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)

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

A post-translational protein modification system involving the polypeptide ubiquitin results in ubiquitin-protein conjugates of various functions. A ubiquitin-conjugating enzyme system was isolated from the epithelial tissue of bovine eye lens by DEAE-Sepharose and Bio-Gel A-1.5m column chromatography. The lens system shows similar enzymatic properties to the one from rabbit reticulocytes: requirement for ATP and sensitivity to thiol reagents. Two sets of prominent ubiquitin conjugates were formed with endogenous ubiquitin-acceptor proteins from fractions of the Bio-Gel column: a pair of ubiquitin conjugates of approximately 130 kDa and others with very high molecular mass. Extreme specificity is indicated by the ability of the lens system to catalyze conjugation of ubiquitin to the few endogenous acceptor proteins, or to histone H2B, but not to lysozyme, S-carboxymethylated bovine serum albumin, or native or heat-denatured lens alpha crystallin.
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PMID:Properties of the ubiquitin conjugation system from bovine eye lens. 284 35

Soluble, cell-free extracts of BHK 21/C13 fibroblasts degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated the rates of proteolysis. Both the absolute rate of proteolysis and the magnitude of the ATP effect depended on the specific substrate. For example, casein was degraded approximately 10-fold faster than lysozyme, but lysozyme degradation was more highly stimulated by ATP than was casein degradation. Ubiquitin enhanced the ATP-stimulated proteolysis of each substrate in both postmicrosomal extracts and DEAE-cellulose fractionated extracts. In each extract, ubiquitin enhanced the ATP-stimulated degradation of lysozyme to a greater degree than that of casein. These results suggested that lysozyme was degraded by a pathway that was more dependent upon ubiquitin than was casein. Further evidence for this conclusion was obtained in studies using substrates whose amino groups were blocked by extensive methylation or carbamoylation. The high molecular weight proteinase, macropain, appears to be involved in the ATP-stimulated degradation of both substrates. Specific immunoprecipitation of macropain with polyclonal antibodies resulted in the inhibition of ATP-stimulated proteinase activity both in the absence and presence of ubiquitin. These results indicate that macropain plays a role in both ubiquitin-mediated and ubiquitin-independent ATP-stimulated proteolysis in BHK cell extracts.
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PMID:An enzyme related to the high molecular weight multicatalytic proteinase, macropain, participates in a ubiquitin-mediated, ATP-stimulated proteolytic pathway in soluble extracts of BHK 21/C13 fibroblasts. 284 2

The proton-translocating, membrane ATPases of oral streptococci have been implicated in cytoplasmic pH regulation, acidurance, and cariogenicity. Membranes were isolated from Streptococcus mutans GS-5 and Streptococcus sanguis NCTC 10904 following salt-induced lysis of cells treated with lysozyme and mutanolysin. The ATPase activities of these membranes were 1.8 and 1.1 units per mg membrane protein, respectively. F1 ATPases were washed free from the membranes and purified by fast protein liquid chromatography (FPLC). Hydrolytic activities of the F1 ATPases were maximal at pH values between 6.0 and 6.6, whereas the membrane-bound enzymes had pH maxima of 7.5 (S. sanguis) and 6.0 (S. mutans). The F1 ATPases of the streptococci were similar to the well-characterized enzyme of Escherichia coli; they consisted of five different polypeptides and had apparent, aggregate molecular weights of from 335 to 350 Kd. The membrane-bound ATPases were characterized biochemically and found to be similar to those of proton-translocating ATPases of E. coli and Streptococcus faecalis. Km values for the membranes with respect to ATP were found to be 0.9 and 1.0 mmol/L for S. mutans and S. sanguis, respectively. Both enzymes had specificities for purine triphosphates and were active with a variety of divalent cations, although optimal activity occurred with ATP and Mg. The membrane-associated enzymes were sensitive to the inhibitors dicyclohexylcarbodiimide (DCCD) and azide, but insensitive to ouabain and vanadate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Membrane-associated and solubilized ATPases of Streptococcus mutans and Streptococcus sanguis. 288 1

The C-terminal two-thirds of the rat liver ATP synthase beta subunit has been overexpressed and exported to the Escherichia coli periplasm under the direction of the alkaline phosphatase (phoA) promoter and leader peptide. The processed soluble protein contains the 358 amino acids from glutamate 122 to the rat liver beta C-terminal serine 479, including all the regions that have been predicted by chemical and genetic modification studies to be involved in nucleotide, Pi, and Mg2+ binding. Through a simple sequence of Tris/EDTA/lysozyme treatment, osmotic lysis, and alkaline pH washes, the processed beta subunit fragment can be prepared in greater than 95% purity and at a yield of greater than 20 mg/liter of culture. It interacts with 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) which exhibits a strong enhancement of fluorescence upon binding. A similar enhancement is observed upon interaction with TNP-ADP. Enhancement observed with both TNP-nucleotides is markedly reduced by prior addition of either ATP or ADP and almost completely prevented by the ATP synthase inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. Both TNP-ATP and TNP-ADP bind at a stoichiometry of approximately 1 mol of nucleotide/mol of beta subunit fragment. Under the same conditions, TNP-AMP does not exhibit a fluorescence enhancement. This work demonstrates that, in the absence of interaction with other ATP synthase subunits, the rat liver beta subunit sequence from glutamate 122 to the C terminus exhibits no more than one readily detectable nucleotide binding domain. This success in producing a "functional" beta subunit fragment has significance for the pursuit of genetic and physical studies focused on the structure and function of the rat liver ATP synthase beta subunit.
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PMID:Mitochondrial ATP synthase. Overexpression in Escherichia coli of a rat liver beta subunit peptide and its interaction with adenine nucleotides. 290 92

A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells. We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system. The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold. Nonhydrolyzable ATP analogs had no effect. In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates. MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown. After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect. In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin. After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts. For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly. The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP. Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease. Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria.
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PMID:A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells. Evidence for a protease which requires ATP hydrolysis but not ubiquitin. 299 55

To produce ubiquitinated substrates for studies on ATP-dependent proteolysis, 125I-lysozyme was incubated in hemin-inhibited rabbit reticulocyte lysates. A portion of the labeled molecules became linked to ubiquitin in large covalent complexes. When these were partially purified and returned to uninhibited lysates containing ATP, the conjugated lysozyme molecules were degraded 10 times faster than free lysozyme. Purification of covalently modified lysozyme from hemin-inhibited lysates containing 125I-ubiquitin and 131I-lysozyme confirmed that both molecules were present in the complexes. The doubly labeled conjugates also permitted us to determine the fate of each molecule in uninhibited lysates. Besides degradation of lysozyme, there was a progressive release of intact lysozyme molecules from the complexes. This disassembly, which was the only fate of the complexes in the absence of ATP, proceeded through a series of smaller intermediates, several having molecular weights expected for ubiquitin-lysozyme conjugates, and eventually free lysozyme was regenerated. The behavior of labeled ubiquitin was similar, though not identical, to that of lysozyme. Even in lysates containing ATP ubiquitin emerged from the complex undegraded. Furthermore, ubiquitin was present in a greater number of species than was lysozyme. The demonstration that ubiquitin-lysozyme conjugates are rapidly degraded provides support for the hypothesis of Hershko, Rose, Ciechanover, and their colleagues that a key function of ubiquitin is to modify the proteolytic substrate. Further support for the hypothesis is presented in the following paper where we show that the conjugated lysozyme molecules are substrates for an ATP-dependent protease that does not degrade free lysozyme.
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PMID:Ubiquitin-lysozyme conjugates. Purification and susceptibility to proteolysis. 300 13

Ubiquitin-lysozyme conjugates have been used as substrates to identify an ATP-dependent protease from rabbit reticulocyte lysates. The enzyme, which has been partially purified by DEAE chromatography and glycerol gradient centrifugation, has an apparent molecular weight greater than 600,000 based on sedimentation and gel filtration. Whereas it degrades conjugated lysozyme molecules in the presence of ATP, the protease does not degrade free lysozyme molecules even upon addition of ubiquitin, lysozyme-ubiquitin conjugates, and ATP. Degradation of lysozyme conjugates is independent of added ubiquitin and occurs in fractions incapable of ubiquitin conjugation. Proteolysis is maximal at pH 7.8, inhibited by hemin, N-ethylmaleimide, or aurintricarboxylic acid, and proceeds with an apparent Arrhenius activation energy in the range of 27 +/- 5 kcal/mol. These properties are similar to those observed for the degradation of lysozyme conjugates in lysates indicating that the partially purified protease catalyzes the "second" ATP-utilizing reaction identified previously (Hough, R., and Rechsteiner, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 90-94; Hershko, A., Leshinsky, E., Ganoth, D., and Heller, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1619-1623; Tanaka, K., Waxman, L., and Goldberg, A. L. (1983) J. Cell Biol. 96, 1580-1585).
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PMID:Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates. 300 14

ATP and AMP were immediately converted into ADP by intact cells of Escherichia coli in the presence of Mg2+, while ADP was also rapidly converted into ATP and AMP under the same conditions. Adenylate kinase was released when E. coli cells were converted to spheroplasts by treatment with lysozyme-EDTA or osmotic shock. Adenylate kinase activities detected in the cytoplasm, periplasm and membrane fractions were approximately 58%, 36% and 6% of the total cellular activity, respectively. These results indicate that adenylate kinase in E. coli occurs in the periplasm as well as the cytoplasm.
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PMID:Intracellular localization of ATP:AMP phosphotransferase in Escherichia coli. 300 52


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