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 steady-state kinetics of the alkaline mesentericopeptidase-catalysed hydrolysis of esters of the general formula Ac-X-OMe(OEt) has been studied, "X" being an amino acid residue (Ala, Val, Leu, Ile, Phe, Tyr, Trp, Lys). The values of the specificity ratio kcat/Km indicate that the bonds involving the carboxyl group of amino acids with aromatic and bulky aliphatic side chain are hydrolysed most effectively. On account of this, alkaline mesentericopeptidase is classified as a chymotrypisn-like alkaline protease. The primary specificity of mesentericopeptidase reveals the similarity of this enzyme to the group of subtilisins, as well as the distinctive characteristic feature of the enzyme to hydrolyse Ac-Leu-OMe with an efficiency practically equal to that of aromatic amino acid derivatives. Suggestions are made about the nature of the substrate-binding centre, taking into consideration Schechter's and Berger's concept of the secondary specificity.
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PMID:Primary specificity of alakaline mesentericopeptidase. Kinetic parameters for the hydrolysis of alpha-N-acetyl-L-amino acid methyl esters. 63 84

The sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase (EC 2.4.1.1) has been determined. Limited proteolysis of native phosphorylase b (841 residues, subunit molecular weight 97 412) by subtilisin BPN', Streptomyces alkaline protease, or elastase yielded two large segments (light and heavy). The light segment isolated from the subtilisin digest was cleaved at methionyl bonds with cyanogen bromide to yield eight major fragments and two minor overlapping fragments. The alignment of the major fragments was obtained by analysis of the two minor fragments, of five tryptic peptides containing methionine and of one large fragment generated by cleavage of an aspartylproline bond. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule identified the sites susceptible to limited proteolysis and the overlap between the light and the heavy segments. Serine-14 and tyrosine-155 were identified as the residues involved in the covalent and allosteric controls of the enzyme, respectively. Residues 108 and 142 were identified as the cysteine residues reported to be involved in the aggregation of subunits.
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PMID:Sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase including the sites of covalent and allosteric control. 72 24

The fine purification of an alkaline protease (thermitase) from Thermoactinomyces vulgaris by means of isoelectrical focussing in the flat-bed procedure using granulated gel is reported. An Na2SO4-precipitated crude product serves as the starting material. Isoelectrical focussing leads in a single step to a highly purified protein with an uniform N-terminal end group. The enzyme has an IP at 9.0 and a mol. wt. of 37,400; it consists of a polypeptide chain with arginine as the N-terminal, and tyrosine as the C-terminal end group. In addition to an essential serine residue, a SH group could be demonstrated which is hardly accessible in the native enzyme. Furthermore, the influence of different protease inhibitors was studied.
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PMID:[Characterization of a protease from Thermoactinomyces vulgaris (thermitase). 2. Single-step fine purification and protein-chemical characterization]. 74 56

Screening of a lambda gt11 cDNA expression library of the HeLa cell genome with a monoclonal antibody that specifically recognizes prosomal 30-33-kDa proteins, allowed isolation of a 1264-nucleotide (nt) recombinant cDNA containing a 327-nt untranslated 5'-end. The amino acid (aa) sequence deduced from this cDNA revealed a protein of 269 aa (M(r) of 30,227) that includes a consensus box characteristic for Tyr phosphorylation, also observed in other prosomal proteins. Comparison with another prosomal 27-kDa protein, cloned in our laboratory, indicated the presence of three prosome-specific homology boxes observed in these proteins from archaebacteria to man. Interestingly, except for the untranslated 5'-end, as well as the sequence coding for the N-terminal six aa, this cDNA is identical to two recently published cDNAs encoding subunit C2 of human liver proteasome [Tamura et al., Biochim. Biophys. Acta 1089 (1991) 95-102] and subunit NU of human erythrocyte macropain [DeMartino et al., Biochim. Biophys. Acta 1079 (1991) 29-38]. Primer extension and Northern blot analysis using two specific 18-mer oligodeoxyribonucleotides indicated the presence of two mRNAs that have divergent 5'-ends. These results, as confirmed by the polymerase chain reaction, establish the existence of two distinct Hs PROS-30 mRNAs, differing in their 5'-noncoding regions and in the N-terminal six aa of their protein products.
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PMID:Two mRNAs exist for the Hs PROS-30 gene encoding a component of human prosomes. 139 36

The multicatalytic proteinase (MCP) complex catalyses cleavage of bonds on the carboxy-group side of basic, hydrophobic or acidic amino acid residues. Originally, it was proposed that the complex contained three distinct types of catalytic component. MCP from rat liver has been assayed for so-called trypsin-like activity with Boc-Leu-Ser-Thr-Arg-NH-Mec (Mec, 4-methylcoumarin; Boc, t-butoxycarbonyl), for chymotrypsin-like activity with Ala-Ala-Phe-NH-Mec and Suc-Leu-Leu-Val-Tyr-NH-MEc (Suc, succinyl), and peptidyl-glutamylpeptide hydrolase activity with Cbz-Leu-Leu-Glu-Nap (Nap, naphthylamide; Cbz, benzyloxycarbonyl). Results of these studies suggest that as many as five distinct components can be distinguished, one for the trypsin-like activity and two for each of the others. The activities were tested with a variety of serine-protease inhibitors, and other novel effectors have also been identified. The two most effective inhibitors were 4-(2-amino-ethyl)benzenesulphonyl fluoride, which selectivity inactivates the trypsin-like activity, and 3,4-dichloroisocoumarin which inhibits chymotrypsin-like activity and the second, cooperative component [Djaballah, H. & Rivett, A. J. (1992) Biochemistry 31, 4133-4141] of peptidylglutamylpeptide hydrolase activity. The three activities inhibited by 3,4-dichloroisocoumarin can easily be distinguished by the effects of chymostatin analogues, diisopropylfluorophosphate, guanidine/HCl and casein. The results support the view that the enzyme is a novel type of serine protease and suggest that it may contain at least five distinct catalytic components. Marked differences in the reactivities of the different catalytic sites with different reagents can be used to distinguish between them.
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PMID:Use of serine-protease inhibitors as probes for the different proteolytic activities of the rat liver multicatalytic proteinase complex. 142 69

The crystal structure of subtilisin BL, an alkaline protease from Bacillus lentus with activity at pH 11, has been determined to 1.4 A resolution. The structure was solved by molecular replacement starting with the 2.1 A structure of subtilisin BPN' followed by molecular dynamics refinement using X-PLOR. A final crystallographic R-factor of 19% overall was obtained. The enzyme possesses stability at high pH, which is a result of the high pI of the protein. Almost all of the acidic side-chains are involved in some type of electrostatic interaction (ion pairs, calcium binding, etc.). Furthermore, three of seven tyrosine residues have potential partners for forming salt bridges. All of the potential partners are arginine with a pK around 12. Lysine would not function well in a salt bridge with tyrosine as it deprotonates at around the same pH as tyrosine ionizes. Stability at high pH is acquired in part from the pI of the protein, but also from the formation of salt bridges (which would affect the pI). The overall structure of the enzyme is very similar to other subtilisins and shows that the subtilisin fold is more highly conserved than would be expected from the differences in amino acid sequence. The amino acid side-chains in the hydrophobic core are not conserved, though the inter-residue interactions are. Finally, one third of the serine side-chains in the protein have multiple conformations. This presents an opportunity to correlate computer simulations with observed occupancies in the crystal structure.
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PMID:The crystal structure of the Bacillus lentus alkaline protease, subtilisin BL, at 1.4 A resolution. 145 65

We have reinvestigated the recent proposal that the multicatalytic proteinase, together with other components of reticulocyte lysate, may become incorporated into a very large, "26 S" proteinase complex via an ATP-dependent process. Different from these published results, we consistently isolate the multicatalytic proteinase as a 650,000 Da "20 S" multisubunit proteinase. Analysis on nondenaturing polyacrylamide gels of reticulocyte fractions containing the putative complexed form of the multicatalytic proteinase reveal that activity against succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin is associated with two groups of protein of different molecular mass. One migrates like multicatalytic proteinase purified to homogeneity, displays, on sodium dodecyl sulfate gels, a set of protein species in the range of 23,000-32,000 Da, characteristic of the multicatalytic proteinase, and is recognized by a monospecific antibody to the enzyme. In contrast, the activity associated with the higher molecular mass (26 S) proteinase complex lacks the typical multicatalytic proteinase subunits and is devoid of antigenic material, when tested with the antibody. These results confirm and extend our recent findings in mouse liver by showing that the multicatalytic proteinase is not a constituent of a 26 S proteinase complex.
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PMID:Evidence indicating that the multicatalytic proteinase of rabbit reticulocytes is not incorporated as a core enzyme into a 26 S proteinase complex. 157 17

On the basis of recent reports that suggested that proteasomes, via an ATP-dependent process, become integral components of a '26 S' complex possessing 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity, we have investigated the molecular interaction of proteasomes in ATP-stabilized fraction II (proteins absorbed on DEAE-matrix and eluted with 0.5 M-KCl) of rabbit reticulocytes and mouse liver. Analysis of the various extracts by (NH4)2SO4 fractionation, velocity-gradient centrifugation, non-denaturing PAGE and SDS/PAGE and immunoblotting with proteasome-specific antisera failed to identify the proteasome as part of a higher-molecular-mass '26 S' multienzyme complex. In all instances proteasomes are identified in their 'free' 650 kDa '20 S' form. In addition to the proteasome and independent of the presence of MgATP, we isolated a high-molecular-mass proteinase whose electrophoretic migration behaviour and sedimentation rate correspond to that of the previously described '26 S' proteinase. This '26 S' proteinase possesses a strong 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity and is composed of several non-identical polypeptides in the molecular-mass range 20-150 kDa. Despite its similarity to proteasomal enzyme activity, protein analysis and immunoblotting experiments demonstrate that neither the intact proteasome nor subunits thereof are components of the '26 S' proteinase complex.
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PMID:Molecular interaction of the proteasome (multicatalytic proteinase). Evidence that the proteasome is not a constituent of the '26 S' multienzyme complex. 174 50

During a three-day fast, followed by four days of refeeding, the content of the multicatalytic proteinase as well as hydrolyzing activity towards Suc-Leu-Leu-Val-Tyr-7-amino-4-methylocoumarin (SLLVT-MCA) was measured in various rat tissues. When compared with normal rats, the MCP content, as determined by immunochemical techniques, was unchanged over the entire experimental period in the three tissues examined: gastrocnemius muscle, thymus and testis. By contrast, a differential response was observed in the three tissues with respect to specific and total SLLVT-MCA splitting activity: for thymus and testis, these values were again unchanged, whereas in gastrocnemius muscle, both specific and total enzyme activity fell by almost 70% on day three of fasting but returned to control values on day four of refeeding. This change in activity was not due to the accumulation or degradation of a specific proteinase inhibitor. Data demonstrate that, in association with the insulin-deficient state of starvation, the activity of the multicatalytic proteinase shows an adaptive behaviour which becomes manifest in some but not in other tissues.
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PMID:Tissue-specific changes of multicatalytic proteinase activity in the fasted rat. 184 9

The proteolytic activities of the 20 S proteasome were found to change in their levels during the development of chick embryonic muscle. The peptide-cleaving activities against N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy-beta-naphthylamide gradually decreased with the time of development. On the other hand, the casein-degrading activity in the presence of poly-L-lysine markedly increased from embryonic day 11 and reached a maximal level by day 17. These changes appeared to be tissue-specific because little or no change in any of the proteolytic activities was observed with developing embryonic brain, while dramatic alterations occurred in the extents of the peptide hydrolyses in liver. Furthermore, a number, but not all, of the proteasome subunits in embryonic muscle were changed in their amounts during the development. These results suggest that the alterations in the proteasome activities and subunit pattern are developmentally regulated and may be correlated.
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PMID:Developmental regulation of proteolytic activities and subunit pattern of 20 S proteasome in chick embryonic muscle. 187 33


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