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:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The underlying basis of the main chain directed (MCD) resonance assignment strategy for the analysis of 1H NMR spectra of proteins is reexamined. The criteria used in the construction of the patterns used in the MCD method have been extended to increase the robustness of the approach to the presence of variable protein secondary structure and significant spectral degeneracy. These criteria have led to the development of several dozen patterns exclusively involving the short distance relationships between main chain amide NH-C alpha-H-C beta H (NAB) J-coupled subspin systems of the amino acid residues. The MCD patterns have been examined for fidelity and frequency of occurrence in a database composed of the high resolution crystal structures of 39 proteins. The analysis has identified several extremely robust patterns, suitable for initiating a hierarchical construction of units of secondary structure based upon a systematic analysis of two-dimensional nuclear Overhauser effect spectra. A formal procedure, suitable for the computer assisted application of the MCD strategy, is developed. This procedure, termed MCDPAT, has been applied to the analysis of the crystal structures of human ubiquitin, T4 lysozyme, and ribonuclease A. It has been found that the MCDPAT procedure is conservative producing no significant errors and is globally successful in correctly identifying the appropriate units of secondary structure contained in these three proteins.
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PMID:Refinement of the main chain directed assignment strategy for the analysis of 1H NMR spectra of proteins. 186 55

Mammalian cells contain two large proteolytic complexes, the 650-kDa proteasome (or multicatalytic protease) and the 1500-kDa (26 S) Ubiquitin-conjugate-degrading enzyme. Since the proteasome is also required for the ATP-dependent degradation of ubiquitinated proteins, we tested whether it may be a component of the larger complex. The proteasome normally is soluble in 38% ammonium sulfate. However, after preincubation of reticulocyte extracts with ATP, several proteasome activities appeared in the 38% ammonium sulfate pellet, including the ability to degrade hydrophobic peptides and 14C-casein. Also, following preincubation with ATP, the precipitable fraction could degrade 125I-lysozyme-ubiquitin (Ub) conjugates. The activities were not present after incubation without ATP or with a nonmetabolizable ATP analog. Nondenaturing gel electrophoresis indicated the ATP-dependent appearance of a new band which degraded proteasome substrates, and reacted with an anti-proteasome monoclonal antibody on Western blot. This new band appeared larger than the proteasome and migrated similarly to the larger Ub-conjugate-degrading complex. The formation of the larger complex required factor(s) present in the 38% ammonium sulfate pellet and either the 40-80% fraction or the purified proteasome from reticulocytes or muscle. After complex formation, hydrolysis of Ub-protein conjugates and also the non-ubiquitinated substrate, casein, was stimulated severalfold by ATP, but non-metabolizable ATP analogs had little or no effect. Thus, the proteasome corresponds to component CF-3 of Ganoth et al. (Ganoth, D., Leshinisky, E., Eytan, E., and Hershkov, A. (1989) J. Biol. Chem. 263 12412-12419) and undergoes an energy-dependent association with other factors to form the 1500-kDa, ATP-requiring proteolytic complex.
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PMID:The proteasome (multicatalytic protease) is a component of the 1500-kDa proteolytic complex which degrades ubiquitin-conjugated proteins. 218 Sep 50

Immobilized metal ion affinity chromatography (IMAC) has been explored as a probe into the topography of histidyl residues of a protein molecule. An evaluation of the chromatographic behavior of selected model proteins--thioredoxin, ubiquitin, calmodulin, lysozyme, cytochrome c, and myoglobin on immobilized transition metal ions (Co2+, Ni2+, Cu2+, and Zn2+)--allows establishment of the following facets of the histidyl side chain distribution: (i) either interior or surface; (ii) when localized on the surface, accessible or unaccessible for coordination; (iii) single or multiple; (iv) when multiple, either distant or vicinal. Moreover, proteins displaying single histidyl side chains on their surfaces may, in some instances, be resolved by IMAC; apparently, the microenvironments of histidyl residues are sufficiently diverse to result in different affinities for the immobilized metal ions. IMAC, previously introduced as an approach to the fractionation of proteins, has become also, upon closer examination, a facile probe into the topography of histidyl residues. This is possible because of the inherent versatility of IMAC; an appropriate metal ion (M2+) can be selected to suit the analytical purpose and a particular chromatographic protocol can be applied (isocratic pH, falling pH, and imidazole elution).
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PMID:Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. 253 16

Eukaryotic cells contain a 700-kDa proteolytic complex (the "proteasome" or multicatalytic endopeptidase complex), whose role in intracellular protein breakdown is unclear. It has been suggested that the proteasome functions in the rapid degradation of oxidant-damaged proteins and in the ATP-dependent proteolytic pathway. To test these possibilities, oxidant-damaged hemoglobin and albumin were produced by treating hemoglobin and albumin with phenylhydrazine, with hydroxyl radicals, or with both hydroxyl and superoxide radicals. After oxidant damage, these proteins were degraded more rapidly in erythrocyte extracts and also by the purified proteasome. However, complete removal of proteasomes from these extracts by immunoprecipitation (or inhibitors of its proteolytic activity) did not reduce the breakdown of oxidant-damaged hemoglobin and decreased degradation of hydroxyl- and superoxide-treated proteins by only 30-40%. Thus, erythrocytes must contain another proteolytic system for degradation of oxidant-damaged proteins. In contrast, immunoprecipitation of proteasomes with polyclonal or monoclonal antibodies prevented the ATP/ubiquitin-dependent degradation of lysozyme and also blocked the ATP-stimulated degradation of ubiquitin-conjugated lysozyme in reticulocyte and skeletal muscle extracts. These data indicate a critical role of the proteasome in the degradation of ubiquitin-conjugated proteins and suggest that the proteasome is associated with or is a component of the larger ubiquitin-conjugate-degrading enzyme complex.
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PMID:Involvement of the proteasome in various degradative processes in mammalian cells. 253 95

When intact HeLa cells were incubated at 45 degrees C, there was progressive inactivation of proline endopeptidase. Rapid loss of the enzyme did not occur in extracts maintained at 45 degrees C. Since Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no decrease in the immunoreactive 70-kDa proline endopeptidase band, its in vivo disappearance apparently results from irreversible denaturation or modification. Loss of proline endopeptidase activity was paralleled by reduced degradation of injected ubiquitin and bovine serum albumin. In contrast, proteolysis of injected lysozyme or pancreatic trypsin inhibitor was barely affected. Electrophoretic analysis of ubiquitin or bovine serum albumin retrieved from heated HeLa cells showed that the injected proteins were intact. Thus, the presence of proline endopeptidase appears to be required for initial cleavage of these two substrates, but it has not been shown that the enzyme is directly responsible. Selective stabilization of a subset of the injected proteins does, however, demonstrate the existence of distinct proteolytic pathways in HeLa cytosol.
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PMID:Proteolysis in heat-stressed HeLa cells. Stabilization of ubiquitin correlates with the loss of proline endopeptidase. 254 10

In the multienzyme ubiquitin-dependent proteolytic pathway, conjugation of ubiquitin to target proteins serves as a signal for protein degradation. Rabbit reticulocytes possess a family of proteins, known as E2's, that form labile ubiquitin adducts by undergoing transthiolation with the ubiquitin thiol ester form of ubiquitin activating enzyme (E1). Only one E2 appears to function in ubiquitin-dependent protein degradation. The others have been postulated to function in regulatory ubiquitin conjugation. We have purified and characterized a previously undescribed E2 from rabbit reticulocytes. E2(230K) is an apparent monomer with a molecular mass of 230 kDa. The enzyme forms a labile ubiquitin adduct in the presence of E1, ubiquitin, and MgATP and catalyzes conjugation of ubiquitin to protein substrates. Exogenous protein substrates included yeast cytochrome c(Km = 125 mu M; kcat approximately 0.37 min-1) and histone H3 (Km less than 1.3 mu M; kcat approximately 0.18 min-1) as well as lysozyme, alpha-lactalbumin, and alpha-casein. E2(230K) did not efficiently reconstitute Ub-dependent degradation of substrates that it conjugated, either in the absence or in the presence of the ubiquitin-protein ligase that is involved in degradation. E2(230K) may thus be an enzyme that functions in regulatory Ub conjugation. Relative to other E2's, which are very iodoacetamide sensitive, E2(230K) was more slowly inactivated by iodoacetamide (k(obs) = 0.037 min-1 at 1.5 mM iodoacetamide; pH 7.0, 37 degrees C). E2(230K) was also unique among E2's in being subject to inactivation by inorganic arsenite (k(i)max = 0.12 min-1; K(0.5) = 3.3 mM; pH 7.0, 37 degrees C). Arsenite is considered to be a reagent specific for vicinal sulfhydryl sites in proteins, and inhibition is usually rapidly reversed upon addition of competitive dithiol compounds. Inactivation of E2(230K) by arsenite was not reversed within 10 min after addition of dithiothreitol at a concentration that blocked inactivation if it was premixed with arsenite; inactivation is therefore irreversible or very slowly reversible. We postulate that a conformation change of E2(230K) may be rate-limiting for interaction of enzyme thiol groups with arsenite.
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PMID:A novel, arsenite-sensitive E2 of the ubiquitin pathway: purification and properties. 255 69

It was found previously that the enzyme ubiquitin-protein ligase (E3) contains specific protein substrate binding sites that are responsible for the selection of proteins for degradation by the ubiquitin system. In the present study, we have tried to gain more insight into the mode of action of E3 by the characterization of other binding sites of this enzyme. Following the ligation of ubiquitin to 125I-lysozyme, the conjugates produced are very tightly bound to E3, as indicated by size analysis on glycerol density gradient centrifugation. The strong binding of ubiquitin-protein conjugates to the enzyme may account for the apparently processive addition of multiple molecules of ubiquitin to the protein substrate. Both the protein substrate moiety and the ubiquitin moiety participate in the interaction of ubiquitin-protein conjugates with E3, as indicated by competition with specific agents and by the comparison of the binding of ubiquitin-conjugated protein to that of free protein. In addition to the binding of its substrates and products, E3 also appears to interact with some of the enzymes with which it acts in concert. When E3 is incubated with the ubiquitin-carrier protein E2, a complex is formed between the two enzymes as analyzed on glycerol gradients. The formation of an E2.E3 complex may facilitate the transfer of activated ubiquitin from E2 to the protein substrate bound to the ligase.
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PMID:Binding sites of ubiquitin-protein ligase. Binding of ubiquitin-protein conjugates and of ubiquitin-carrier protein. 273 27

Although protein breakdown in most cells seems to require metabolic energy, it has only been possible to establish a soluble ATP-dependent proteolytic system in extracts of reticulocytes and erythroleukemia cells. We have now succeeded in demonstrating in soluble extracts and more purified preparations from rabbit skeletal muscle a 12-fold stimulation by ATP of breakdown of endogenous proteins and a 6-fold stimulation of 125I-lysozyme degradation. However, it has still not been possible to demonstrate such large effects of ATP in similar preparations from liver. Nevertheless, after fractionation by DEAE-chromatography and gel filtration, we found that extracts from liver as well as muscle contain both the enzymes which conjugate ubiquitin to 125I-lysozyme and an enzyme which specifically degrades the ubiquitin-protein conjugates. When this proteolytic activity was recombined with the conjugating enzymes, ATP + ubiquitin-dependent degradation of many proteins was observed. This proteinase is unusually large, approx. 1500 kDa, requires ATP hydrolysis for activity and resembles the ubiquitin-protein-conjugate degrading activity isolated from reticulocytes. Thus the ATP + ubiquitin-dependent pathway is likely to be present in all mammalian cells, although certain tissues may contain inhibitory factors.
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PMID:Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system. 282 Mar 75

In eukaryotes, a major route for ATP-dependent protein breakdown proceeds through covalent intermediates of target proteins destined for degradation and the highly conserved, 76 amino acid protein ubiquitin. In rabbit reticulocytes, it has been shown that hemin effectively inhibits this pathway by blocking the catabolism of ubiquitin-protein conjugates [KI = 25 microM (Haas, A. L., & Rose, I. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6845-6848)]. Here, we demonstrate that hemin is also an effective inhibitor of the ubiquitin-dependent proteolytic pathway in both a higher plant, oats (Avena sativa), and yeast (Saccharomyces cerevisiae). Hemin inhibits all stages of the pathway in vitro, including ATP-dependent formation of ubiquitin-protein conjugates, disassembly of conjugates by ubiquitin-protein lyase(s) (or isopeptidases), and degradation of ubiquitin-protein conjugates by ATP-dependent protease(s). Using ubiquitin-125I-lysozyme conjugates synthesized in vitro as substrates, we determined the specific effects of hemin on the rates of disassembly and degradation separately. The concentration of hemin required for half-maximal inhibition of both processes was identical in each species, approximately 60 microM in oats and approximately 50 microM in yeast. Similar inhibitory effects were observed when two hemin analogues, mesoheme or protoporphyrin IX, were employed. These results demonstrate that the effect of hemin on ubiquitin-dependent proteolysis is not restricted to erythroid cells and as a result hemin may be a useful tool in studies of this pathway in all eukaryotic cells. These results also question models where hemin serves as a specific negative modulator of proteolysis in erythroid cells.
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PMID:Hemin inhibits ubiquitin-dependent proteolysis in both a higher plant and yeast. 283 30

It was found previously that proteins conjugated to ubiquitin are degraded by an ATP-dependent enzyme system, but the mode of action of this system was unknown. We have resolved from reticulocyte extracts three factors that are required for the ATP-dependent breakdown of 125I-lysozyme-ubiquitin conjugates. Two of the factors interact with ATP, as shown by their protection against heat inactivation by the nucleotide. When the three factors are incubated with 125I-lysozyme-ubiquitin conjugates and ATP, there is a lag of 4-6 min in the formation of acid-soluble products before the onset of rapid proteolysis. The lag can be abolished by incubation of the three factors with MgATP prior to the addition of the substrate. This "activation" process does not take place if any of the three factors is omitted from preincubation (and added subsequently) or when ATP is replaced by a nonhydrolyzable analog. Analysis of size distribution by glycerol density gradient centrifugation showed that following incubation of the three factors with MgATP, a high molecular mass (greater than 1000 kDa) activity is formed. That the high molecular weight form is a complex of the three factors is indicated by the finding that its formation is accompanied by a corresponding decrease in the levels of the free forms of all three factors. Complex formation seems to be similar to the activation process with regard to time course, requirements for ATP and Mg2+, partial effect of CTP, and lack of effect of nonhydrolyzable ATP analogs. It is suggested that one role of ATP in conjugate breakdown is the formation of an active multienzyme complex.
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PMID:A multicomponent system that degrades proteins conjugated to ubiquitin. Resolution of factors and evidence for ATP-dependent complex formation. 284 33


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