SEARCH · PubMed Health

Results for “Ligases”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed1978

Simultaneous purification and some properties of aspartate: tRNA ligase and seven other amino-acid:tRNA ligases from Escherichia coli.

A procedure is described for the purification of the aspartate:tRNA ligase from Escherichia coli to a stage where it was homogeneous by polyacrylamide gel electrophoresis. From the same batch of E. coli the lysine, phenylalanine and serine ligases were obtained in an apparently homogeneous form while the alanine, glutamine, leucine and valine enzymes had a purity varying from 20% to 80%. Aspartate: tRNA ligase, which has not been obtained in a highly purified form before, has been characterized in terms of its molecular parameters.

Aspartic Acid↗

PubMed1975

RNA nicking activity associated with DNA ligase of T4 infected E. coli: properties and influence on in vitro reactions of ligase.

Highly purified DNA ligase from T4 infected E. coli displays an RNA nicking activity which cleaves endonucleolytically the RNA of ribo-desoxy-and ribo-ribo type doublestranded structures to oligonucleotides with 5'phosphoryl-and 3'hydroxy termini. In the presence of ATP the generated nicks are repaired by the ligase except at the ends of the doublestranded regions where some short oligonucleotides are released before ligation can occur. As judged from its behaviour during the various purification steps and from some of its properties, the nicking activity seems to be different from known nicking enzymes.

Alkaline Phosphatase↗

PubMed1977

Characterization of liver cholic acid coenzyme A ligase activity. Evidence that separate microsomal enzymes are responsible for cholic acid and fatty acid activation.

Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.

Adenosine Triphosphate↗

PubMed1977

Interaction of bacteriophage T4 RNA and DNA ligases in joining of duplex DNA at base-paired ends.

The joining of duplex DNA at base-paired ends by bacteriophage T4 DNA ligase was confirmed using either a synthetic duplex decamer or restriction endonuclease fragments of ColE1 DNA as substrates. The reaction was not linearly dependent on enzyme concentration but increased markedly at high enzyme concentrations. Although T4 RNA ligase did not catalyze this blunt end joining, it makedly stimulated the DNA ligase reaction particularly at low DNA ligase concentrations. The apparent Km for the decamer was 50 micronM in the presence or absence of RNA ligase. In the presence of RNA ligase, T4 DNA ligase had about the same turnover number for blunt end and cohesive end joining. The joining of duplex DNA at base-paired ends was proven by several techniques including restriction endonuclease cleavage of the products. The products of the ligation reaction using restriction enzyme fragments were mostly linear oligomers but included some circular duplexes. Escherichia coli DNA ligase in the presence or absence of RNA ligase did not catalyze blunt end joining. RNA ligase only moderately affected the joining of cohesive ends by T4 DNA ligase or E. coli DNA ligase and did not itself catalyze this reaction.

Coliphages↗

PubMed1975

Mammalian DNA ligases. Serological evidence for two separate enzymes.

Mammalian cells contain two DNA ligase activities with different chromatographic properties, referred to as DNA ligase I and II. The major ligase activity present in calf thymus cell extracts, DNA ligase I, has been purified 1000-fold. After repeated injections of this enzyme with complete Freund's adjuvant into a rabbit, antibodies were induced that inhibit DNA ligase I from calf, human, mouse, and rabbit tissues. This antiserum did not affect DNA ligase II from the same sources to a detectable extent, even at a concentration 10-fold higher than that required for 98% inhibition of DNA ligase I. These data strongly indicate that the two mammalian DNA ligase activities are due to two separate enzymes, and not to two forms of the same enzyme. Both enzymes are present in the nuclear fraction, but are also found in the cytoplasmic fraction. Rapidly dividing cells (mouse ascites tumor cells and calf thymus) contain higher amounts of DNA ligase I than other cells (calf liver and spleen, human placenta, and rabbit spleen), while no such correlation was observed for DNA ligase II.

Animals↗

PubMed1976

Phenolic metabolism in petunia tissues. IV. - Properties of p-coumarate : coenzyme A ligase isoenzymes.

Three p-coumarate: CoA ligases were separated from Petunia leaves. There was no interconversion from one form to another. The isoenzymes had a number of common properties: optimum pH, instability in the absence of polyols, action on p-coumaric acid as the common substrate. These enzymes differed significantly with respect to: --their substrate specificity towards the other C6-C3 units of Petunia. Form Ia (caffeate: CoA ligase) acted on caffeic acid, form Ib (sinapate: CoA ligase) on sinapic acid form II (ferulate: CoA ligase) on ferulic acid. --their thermal stability. --their sensitivity to phenolics: (a) caffeate: CoA ligase was inhibited by p-coumaroyl and caffeoyl quinic esters. It was insensitive to p-coumaroyl-glucose, on one hand and to a number of flavonoids on the other. (b) ferulate: CoA ligase was specifically inhibited by naringenin. (c) sinapate: CoA ligase was not inhibited by the selected compounds. In all cases, the inhibition was of the non competitive type and the enzymes were desensized to the modifier action by thermal treatment independently from the enzyme activity. These results suggest the occurrence of distinct sites of reception for the substrate and the inhibitor on the enzyme molecule. All these data are consistent with the hypothesis of the possible participation of each individual form in a limited number of pathways. This would be of physiological interest since the metabolic fate of the different cinnamic acids could be independently controlled at the p-coumarate: CoA ligase level.

Coenzyme A Ligases↗

PubMed1992

Inhibition of rat peroxisomal palmitoyl-CoA ligase by xenobiotic carboxylic acids.

ATP-dependent coenzyme A (CoA) ligases catalyse the formation of the acyl-CoA thioesters of xenobiotic carboxylic acids and the formation of xenobiotic-CoAs has been implicated as being a causative factor in peroxisomal proliferation. In this study we have demonstrated using rat liver peroxisomes that the formation of palmitoyl-CoA is inhibited by a variety of xenobiotic carboxylic acids. Palmitoyl-CoA formation exhibited biphasic kinetics indicative of two isoforms, a high affinity (Km1 2.3 microM) low capacity form and a low affinity (Km2 831 microM) high capacity form. These forms were differentially inhibited by a range of xenobiotics. However, it would appear that the low affinity component may not contribute to any major extent to the formation of xenobiotic-CoAs in vivo. At a concentration of 1 mM, greater than 20% inhibition of the high affinity form was observed with the 2-arylpropionates, ibuprofen, naproxen, benoxaprofen, fenoprofen, indoprofen, ketoprofen, tiaprofenic acid and cicloprofen, the hypolipidaemics, nafenopin and ciprofibrate, and the herbicides, silvex and 2,4,5-trichlorophenoxyacetate. Valproic acid, clofibric acid, salicylic acid and 2,4-dichlorophenoxy-acetate were non-inhibitory at all concentrations studied (0.1-2.5 mM). Analysis of the type of inhibition established that only nafenopin (Ki 430 microM) and ciprofibrate (Ki 97 microM) were competitive inhibitors of palmitoyl-CoA formation suggesting that they bind at the active site and thus potentially function as alternative substrates for the peroxisomal ligase. Notably, clofibric acid which has previously been shown to form clofibroyl-CoA in peroxisomes did not interact with the palmitoyl-CoA ligase thereby suggesting that activation is mediated via an alternative peroxisomal CoA ligase. In addition, the xenobiotic inhibitors of the peroxisomal palmitoyl-CoA ligase differed from those previously reported for the equivalent microsomal enzyme suggesting that the organellar forms may be functionally distinct. This study establishes that numerous xenobiotic carboxylic acids interact with the peroxisomal palmitoyl-CoA ligase; however, it would appear that relatively few function as alternative substrates. The toxicological ramifications of peroxisomally mediated xenobiotic-CoA formation and the identification of other peroxisomal xenobiotic-CoA ligase(s) remain to be elucidated.

Animals↗

PubMed1979

RNA ligase reaction products in plasmolyzed Escherichia coli cells infected by T4 bacteriophage.

Searching for a physiological role of T4 RNA ligase [polyribonucleotide synthetase (ATP); poly(ribonucleotide):poly(ribonucleotide) ligase (AMP-forming), EC 6.5.1.3] activity, we developed an acellular system of plasmolyzed Escherichia coli cells infected by T4 bacteriophage. Upon incubation of this system with [gamma-32P]ATP, 32P was transferred into a large number of polyribonucleotides, mostly up to 300-400 residues long. The bulk of 32P in the product polyribonucleotides was found in 5'-terminal phosphate groups, suggesting that they originated by a phosphorylation reaction catalyzed by the endogenous polynucleotide kinase (EC 2.7.1.78). Indeed, these products were not seen in an acellular system from uninfected cells, and their amount and complexity increased with the progress of infection. Analysis of the 32P-labeled polyribonucleotide products by gel electrophoresis, either before or after digestion with alkaline phosphatase (EC 3.1.3.1), revealed that a small fraction of the 32P resided in phosphodiester bonds of several tRNA-sized chains. This specific 32P transfer from [gamma-32P]ATP into phosphodiester bonds was apparently catalyzed by successive polynucleotide kinase and RNA ligase reactions. The possible relationship of the 32P transfer to RNA ligase was investigated next by using a system from cells infected with T4 am M69 (an amber mutant deficient in RNA ligase). Transfer of 32P from [gamma-32P]ATP into phosphodiester bonds was not detected in the am M69 system. However, addition of purified RNA ligase to the am M69 system restored the specific 32P transfer. A system from cells infected with T4 psu-b delta 33 (a deletion mutant lacking the entire tRNA region) sustained the specific 32P transfer into tRNA-sized products, indicating that they were not derived from transcripts of T4 tRNA genes. These data may reflect a role of RNA ligase in posttranscriptional conversion of presumably host polyribonucleotides into novel tRNA species during T4 infection.

Adenosine Triphosphate↗

PubMed1979

Functional interactions beween the DNA ligase of Escherichia coli and components of the DNA metabolic apparatus of T4 bacteriophage.

T4 phage completely defective in both gene 30 (DNA ligase) and the rII gene (function unknown) require at least normal levels of host-derived DNA ligase (E. coli lig gene) for growth. Viable E. coli mutant strains that harbor less than 5% of the wild-type level of bacterial ligase do not support growth of T4 doubly defective in genes 30 and rII (T4 30- rII- mutants). We describe here two classes of secondary phage mutations that permit the growth of T4 30- rII- phage on ligase-defective hosts. One class mapped in T4 gene su30 (Krylov 1972) and improved T4 30- rII- phage growth on all E. coli strains, but to varying degrees that depended on levels of residual host ligase. Another class mapped in T4 gene 32 (helix-destabilizing protein) and improved growth specifically on a host carrying the lig2 mutation, but not on a host carrying another lig- lesion (lig4). Two conclusions are drawn from the work: (1) the role of DNA ligase in essential DNA metabolic processes in T4-infected E. coli is catalytic rather than stoichiometric, and (2) the E. coli DNA ligase is capable of specific functional interactions with components of the T4 DNA replication and/or repair apparatus.

Coliphages↗

PubMed1975

DNA ligase activity in chromatin and its analogs. Rejoining of DNA strands in polylysine-DNA complexes and in reconstituted chromatins.

A highly purified DNA ligase from rat liver nuclei has been tested on DNA containing single-strand breaks ("nicks"); the DNA was present in several types of complexes which were chosen to serve as models for chromatin. These model systems included complexes of polylysine or histones with DNA as well as reconstituted chromatin preparations. In all these cases, the limit of ligase sealing was measured as a function of the ratio of polypeptide or protein to DNA. With an excess of either polylysine or histones, the ligase is totally prevented from sealing nicks in the DNA. However, at ratios of histones to DNA similar to those occurring in chromatin, about half of the nicks are accessible to the ligase. In the reconstitution of chromatin, the proteins are dissociated from the DNA by exposure to high ionic strength either with or without urea. If such procedures are carried out in the presence of labeled nicked DNA, the proteins will redistribute over this ligase substrate as well. When the chromatin is reconstituted at protein/DNA ratios similar to those occurring in chromatin, once more only about half of the nicks are accessible to the ligase. Similar results were obtained with preparations reconstituted with either rat liver or duck reticulocyte chromatin. The rate of ligase action has been measured on a variety of the complexes. While the rate falls as the DNA is increasingly covered with polylysine or histones, this is largely or entirely due to the decrease in concentration of sealable sites. At saturating concentrations of these DNA complexes, the original rate on uncovered DNA is approached.

Animals↗

PubMed1976

Triacylglycerol synthesis in isolated fat cells. An effect of insulin on microsomal fatty acid coenzyme A ligase activity.

Fatty acid CoA ligase (AMP) (EC 6.2.1.3) specific activity was increased approximately 2-fold in microsomes prepared from isolated rat fat cells incubated with 400 microunits of insulin/ml (2.9 nM) for 45 to 60 min compared to paired controls using an assay based on the conversion of [3H]oleic acid to [3H]oleoyl-CoA. Similar insulin-dependent increases in microsomal fatty acid CoA ligase specific activities were observed using an assay based on the conversion of [3H]CoA to fatty acyl-[3H]CoA. Fatty acid CoA ligase activity was predominately (about 80%) associated with the microsomal fraction. The insulin-dependent increase in microsomal fatty acid CoA ligase specific activity was maximal in 2 to 5 min at 400 microunits/ml. At 10 min, 80 to 100 microunits of insulin/ml caused a maximal increase in fatty acid CoA ligase specific activity. Similar apparent Km values for ATP, CoA, and fatty acid were observed for fatty acid CoA ligase activity in microsomal preparations from control and insulin-exposed cells. These data suggest that fatty acid CoA ligase activity is regulated in adipose tissue by insulin. Such regulation may serve to promote the capture of fatty acid and thereby, triacylglycerol synthesis in adipose tissue.

Adenosine Triphosphate↗

PubMed2026

Post-translational chemical modification of E3 ligase for efficient target protein degradation.

Targeted protein degradation (TPD) has emerged as a powerful therapeutic strategy, with proteolysis-targeting chimeras (PROTACs) leading efforts to address previously undruggable targets. However, PROTACs face challenges such as low bioavailability and poor pharmacokinetic properties which limit their biological applications. Here, we report a strategy termed post-translational chemical modification targeting chimera (PTcM-TAC), which integrates ligand-directed chemistry into the PROTAC framework to achieve sustained target protein degradation through covalent modification of E3 ligases. PTcM-TAC incorporates an electrophilic dibromophenyl benzoate warhead into the linker connecting the E3 ligase ligand and the protein-of-interest (POI) ligand, enabling selective transfer of the POI ligand onto the recruited E3 ligase while releasing the E3-binding moiety. Mechanistic studies, including LC-MS/MS peptide mapping, pull-down assays, and structural modeling, demonstrated site-selective modification of CRBN by the PTcM-TAC. The resulting ligand-labeled E3 ligase enables sustained pseudo-catalytic target recognition through a simplified binary interaction, thereby maintaining degradation activity even after compound washout. Furthermore, we successfully applied the PTcM-TAC strategy to another representative E3 ligase, von Hippel-Lindau (VHL), which exhibited substantially sustained degradation activity compared with conventional PROTACs. To our knowledge, PTcM-TAC represents the first ligand-directed chemical strategy that converts transient PROTAC-mediated ternary complex formation into binary target recognition via post-translational chemical modification of an E3 ligase. We believe that PTcM-TAC could provide a platform for next-generation targeted protein degraders to overcome the current limitation of PROTAC approach.

Ubiquitin-Protein Ligases↗

PubMed1992

Dual mode of inhibition of purified DNA ligase I from human cells by 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (F-ara-A) is an analogue of adenosine and deoxyadenosine with potent anti-tumor activity. The mechanism of action for this compound has been elucidated as the inhibition of DNA and RNA synthesis, induction of DNA fragmentation, and genetic damage. This study demonstrated that DNA ligase I, an enzyme involved in DNA replication, is a target for the drug action. F-ara-adenine triphosphate (F-ara-ATP) at 80 microM inhibited the activity of DNA ligase I by more than 90%. In contrast, eight other related nucleoside analogues showed no effect on the enzyme activity at 200 microM. F-ara-ATP inhibited DNA ligation in two distinct ways. First, F-ara-ATP directly interacted with DNA ligase I and inhibited the formation of the ligase-AMP complex. This inhibition could not be reversed when free F-ara-ATP was eliminated from the treated enzyme; however, the addition of pyrophosphate, followed by gel filtration chromatography, restored enzyme activity, indicating that F-ara-ATP bound to the enzyme and altered the AMP-binding site. Secondly, the activity of DNA ligase I was inhibited when F-ara-ATP was incorporated into the 3' terminus of the DNA substrate. The dual mode of inhibition of DNA ligase I by F-ara-ATP indicates that its effect on DNA ligation may be important in the inhibition of DNA synthesis and the cytotoxicity of F-ara-A.

Adenosine Monophosphate↗

PubMed1975

Genetic evidence for an additional function of phage T4 gene 32 protein: interaction with ligase.

Gene 32 of bacteriophage T4 is essential for DNA replication, recombination, and repair. In an attempt to clarify the role of the corresponding gene product, we have looked for mutations that specifically inactivate one but not all of its functions and for compensating suppressor mutations in other genes. Here we describe a gene 32 ts mutant that does not produce progeny, but in contrast to an am mutant investigated by others, is capable of some primary and secondary DNA replication and of forming "joint" recombinational intermediates after infection of Escherichia coli B at the restrictive temperature. However, parental and progeny DNA strands are not ligated to covalently linked "recombinant" molecules, and single strands of vegetative DNA do not exceed unit length. Progeny production as well as capacity for covalent linkage in this gene 32 ts mutant are partially restored by additional rII mutations. Suppression by rII depends on functioning host ligase [EC 6.5.1.2; poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming, NMN-forming)]. This gene 32 ts mutation (unlike some others) in turn suppresses the characteristic plaque morphology of rII mutants. We conclude that gene 32 protein, in addition to its role in DNA replication and in the formation of "joint" recombinational intermediates, interacts with T4 ligase [EC 6.5.1.1; poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming)] when recombining DNA strands are covalently linked. The protein of the mutant that we describe here is mainly defective in this interaction, thus inactivating T4 ligase in recombination. Suppressing rII mutations facilitate substitution of host ligase. There is suggestive evidence that these interactions occur at the membrane.

Binding Sites↗

PubMed1976

Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12.

Toluene-treated Escherichia coli mutants have been used to study the roles of deoxyribonucleic acid (DNA) polymerases I, II, and III, and of DNA ligase in repair synthesis and strand rejoining following X-irradiation. In cells possessing all three DNA polymerases, both a greater amount of repair synthesis ("exaggerated" repair synthesis) and failure of ligation are observed when DNA ligase activity is inhibited. In a mutant lacking the polymerizing activity of DNA polymerase I, exaggerated repair synthesis is not observed, and strand rejoining does not occur even if DNA ligase is fully activated. In a mutant possessing the polymerizing activity of DNA polymerase I but lacking its 5'leads to 3' exonuclease activity, exaggerated repair synthesis is minimal. After irradiation, DNA polymerases II and III are capable of carrying out an adenosine 5'-triphosphate-dependent repair synthesis,but rejoining of strand breaks does not occur and exaggerated synthesis is not seen whether DNA ligase is active or not. These results suggest that DNA polymerase I and DNA ligase act together to limit repair synthesis after X irradiation and that both are necessary in toluene-treated cells for strand rejoining. DNA polymerases II and III apparently cannot complete chain elongation and gap filling, and therefore repair carried out by these enzymes does not respond to ligase action.

Adenosine Triphosphate↗

PubMed1977

Bacteriophage T4 RNA ligase: preparation of a physically homogeneous, nuclease-free enzyme from hyperproducing infected cells.

Infection of Escherichia coli by a bacteriophage T4 regA, gene 44 double mutant leads to about a 7-fold increase in the amount of RNA ligase obtained after infection by wild-type phage. Using cells infected by the double mutant, RNA ligase was purified to homogeneity with a 20% yield. Unlike previous preparations of this enzyme, the ligase is free of contaminating nuclease and is therefore suitable for intermolecular ligation of DNA substrates. In the course of these studies it was discovered that adenylalation of the enzyme--a step in the reaction pathway--markedly decreased the electrophoretic mobility of RNA ligase through polyacrylamide gels containing sodium dodecyl sulfate. This behavior allows identification of RNA ligase among a mixture of proteins and was used to demonstrate that virtually all of the purified protein is enzymatically active.

Adenosine Monophosphate↗

PubMed1975

Properties of a DNA-adenylate complex formed in the reaction between mammalian DNA ligase I and DNA containing single-strand breaks.

The major DNA ligase from calf thymus (mammalian DNA ligase I) forms a covalent enzyme-AMP complex on incubation with ATP [Söderhäll & Lindahl, J. Biol. Chem. 248, 672-675, (1973)]. The reaction of this complex with DNA has now been studied. When the ligase-adenylate complex is incubated at 0 degrees C for short time periods with DNA containing single-strand breaks, a DNA-AMP complex can be isolated from the reaction mixture by isopycnic centrifugation in CsCl. Incubation at pH 6.5 increased the amount of DNA-AMP complex that could be isolated 10-20-fold relative to that obtained at pH 7.4. Under the same conditions, incubation of the ligase-AMP complex with DNA free from single-strand breaks did not lead to detectable DNA-AMP formation. The DNA-AMP complex was resistant to treatment with dilute acid and alkali indicating the presence of a covalent linkage. Further, this complex was sensitive to DNase but resistant to pronase and RNase. Free AMP was released on further incubation of the isolated DNA-AMP complex with thymus DNA ligase I and Mg2+, suggesting that the complex is a reaction intermediate. Degradation of the DNA-AMP complex with several reagent enzymes indicated that the AMP residues were bound at the 5' ends of the single-strand breaks in DNA by pyrophosphate bonds.

Adenosine Monophosphate↗

PubMed1977

Isolation and characterization of two methionine: tRNA ligases from wheat germ.

Two methionine: tRNA ligases (here called ligase A and ligase B) with distinctly different enzymatic and molecular properties were isolated in homogenous form from extracts of raw wheat germ. Both the A and B enzyme are composed of single polypeptide chains of Mr 105000 and 70000 respectively. The smaller molecule (B) has been shown not to be a proteolytic fragment of the larger one (A). The catalytic properties of both the A and B enzymes have been established and the Mg2-dependent capacity to charge six purified methionine-accepting tRNAs have been compared to those of the methionine: tRNA ligases from Escherichia coli and bakers' yeast. The possible reasons for the presence of two methionine: tRNA ligases and their unusual monomeric nature are discussed.

Amino Acids↗