysis to possess the same protein content. They differ, however, with respect to their specific proteolytic activity against the substrates benzoyl-Val-Gly-Arg-4-methylcoumaryl-7-amide, succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, and succinyl-Ala-Ala-Phe-4-methylcoumaryl-7-amide and the degree to which their hydrolytic activity can be enhanced by the addition of 30-110 microM sodium dodecyl sulfate (SDS). Our data show that the 200 mM proteasome fraction exhibits the lowest basal specific proteolytic activity but can be stimulated most by SDS. The 300 and 500 mM proteasome subfractions, on the other hand possess considerably higher but similar basal specific proteolytic activity. Of these only the proteolytic activity of the 300 mM subfraction against the substrates benzoyl-Val-Gly-Arg-4-methylcoumaryl-7-amide and succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide can be enhanced by SDS. Our data raise the possibility that the different subpopulations reflect structural differences between the proteasome particles, which in turn may result in different in vivo substrate specificities of the proteasome subpopulations.
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PMID:Identification and characterization of three different subpopulations of the Drosophila multicatalytic proteinase (proteasome). 249 19

In our effort to identify the proteolytic specificity of various hemorrhagic toxins isolated from western diamondback rattlesnake venom, hemorrhagic toxin b was isolated in homogeneous form by previously published methods. Hemorrhagic toxin b hydrolyzed glucagon, producing six fragments. The proteolytic sites were identified as Thr(5)-Phe(6), Thr(10)-Ser(11), Asp(15)-Ser(16), Asp(21)-Phe(22) and Try(25)-Leu(26). When oxidized insulin B chain was used, proteolysis occurred at four sites: Asn(3)-Gln(4), His(10)-Leu(11), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The proteolytic specificity of hemorrhagic toxin b is quite different from those of the nonvenom proteases such as thermomycolin, aspergillopeptidase c, alkaline protease from Aspergillus flavus, elastase, subtilisin and papain.
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PMID:Proteolytic specificity of hemorrhagic toxin b from Crotalus atrox (western diamondback rattlesnake) venom. 286 65

An alkaline proteinase, previously identified in rat liver and heart, has been purified from the soluble fraction of human erythrocytes. The proteinase has an apparent molecular weight of 600 000 and is composed of eight subunits with molecular weights ranging from 32 000 to 21 000. The proteinase degrades both protein and synthetic peptide substrates with a broad pH optimum of 7.5-11.0. Among the synthetic peptides tested, tripeptides with arginine at the P1 position (e.g. Z-Val-Leu-Arg-4-methoxy-2-napthylamine and Boc-Leu-Gly-Arg-4-methylcoumarin-7-amide) are particularly good substrates. The proteinase appears to be sulfhydryl-dependent and is inhibited completely by mersalyl acid and by hemin; inhibitors of serine and metallo-type proteinases have no effect on proteinase activity. Interestingly, a variety of other proteinase inhibitors such as leupeptin, chymostatin and N-ethylmaleimide failed to completely inhibit protein-hydrolyzing activities of the enzyme. These results indicate that these activities may be accounted for by at least two different catalytic sites. Proteinase activity is stable in the presence of 1 M urea, 0.5% Triton X-100 or 0.03% SDS and is not affected by ATP. Based on the high molecular weight and sulfhydryl-dependence, we have named this proteinase macropain.
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PMID:Purification and characterization of a high molecular weight proteinase (macropain) from human erythrocytes. 353 Mar 30

A multicatalytic proteinase from rat skeletal muscle contains active site(s) catalysing the degradation of benzoyl-Val-Gly-Arg 4-methyl-7-coumarylamide, succinyl-Ala-Ala-Phe 4-methylcoumarylamide and [14C]methylcasein as well as benzyloxy-carbonyl-Leu-Leu-Glu 2-naphthylamide. These activities are 7-14-fold activated by 1 mM-sodium dodecyl sulphate. The activation leads to a higher susceptibility to the proteinase inhibitor chymostatin and to a lower ability to be inhibited and precipitated by antibodies raised against the non-activated enzyme. Since no changes in Mr or subunit composition were observed in the SDS-activated form, some conformational changes seem to occur during the activation step. More pronounced activation was observed in the presence of physiological concentrations of fatty acids; oleic acid at 100 microM concentrations stimulated the proteinase about 50-fold. In contrast with the non-activated proteinase, the activated enzyme considerably degrades muscle cytoplasmic proteins in vitro. Thus it is not unlikely that, in vivo, potential activators such as fatty acids can induce the multicatalytic proteinase to participate in muscle protein breakdown.
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PMID:Activation of the multicatalytic proteinase from rat skeletal muscle by fatty acids or sodium dodecyl sulphate. 389 Aug 40

Enzyme-IIIglc is part of the glucose phosphotransferase system of Escherichia coli and Salmonella typhimurium and is phosphorylated by phosphoenolpyruvate in a reaction requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase), and the histidine-containing phospho-carrier protein HPr. In this paper we report the isolation of IIIglc from E. coli and the characterization of the active center. Alkaline hydrolysis of [32P]P-IIIglc and chromatography of the hydrolysate suggested that the phosphoryl group is bound to a histidyl residue in P-IIIglc of S. typhimurium. Here we present 1H-NMR measurements of IIIglc and P-IIIglc from E. coli which further substantiate that the phosphoryl group in P-IIIglc is linked to the N-3 position of a histidyl residue. After phosphorylation of IIIglc with [32P]Phosphoenolpyruvate, enzyme I and HPr, the phosphorylated protein was cleaved with either alkaline protease from Streptomyces griseus or subtilisin from Bacillus subtilis. According to amino acid analysis both proteases produced the same peptide carrying the phosphoryl group. The amino acid sequence of this peptide was found to be Val-His-Phe-Gly-Ile-Asp. The lower electrophoretic mobility of P-IIIglc on dodecylsulfate/polyacrylamide gels and its stronger binding to the hydrophobic matrix of a reversed-phase column compared to unphosphorylated protein may indicate a structural change following phosphoenolpyruvate-dependent phosphorylation.
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PMID:Phosphoenolpyruvate-dependent phosphorylation site in enzyme IIIglc of the Escherichia coli phosphotransferase system. 638 26

A multicatalytic proteinase complex present in the skin secretion of Xenopus laevis was purified and its enzymatic activity towards natural and synthetic peptides was investigated. We identified three activities: i) a C-terminal deamidation enzyme activity which exhibited selectivity for the Asp-Phe-NH2 and Phe-Leu-NH2 motifs of cerulein, minigastrin Leu-enkephalinamide, (des-Tyr1)Leu-enkephalinamide and diaminobenzylthiocyanate-DVDERDVRGFASFLNH2 (DABTC-DR8kermit); ii) an endopeptidase activity that cleaves peptide bonds on the carboxyl side of hydrophobic amino acid residues such as Tyr-Gly of LHRH, Ile-Ala of PGLa and Leu-Ala of buccalin; iii) an enzyme activity that cleaves peptide bonds at the dibasic sites of peptides of the dynorphin family. The molecular weight determined by Sephacryl S-400 molecular sieve filtration indicated an M(r) about 600 kDa. The activities characterized here exhibit an optimal pH of about 7.4. The activities of the multicatalytic complex were differentially inhibited by the classical inhibitors of proteases.
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PMID:Isolation and properties of a multicatalytic proteinase complex from Xenopus laevis skin secretion. 755 6


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