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)

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

In the ubiquitin-mediated pathway for the degradation of intracellular proteins, several molecules of ubiquitin are linked to the protein substrate by amide linkages. It was noted that the number of ubiquitin-protein conjugates and their apparent molecular size are higher than expected from the number of amino groups in the protein. When the amino groups of ubiquitin were blocked by reductive methylation, it was efficiently conjugated to lysozyme, but the higher-molecular-weight conjugates were not formed. This suggests that the higher-molecular-weight conjugates with native ubiquitin contain structures in which one molecule of ubiquitin is linked to an amino group of another molecule of ubiquitin. Methylated ubiquitin stimulated protein breakdown at about one half the rate obtained with native ubiquitin, and isolated conjugates of 125I-lysozyme with methylated ubiquitin were broken down by reticulocyte extracts. These findings indicate that the formation of polyubiquitin chains is not obligatory for protein breakdown, though it may accelerate the rate of this process.
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PMID:Occurrence of a polyubiquitin structure in ubiquitin-protein conjugates. 298 26

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

Degradation of intracellular proteins via the ubiquitin- and ATP-dependent proteolytic pathway involves several steps. In the initial event, ubiquitin, an abundant 76-residue polypeptide is covalently linked to the protein substrate in an ATP-requiring reaction. Proteins marked by ubiquitin are selectively proteolyzed in a reaction that also requires ATP. Ubiquitin conjugation to proteins appears also to be involved in regulation of cell cycle and cell division, and probably in the regulation of gene expression at the level of chromatin structure. We have previously shown (Ciechanover, A., Wolin, S. L., Steitz, J. A., and Lodish, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1341-1345) that transfer RNA is an essential component of the ubiquitin pathway. Ribonucleases strongly and specifically inhibited the degradation of 125I-labeled bovine serum albumin, while tRNA purified from reticulocyte extract could restore the proteolytic activity. Specifically, pure tRNAHis isolated by immunoprecipitation with human autoimmune serum could restore the proteolytic activity. Here we demonstrate that tRNA is required for conjugation of ubiquitin to some but not all proteolytic substrates of the ubiquitin mediated pathway. Conjugation of 125I-labeled ubiquitin to reduced carboxymethylated bovine serum albumin, alpha-lactalbumin, and soybean trypsin inhibitor was strongly and specifically inhibited by ribonucleases. Consequently, the ATP-dependent degradation of these substrates in the cell-free ubiquitin-dependent reticulocyte system was inhibited as well. Addition of tRNA to the ribonuclease inhibited system (following inhibition of the ribonuclease) restored both the conjugation activity and the ubiquitin- and ATP-dependent degradation of these substrates. Conjugation of ubiquitin to some endogenous reticulocyte proteins was also inhibited by ribonucleases and could be restored by the addition of tRNA. In striking contrast, the conjugation of radiolabeled ubiquitin to lysozyme, oxidized RNase A, alpha-casein, and beta-lactoglobulin was not affected by the ribonuclease treatment, and the degradation of these substrates was significantly accelerated by the ribonucleases. These findings indicate that there are at least two distinct ubiquitin conjugation systems. One requires tRNA, and the other is tRNA independent. These pathways, however, must share some common component(s) of the system, since the inhibition of one system accelerates the other. The possible function of tRNA in the selective conjugation reaction and the possible role of the two distinct ubiquitin marking mechanisms are discussed.
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PMID:Transfer RNA is required for conjugation of ubiquitin to selective substrates of the ubiquitin- and ATP-dependent proteolytic system. 300 81

Egg white lysozyme, treated with O-methylisourea to convert lysine to homoarginine residues, was used as a substrate for the ATP-dependent proteolytic pathway in rabbit reticulocyte lysates. Although guanidinated lysozyme was degraded by an ATP-dependent, hemin-sensitive process, ubiquitin conjugates of this protein were present at less than 5% the level of conjugates between ubiquitin and nonguanidinated lysozyme. When lysates were chromatographed on DEAE-cellulose to produce Fractions I and II of (Hershko et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3107), ubiquitin-depleted Fraction II was capable of degrading nonguanidinated lysozyme, but the degradation of guanidinated lysozyme was markedly reduced or abolished. Glycerol-stabilized Fraction II, on the other hand, supported the degradation of both proteins in an ATP-dependent process stimulated by ubiquitin. The degradation of the two proteins differed, however, in that guanidinated lysozyme was more sensitive to competitive substrates, and higher concentrations of ubiquitin were required for its maximal proteolysis. Despite ubiquitin stimulation of guanidinated lysozyme degradation, only trace amounts of higher molecular weight species of guanidinated lysozyme attributable to ubiquitin conjugation were observed in ubiquitin-supplemented, glycerol-stabilized Fraction II even when special precautions were employed to preserve labile covalent bonds. These results indicate that covalent attachment of ubiquitin to the epsilon-amino group of substrate lysines is not mandatory for ATP-dependent proteolysis in rabbit reticulocyte lysates. The observation that ubiquitin stimulates proteolysis of guanidinated lysozyme, without extensive conjugation to it, suggests that ubiquitin may have essential functions for proteolysis other than direct marking of the protein substrate.
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PMID:The degradation of guanidinated lysozyme in reticulocyte lysate. 300 5

Enzyme activities that catalyzed the covalent attachment of ubiquitin to protein substrates (ubiquitin-protein ligase, UbL) were purified from the extracts of human red blood cells. These activities required the presence of ubiquitin-activating enzyme and ATP for activity. Four fractions (UbL A, B1, B2, and C) were resolved and showed different specificities toward added substrates [carboxymethylated bovine serum albumin (CM-BSA), G-actin, lysozyme, and alpha-lactalbumin]. The enzyme fractions gave different products with a given substrate. UbL A and UbL B1 were exclusively active with CM-BSA and alpha-lactalbumin, respectively. UbL B2 was most active toward CM-BSA with substantial activities to G-actin and alpha-lactalbumin and with no activity to lysozyme. UbL C showed significant activities with all four substrates, having a highest activity toward CM-BSA. There were many endogenous proteins present in the erythrocyte extract which were efficient substrates for ubiquitin conjugation. In particular, a pair of substrates were identified from erythrocyte extracts which were far more efficient substrates than the denatured proteins exogenously added.
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PMID:Multiple forms of ubiquitin-protein ligase. Binding of activated ubiquitin to protein substrates. 301 98

In the aged lens postsynthetically altered molecules comprise the majority of lens proteins. Many proteolytic activities have been observed in lens supernatants. Since damaged or altered proteins are usually selectively and rapidly degraded in other cells and tissues, the accumulation of these species in the lens seemed enigmatic. Initiation of proteolysis has been studied most extensively in reticulocytes and ts 85 cells. In these systems proteolysis is absolutely ATP dependent, occurs effectively on high molecular weight substrates and, at least for a majority of proteolytic reactions, requires conjugation of ubiquitin to putative substrates. It seemed plausible that the accumulation of high molecular weight protein aggregates in older lenses might be due to the attenuated function of these ubiquitin- and ATP-dependent components in the initial committing processes of proteolysis. This research shows that: ubiquitin is present in the lens; lens proteins are conjugated to 125I-ubiquitin using reticulocyte conjugating systems; the reaction is ATP dependent; proteins from lens epithelium/outer cortex and core form different ubiquitin conjugates; lens proteins compete with lysozyme and reticulocyte proteins for the ubiquitin conjugating apparatus; most of the conjugates are of very high molecular weight; there is a temporal nature to the pattern of conjugates observed; and the ubiquitin conjugation system shows extreme selectivity.
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PMID:Lens proteins are substrates for the reticulocyte ubiquitin conjugation system. 302 93


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