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 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

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 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

Studies were carried out to characterize further the cytoplasmic ATP- and ubiquitin-independent proteolytic system in red blood cells that degrades hemoglobin damaged by exposure to oxidants (Fagan, J. M., Waxman, L., and Goldberg, A. L. (1986) J. Biol. Chem. 261, 5705-5713). Several proteases were ruled out as having a major role in the degradation of oxidant-treated hemoglobin (Ox-Hb). Acid hydrolases are not active in this process since the degradation of Ox-Hb has a pH optimum between 6 and 8. The calpains are also not involved since inhibitors of cysteine proteases (leupeptin and trans-epoxysuccinyl-L-leucylamido-(3-methyl)butane) did not diminish the increased proteolysis in intact erythrocytes treated with oxidants or in lysates to which Ox-Hb was added. The degradation of Ox-Hb was unaffected by inhibitors of serine and aspartic proteases. Removal of the high M(r) multicatalytic proteinase by immunoprecipitation also did not significantly affect the degradation of Ox-Hb in erythrocyte lysates. The degradation of Ox-Hb was sensitive to metal chelators and sulfhydryl-modifying reagents but not to specific inhibitors of known metalloproteases. Insulin, which is rapidly degraded in lysates, completely blocked the degradation of Ox-Hb. Insulin- and Ox-Hb-hydrolyzing activity was also inhibited following immunoprecipitation of the 100-kDa metalloinsulinase. The metalloinsulinase, which is inhibited by sulfhydryl-modifying reagents and which requires divalent metals, may therefore participate in the degradation of hemoglobin damaged by oxidants in erythrocytes.
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PMID:The ATP-independent pathway in red blood cells that degrades oxidant-damaged hemoglobin. 142 49

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

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

Human granulocytes (polymorphonuclear leucocytes, PMN) possess a membrane cofactor protein (MCP, CD46), which is structurally and functionally distinct from the MCPs of other cell types: it shows a single broad band of 56-80 kDa (without the doublet pattern characteristic of MCP) on SDS/PAGE and has less affinity for complement component C3b. We purified PMN MCP using monoclonal antibodies in order to study the molecular differences between it and other MCPs. Several forms of PMN MCP with size heterogeneity were noted on SDS/PAGE and by immunoblotting. O-Glycanase treatment decreased this heterogeneity, yielding a fast-migrating component identical in position on SDS/PAGE to the O-glycanase-treated MCP of other cells. The cell-specific variation of MCP, therefore, arises from post-translational glycosylation and not from a difference in primary structure. The Factor I cofactor activity of PMN MCP was more efficient in cleaving the methylamine-treated complement components C4/C3 than was MCP from other cells, which shared a similar potency of cofactor activity on a weight basis. Two types of small-form PMN MCP were identified during purification. These were 42 kDa and 30 kDa in size; the former was recognized by M177 (a monoclonal antibody against the active site marker), possessed N-linked sugars [located on the short consensus repeats (SCRs)] but not O-linked ones (on the Ser/Thr-rich region), and retained cofactor activity for C3b/C4b cleavage, similar in potency to that of other MCPs. The functionally active soluble form of MCP was observed specifically in PMN. Protease inhibitors did not inhibit liberation of the fragments, although the generated fragments became susceptible to serine proteases. The findings show that the SCRs are the functional domain of MCP and that the MCP proteolysis found only in PMN may modulate the properties of PMN MCP. In conclusion, the structural features of PMN MCP largely reflect a variability in the O-linked sugars, and the decreased affinity for C3b may be in part attributable to proteolysis.
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PMID:Polymorphism and proteolytic fragments of granulocyte membrane cofactor protein (MCP, CD46) of complement. 173 95

1. Lobster muscles contain a latent multicatalytic proteinase; heating at 60 degrees C for 1-2 min converts the latent form to a heat-activated form with enhanced proteolytic activity. Both forms have three endopeptidase activities, which are classified as the trypsin-like, chymotrypsin-like, and peptidylglutamylpeptide bond hydrolyzing activities. 2. Sulfhydryl reagents (mersalyl acid, N-ethylmaleimide, hemin, iodoacetamide, and p-chloromercurisulfonic acid), benzamidine, and chloromethyl ketones inhibited all three activities of the heat-activated form. Leupeptin and antipain inhibited only the trypsin-like activity, while the chymotrypsin-like activity was the most sensitive to diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, aprotinin, and soybean trypsin inhibitor. Pepstatin and L-trans-epoxysuccinylpeptides had little effect on the peptidase activities. 3. Sodium dodecyl sulfate and oleic acid preferentially activated the peptidylglutamyl-peptide hydrolyzing activity of the latent form, whereas N-ethylmaleimide stimulated both the trypsin-like and peptidylglutamyl-peptide hydrolases. These results suggest that the lobster enzyme is an atypical serine proteinase.
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PMID:Differential effects of oleic acid, sodium dodecyl sulfate, and protease inhibitors on the endopeptidase activities of the lobster multicatalytic proteinase. 176 21

Two metalloendopeptidases, designated as Streptomyces griseus metalloendopeptidases I and II (SGMPI and SGMPII), were isolated from a commercial Pronase P by a method including affinity chromatography on carbobenzoxy-L-alaninyl-triethylenetetraminyl-Sepharose (Z-Ala-T-Sepharose). The two enzymes differed from each other in behavior on ion-exchange chromatography but showed the same amino-terminal sequence at least up to the 20th residue. Their molecular weights were both estimated to be 37,000 by SDS-polyacrylamide gel electrophoresis. Elemental and amino acid composition analyses indicated that both of them contained about 1 g atom of zinc and one cystine residue per mol of protein. Cleavage specificities of the two enzymes toward synthetic peptide-substrates were very similar to those observed with thermolysin. EDTA, o-phenanthroline, and phosphoramidon strongly inhibited these enzymes, while typical serine-protease inhibitors and cysteine-protease inhibitors had no effect. The findings clearly indicate that SGMPI and SGMPII can be classified into the family of zinc-endopeptidases. It was unexpectedly found, however, that these metalloendopeptidases were strongly inhibited by protein serine-protease inhibitors produced by Streptomycetes, such as Streptomyces subtilisin inhibitor (SSI), alkaline protease inhibitor-2c' (API-2c'), and plasminostreptin (PS).
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PMID:Purification and characterization of Streptomyces griseus metalloendopeptidases I and II. 176 59


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