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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An in situ PCR protocol by which we can monitor the presence or absence of lac mRNA in individual cells of a Salmonella typhimurium F' lac+ strain has been developed. In this protocol, fixed cells are permeabilized with lysozyme and subjected to a seminested reverse transcriptase PCR using reporter molecule-labeled primers, and subsequently, intracellular reporter molecules are detected microscopically at the individual-cell level by use of a horseradish peroxidase-conjugated antifluorescein antibody assay. In order to determine the sensitivity of the in situ PCR assay, the ability to detect lac mRNA in suboptimally isopropyl-beta-D-thiogalactopyranoside-induced cells was investigated. By use of a single-cell beta-galactosidase assay, it was confirmed that homogeneous suboptimally induced cultures of S. typhimurium F' lacY cells could be established, and the number of functional lac mRNAs in individual cells was estimated from standard population level beta-galactosidase assays. Cells estimated to contain a single lac mRNA were detected as containing lac mRNA by the in situ PCR method. Conclusively, we demonstrate the potential of in situ PCR for detection of even poorly expressed mRNA in individual bacterial cells.
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PMID:Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR. 936 4

Ubiquitin C-terminal hydrolases (UCH) are deubiquitinating enzymes which hydrolyze C-terminal esters and amides of ubiquitin. Here we report the processing of a number of ubiquitin derivatives by two human UCH isozymes (isozymes L1 and L3) and find that these enzymes show little discrimination based on the P1' amino acid, except that proline is cleaved slowly. Ubiquitinyllysine derivatives linked by the alpha- or epsilon-amino group are hydrolyzed at identical rates. Isozyme-specific hydrolytic preferences are only evident when the leaving group is large. The ubiquitin gene products can be cotranslationally processed by one or both of these UCH isozymes, and purified UbCEP52 can be hydrolyzed by UCH isozyme L3. Binding of nucleic acid by UbCEP52 converts it to a form resistant to processing by these enzymes, apparently because of the formation of a larger, more tightly folded substrate. Consistent with this postulate is the observation that these enzymes do not hydrolyze large ubiquitin derivatives such as N epsilon-ubiquitinyl-cytochrome-c, N epsilon-K48polyubiquitinyl-lysozyme, or an N alpha-ubiquitinyl-beta-galactosidase fusion protein. Thus, these enzymes rapidly and preferentially cleave small leaving groups such as amino acids and oligopeptides from the C-terminus of ubiquitin, but not larger leaving groups such as proteins. These data suggest that the physiological role of UCH is to hydrolyze small adducts of ubiquitin and to generate free monomeric ubiquitin from ubiquitin proproteins, but not to deubiquitinate ubiquitin-protein conjugates or disassemble polyubiquitin chains.
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PMID:Substrate specificity of deubiquitinating enzymes: ubiquitin C-terminal hydrolases. 952 56

The behaviors of heat-induced translocation of cytoplasmic beta-galactosidase to periplasm across the inner membrane of Escherichia coli cells were investigated in order to apply such phenomena to the process for production and separation of intracellular biomolecules. The heat stress was found to induce translocation of cytoplasmic beta-galactosidase (beta-gal) together with reduction of the amounts of intracellular soluble proteins and formation of their inactive aggregates. The translocation of beta-gal was then analyzed using (a) the location factor of beta-gal (LFG), which meant enzyme location in the cells and could be determined from the kinetic analysis of enzyme release process, and (b) the percentage of beta-gal activity in periplasm after solublizing the outer membrane of E. coli cells by lysozyme/EDTA treatment. The LFG values were maximized when cells were stressed at the temperature of 42-47 degrees C. From the results on the surface properties of both beta-gal and cell membrane under the heat stress, it is suggested that (1) the conformational change of cytoplasmic oligomeric beta-gal to the partially dissociated and/or unfolded state with higher local hydrophobicity, (2) the increase in membrane fluidity of inner membrane, (3) the enhancement of hydrophobic interaction between lipid and protein, and (4) the inhibition of its translocation by GroEL restabilizing the proteins could underlie the heat-induced translocation of beta-gal across the inner membrane. The possibility to apply the heat-induced translocation of beta-gal for the enhancement of the target selectivity at the process upstream is finally presented.
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PMID:Heat-induced translocation of cytoplasmic beta-galactosidase across inner membrane of Escherichia coli. 954 71

We have recently identified a cDNA for a ubiquitin-specific protease (UBP), UBP41, that encodes the smallest functional UBP identified to date, using an Escherichia coli-based in vivo screening method. In the present study we isolated highly related cDNAs encoding a new family of UBP enzymes, named UBP46, UBP52 and UBP66. These UBPs have virtually identical catalytic domains spanning the sequence of UBP41 between the active-site Cys and the His box (95% identity). However, they possess distinct N- and/or C-terminal extensions. Moreover, they are more closely related to each other than to any other members of the UBP family. Thus these chick UBPs must define a novel family of de-ubiquitinating enzymes and should represent the first example among the UBP family enzymes, whose multiplicity is achieved by variation in their N- and C-terminal extensions. The chick UBPs were expressed in E. coli, and purified from the cells to apparent homogeneity using 125I-labelled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate. Each of the purified UBP46, UBP52 and UBP66 enzymes behaved as proteins of similar sizes under both denaturing and non-denaturing conditions, suggesting that all of them consist of a single polypeptide chain. The UBP enzymes cleaved the C-terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes and thus are active against ubiquitin-beta-galactosidase as well as a ubiquitin C-terminal extension protein of 80 amino acids. All UBPs except UBP66 released free ubiquitin from poly-His-tagged di-ubiquitin. However, the isopeptidase activity for hydrolysing polyubiquitinated lysozyme conjugates was not detected from these UBPs, which makes these UBPs distinct from UBP41. These results suggest that the chick UBPs may play an important role in production of free ubiquitin from linear polyubiquitin chains and of certain ribosomal proteins from ubiquitin fusion proteins.
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PMID:A novel family of ubiquitin-specific proteases in chick skeletal muscle with distinct N- and C-terminal extensions. 972 77

In the present study the level of enzyme hydrolases (alkaline phosphatase, myeloperoxidase, elastase, arginase, lysozyme and beta-galactosidase) of polymorphonuclear cell (PMN) granules in different ruminant species and their release in response to activation was studied. Buffalo PMN alkaline phosphatase activity was higher (P < 0.01) than in PMNs of cattle and goats. Interestingly, myeloperoxidase was higher in cattle PMNs and least in goat PMNs (P < 0.01), a similar pattern was observed in the distribution of enzyme arginase. As far as lysozyme is concerned, its activity was significantly higher (P < 0.01) in PMNs of buffaloes than in the case of cattle and goat PMNs. On activation, these cells released MPO and elastase, in all the species studied, while lysozyme was secreted only in buffalo PMN cells. Activity of certain enzymes related to oxidant defence systems such as glutathione peroxidase and glutathione reductase were higher in cattle and goats compared to that in buffaloes. These observations are likely to have bearing on immunodefense roles played by PMNs and reflected differences among the ruminant species studied.
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PMID:A comparative study on certain enzymes of the granulocyte from different ruminant species. 977 61

Effects of the water activity (a(w)) and the solvent ordering, as determined by the activity coefficient of water, were investigated on the enzyme kinetics of alcohol dehydrogenase, lysozyme, and beta-galactosidase in various aqueous solutions. The water activity and the solvent ordering were adjusted by addition of electrolytes (NaCl, KCl, CsCl, etc.) or nonelectrolytes (sugars, alcohols, urea, etc.) at various concentrations. Although the enzyme kinetics were strongly dependent on a(w), a(w) was not a complete determinant of the enzyme behavior in aqueous solutions. Enzyme kinetics were also dependent on the solvent ordering. At a fixed a(w), all the enzyme kinetic parameters tested had a good correlation with the solvent ordering parameter as represented by the parameter alpha, an index of the deviation of the water state from the ideal solution, determined from the activity coefficient of water in solutions. Solvent ordering was expected to affect the enzyme kinetics through its effect on the hydrophobic interaction between the enzyme and the substrate and also on the thermal fluctuation.
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PMID:Influence of water activity and aqueous solvent ordering on enzyme kinetics of alcohol dehydrogenase, lysozyme, and beta-galactosidase. 1071 6

A continuous integrated process for on-line quantification of intracellular components has been developed. By applying the concept of expanded micro-beds in a flow injection system it was possible to first perform on-line cell disintegration followed by an on-line binding assay for quantification of a reporter protein (beta-galactosidase) from the cell interior. The disintegration process involved the use of an expanded bed with immobilised lysozyme followed by ultrasonic treatment in a flow-through cell. The cell debris does not interfere in the binding assay as it is carried out in an expanded bed. The time for an assay cycle is at present approx. 35 min. This integrated system can be used for quantification of proteins down to at least 10(-7) mol/L.
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PMID:Flow injection analysis of intracellular beta-galactosidase in Escherichia coli cultivations, using an on-line system including cell disruption, debris separation and immunochemical quantification. 1073 78

Previously we have shown that chicken egg white lysozyme, an efficient bactericidal agent, affects both gram-positive and gram-negative bacteria independently of its muramidase activity. More recently we reported that the digestion of lysozyme by clostripain yielded a pentadecapeptide, IVSDGNGMNAWVAWR (amino acid 98-112 of chicken egg white lysozyme), with moderate bactericidal activity but without muramidase activity. On the basis of this amino acid sequence three polypeptides, in which asparagine 106 was replaced by arginine (IVSDGNGMRAWVAWR, RAWVAWR, RWVAWR), were synthesized which showed to be strongly bactericidal. To elucidate the mechanisms of action of lysozyme and of the modified antimicrobial polypeptides Escherichia coli strain ML-35p was used. It is an ideal organism to study the outer and the inner membrane permeabilization since it is cryptic for periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless the outer or inner membrane becomes damaged. For the first time we present evidence that lysozyme inhibits DNA and RNA synthesis and in contrast to the present view is able to damage the outer membrane of Escherichia coli. Blockage of macromolecular synthesis, outer membrane damage and inner membrane permeabilization bring about bacterial death. Ultrastructural studies indicate that lysozyme does not affect bacterial morphology but impairs stability of the organism. The bactericidal polypeptides derived from lysozyme block at first the synthesis of DNA and RNA which is followed by an increase of the outer membrane permeabilization causing the bacterial death. Inner membrane permeabilization, caused by RAWVAWR and RWVAWR, follows after the blockage of macromolecular synthesis and outer membrane damage, indicating that inner membrane permeabilization is not the deadly event. Escherichia coli bacteria killed by the substituted bactericidal polypeptides appeared, by electron microscopy, with a condensed cytoplasm and undulated bacterial membrane. So the action of lysozyme and its derived peptides is not identical.
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PMID:Effect of lysozyme or modified lysozyme fragments on DNA and RNA synthesis and membrane permeability of Escherichia coli. 1095 Jan 88

Alcoholysis reactions were performed in organic one-phase liquid systems with E. coli beta-galactosidase to produce heptyl-beta-galactoside from lactose and 1-heptanol. The reaction rate was highly dependent on the amount of water solubilized in the alcohol. A larger amount of water led to a system of two liquid phases in which the alcoholysis rate was 73% faster than in the one-phase system. No hydrolysis reaction of either lactose or product was observed in one-phase liquid systems up to 20 h, independent of the water content. Solubility of lactose in the organic phase increased with the water content in the system and the reaction followed the Michaelis-Menten model. Water activity was calculated for heptanol containing different amounts of water and the obtained values were used to estimate the hydration of beta-galactosidase from known models. Enzyme activity correlated with sorbed water, similar to the behavior reported for lysozyme in low water environments. It is concluded that water contribution to enzyme hydration dominates the rate of reaction compared to its effect on lactose solubilization.
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PMID:Effect of beta-galactosidase hydration on alcoholysis reaction in organic one-phase liquid systems. 1106 33

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection procedure. Given the variation in the expression levels of the same construct in individual clones, hundreds of clones must be isolated and tested to identify one or more with the desired characteristics. Various boundary elements (BEs), matrix attachment regions, and locus control regions (LCRs) were screened for their ability to augment the expression of heterologous genes in Chinese hamster ovary (CHO) cells. Of the chromatin elements assayed, the chicken lysozyme matrix-attachment region (MAR) was the only element to significantly increase stable reporter expression. We found that the use of the MAR increases the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits are observed both for constructs with MARs flanking the transgene expression cassette, as well as when constructs are co-transfected with the MAR on a separate plasmid. Moreover, the MAR was co-transfected with a multicomponent regulatable beta-galactosidase expression system in C2C12 cells and several clones exhibiting regulated expression were identified. Hence, MARs are useful in the development of stable cell lines for production or regulated expression.
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PMID:Development of stable cell lines for production or regulated expression using matrix attachment regions. 1126 97


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