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PubMed2002

Histone acetyl transferases: a role in DNA repair and DNA replication.

In eukaryotic cells DNA is associated with proteins to form a complex known as chromatin. The dominant proteins within this chromatin complex are the histones, which are subject to a wide variety of covalent and reversible posttranslational modifications such as acetylation. A specialized family of enzymes, the histone acetyl transferases, catalyzes the transfer of acetyl groups from their cosubstrate acetyl-coenzyme A to lysine residues of histones. Acetylation of histone N-terminal lysine residues induces chromosomal changes and results in the loss of chromosomal repression that allows the successful transcription of the underlying genes. Analogously, in DNA repair and also DNA replication the chromosomal repression is thought to be relieved by such mechanisms. Recently several publications have provided evidence that histone acetyl transferases also modify nonhistone proteins and thereby regulate their activities. This review discusses various aspects of histone acetyl transferases and summarizes recent findings which suggest a role for histone acetyl transferases in DNA repair and DNA replication.

Acetyltransferases↗
PubMed1980

Role of galactosyl-transferases in rat gastric epithelial glycoprotein synthesis.

Two galactosyl-transferases have been found in the Golgi-enriched subcellular fractions derived from rat gastric mucosa. One incorporates galactose into ovomucoid at optimal pH 6.8. The reaction can be completely inhibited by acetylglucosamine. The apparent Km for UDPgalactose is 0.024 mM. The other galactosyl-transferase incorporates galactose into desialated ovine submaxillary mucin at optimal pH 7.5 and the transfer cannot be inhibited by acetylglucosamine. The apparent Km for UDPgalactose is 0.191 mM. Both enzymes require Mn2+ and Triton X-100 for optimal galactose incorporation. The enzymes could be separated by polyacrylamide gel electrophoresis. Incorporation into endogenous glycoprotein was studied in conditions optimal for the two galactosyl-transferases: (1) at pH 6.8, using Mes as buffer system, and (2) at pH 7.5, using Tris-HCl in the presence of an inhibitory excess of acetylglucosamine. In both cases, most radioactive galactose is incorporated into macromolecules, which could be identified as epithelial glycoprotein. Endogenous incorporation in the presence of excess acetylglucosamine results in the formation of a substantial amount of a disaccharide (probably galactose-beta-(1-3)acetylgalactosamine), whereas upon incorporation at pH 6.8 almost no disaccharide is formed. Quantitative immunoprecipitation experiments with specific antibodies to the endogenous product, labelled by [3H]galactose in the presence of varying amounts of desialated ovine submaxillary mucin and/or acetylglucosamine, indicated that other galactosyl-transferases are involved in the biosynthesis of epithelial glycoprotein.

Animals↗
PubMed1979

Characterization of the forward and reverse reactions catalyzed by CDP-diacylglycerol:inositol transferase in rabbit lung tissue.

CDPdiacylglycerol:inositol transferase activity in rabbit lung tissue has been characterized and the optimum conditions for assaying this enzyme in vitro were determined. Rabbit lung tissue CDPdiacylglycerol:inositol transferase activity was found primarily in the microsomal fraction. The pH optimum of the enzyme activity was between 8.8 and 9.4, and the reaction was dependent on either Mn2+ or Mg2+. Detergents and Ca2+ inhibited the activity of the enzyme. The apparent Km values of the enzyme for CDPdioleoylglycerol and myoinositol were 0.18 mM and 0.10 mM, respectively. The reversibility of the reaction catalyzed by CDPdiacylglycerol:inositol transferase in microsomes prepared from rabbit lung tissue was demonstrated by the synthesis of [3H]CMPdiacylglycerol when [3H]CMP and phosphatidylinositol were present in the incubation mixture. The reverse reaction was characterized and its importance in the regulation of the acidic phospholipid composition of surfactant during lung development is discussed. The pH optimum for the reverse reaction was 6.2, and the reverse reaction was also dependent on Mn2+ or Mg2+. The apparent Km value of CDPdiacylglycerol:inositol transferase for CMP was found to be 2.8 mM.

Animals↗
PubMed1976

Antigenic relationships in calf thymus and human leukemic cell terminal deoxynucleotidyl transferase.

Antibody to purified terminal deoxynucleotidyl transferase (nucleosidetriphosphate : DNA deoxy-nucleotidylexotransferase, E.C. 2.7.7.31) from calf thymus was prepared in rabbits using terminal deoxynucleotidyl transferase crosslinked to bovine serum albumin. These antibodies, partially purified by 60% ammonium sulfate precipitation and Sephadex G-200 column chromatography, produced one precipitation band with calf thymus terminal deoxynucleotidyl transferase on immunodiffusion. This antibody preparation also inhibited the in vitro activity of terminal deoxynucleotidyl transferase from calf thymus, acute leukemic lymphoblasts and Molt-4 cells but not that of DNA polymerases alpha, beta and psi from these cells

Animals↗
PubMed1980

Terminal transferase in acute lymphoblastic leukemia in gibbons.

Terminal deoxynucleotidyl transferase, an enzyme which catalyzes the polymerization of deoxyribonucleoside triphosphates on a 3'-OH end of an initiator molecule in the absence of a template, has been suggested as a biological marker for human acute lymphoblastic leukemia. Examination of a cell line, 6G1, recently established from the peripheral blood of a gibbon ape with acute lymphoblastic leukemia showed the presence of terminal deoxynucleotidyl transferase. This enzyme after purification by successive column chromatography on DEAE-cellulose, phosphocellulose and hydroxyapatite, was found to have biochemical properties similar to those reported for terminal transferase from calf thymus and human leukemic cells. These studies suggest that terminal transferase can be used as a useful biological marker for acute lymphoblastic leukemia in both humans and subhuman primates.

Animals↗
PubMed1981

Cooperation of terminal deoxynucleotidyl transferase with DNA polymerase alpha in the replication of ultraviolet-irradiated DNA.

The amount of DNA synthesis in vitro with the ultraviolet-irradiated poly-(dT) . oligo(rA) template initiators catalysed by DNA polymerase alpha (Masaki, S. and Yoshida, S., Biochim. Biophys. Acta 521, 74--88) decreased with the dose of ultraviolet-irradiation. The ultraviolet irradiation to the template, however did not affect the rate of incorporation of incorrect deoxynucleotides into the newly synthesized poly(dA). The addition of terminal deoxynucleotidyl transferase to this system enhanced the DNA synthesis to a level which is comparable to that of the control and it concomitantly increased the incorporation of the mismatched deoxynucleotide into the newly synthetized poly(dA) strands. On the other hand, with an unirradiated template initiator, the misincorporation was only slightly enhanced by the addition of terminal deoxynucleotidyl transferase. The sizes of newly synthetized DNA measured by sedimentation velocities were found to be smaller with the ultraviolet-irradiated templates but they increased to the control level with the addition of terminal deoxynucleotidyl transferase to the systems. These results suggest that terminal deoxynucleotidyl transferase can help DNA polymerase alpha to "bypass" thymine dimers in vitro by the formation of mismatched regions at the positions opposite to pyrimidine dimers on the template.

Animals↗
PubMed1984

Ornithine carbamyl transferase in Reye's syndrome.

Serum levels of ornithine carbamyl transferase activities were determined in the acutely ill and convalescent Reye's syndrome patients and in their parents. Acutely ill Reye's syndrome patients had elevated levels of serum ornithine carbamyl transferase activities as compared to those in controls. The convalescent Reye's syndrome patients and their parents had normal levels of serum ornithine carbamyl transferase activities. These results suggest that an inborn metabolic defect was not responsible for the increase in serum ornithine carbamyl transferase activities in Reye's syndrome.

Child↗
PubMed1977

Starch gel electrophoresis for galactose-1-phosphate uridylyl-transferase applied to dried filter paper blood specimens.

Starch gel electrophoresis of galactose-1-phosphate uridylyl transferase has been adapted for use on dried filter paper blood specimens submitted for the purposes of routine newborn screening for galactosemia and other inborn errors of metabolism. Selected for study were those specimens with reduced transferase activity, as determined by the Beutler enzyme spot screening test. Clinically benign low activity enzyme variants were readily identified, thus reducing significantly the number of requested additional blood specimens. Transferase-deficient specimens with potential clinical significance had too little activity for transferase visualization and thus could be separated from the benign variants. This technique would facilitate routine newborn screening for galactosemia.

Carrier State↗
PubMed1987

Base substitution mutagenesis by terminal transferase: its role in somatic mutagenesis.

We have addressed the possibility of terminal transferase involvement in somatic mutagenesis and the creation of N-region diversity, by measuring the ability of TdT to enhance single-base substitution mutagenesis during in vitro DNA synthesis. Using 3 independent assays we find that terminal transferase produces only a small increase in base-substitution mutagenesis when assayed in the presence of DNA polymerase-beta. In the presence of either polymerase-alpha or E. coli polymerase-I, however, no detectable increase in TdT-induced mutagenesis is seen. Furthermore, in an assay capable of detecting a variety of mutational events, terminal transferase primarily produces complex addition/deletion mutations, as well as a few multiple, tightly-clustered, single-base mutations. We conclude that the majority of the scattered single-base changes that occur during antibody gene differentiation are not catalyzed by terminal transferase, but instead result from another error-prone DNA synthetic process (possibly utilizing DNA polymerase-beta).

Bacteriophage phi X 174↗
PubMed1981

Subcellular distributions of UDP-galactose:polysaccharide transferase and UDP-glucose pyrophosphorylase involved in biosynthesis of prespore-specific acid mucopolysaccharide in Dictyostelium discoideum.

During the development of a cell aggregate of Dictyostelium discoideum into a fruiting body, an antigenic acid mucopolysaccharide is synthesized only in the prespore cells of a cell mass. In this study, the subcellular distributions of UDPgalactose:polysaccharide transferase and UDPglucose pyrophosphorylase involved in biosynthesis of the mucopolysaccharide were determined. The transferase was specifically localized in the smaller vesicles with lighter density than the prespore-specific vacuoles identifiable electronmicroscopically. In contrast to the enzyme, the antigenic mucopolysaccharide was exclusively localized in the prespore-specific vacuoles. Unlike the transferase, UDPglucose pyrophosphorylase was confined to the soluble fraction. The sucrose gradient profiles of the transferase activity in the 5000 X g supernatant gave two main peaks. When the profiles wee compared among standing and migrating slugs and culminating cell mass, the difference in the profiles closely reflected the state of biosynthesis of the acid mucopolysaccharide in each developmental stage.

Antigens, Fungal↗
PubMed2004

Spectroscopic study of fluorescent peptides for prenyl transferase assays.

A study of the prenyl transferase reactions was performed by fluorescence using rat brain cytosol fractions as an enzyme source. Four dansylated peptides corresponding to the C-terminal sequence of Ras isoforms were synthesised. The effects of different detergents on the farnesylation or geranylgeranylation of the four peptides were evaluated. Dose-dependent effects of dodecyl-maltoside, a non-ionic detergent, on the farnesyl transferase or geranylgeranyl transferase activities were observed with all peptide substrates. Additionally, the effect of temperature was investigated and these assays were applied to determine Michaelis-Menten constants (K(m)) of the substrates: dansyl-GCVLS (1.8 microM), dansyl-GCVVM (3.2 microM), dansyl-CVIM (3.4 microM) and dansyl-GCVLL (8.4 microM) and FPP (22.6 microM) for FTase activity. Using GGPP as co-substrate, GGTase activity was measured with K(m) values superior to 50 microM for all the three substrate dansyl-GCVLS, dansyl-GCVVM, or dansyl-CVIM, whereas values of 7.6 and 5.4 microM were calculated for the dansyl-GCVLL sequence and GGPP co-substrate, respectively. IC50 values of selective prenyl transferase inhibitors, B-581, FTI 276 and GGTI 287 have been measured to 34, 0.8 and 18 nM, respectively, using dansyl-GCVLS as substrate (FTase inhibition). When dansyl-GCVLL is used as substrate (GGTase inhibition) the IC50 values are 5100, 75 and 5 nM for B-581, FTI 276 and GGTI 287, respectively. Then, this developed method allowed to evaluate the selectivity of all the three inhibitors tested.

Animals↗
PubMed1985

Inhibition of carnitine palmitoyl-CoA transferase activity and fatty acid oxidation by lactate and oxfenicine in cardiac muscle.

High concentrations of lactate and oxfenicine inhibit fatty acid oxidation in cardiac muscle. The site of this inhibition was investigated in isolated perfused rat hearts. In hearts perfused with glucose (11 mM) and [U-14 C]palmitate (1.0 mM), addition of 5 mM lactate caused a 38% reduction in 14CO2 production. Tissue levels of long-chain acyl carnitine decreased suggesting that inhibition occurred at either fatty acyl CoA synthetase or carnitine-acyl CoA transferase. Cytosolic levels of acyl-CoA are low compared with mitochondrial levels and changes in acyl-CoA within the cytosolic compartment cannot be estimated directly. Consequently, the rate of conversion of 14C-palmitate to neutral lipids was used as an indicator of cytosolic acyl CoA levels. Lactate caused a 100% increase in 14C-fatty acid conversion to triglycerides suggesting that cytosolic levels of acyl-CoA increased in association with decreased acyl-carnitine. This indicates that lactate inhibited FFA oxidation at the level of carnitine-acyl CoA transferase. Oxfenicine (2 mM) reduced fatty acid oxidation by 45%, decreased acyl-carnitine levels by 80%, and increased conversion of 14C-palmitate to neutral lipids by 44%, suggesting that oxfenicine also inhibits fatty acid oxidation at the level of carnitine-acyl CoA transferase. These data further indicate that carnitine-acyl CoA transferase is an important site of control in the pathway of fatty acid oxidation.

Acyl Coenzyme A↗
PubMed1997

The delta 5-3 beta-hydroxy steroid acyl transferase activities in tissues of the male rat and sheep.

Pregnenolone and dehydroepiandrosterone and their sulphate and fatty acid ester derivatives are concentrated in the mammalian brain, as compared to the peripheral organs. Although biological functions have been postulated for the free steroid and sulphate conjugates, the role of steroid fatty acid esters in the central nervous system (CNS) has yet to be established. A comparison of the esterifying capacities of brain and peripheral organs of the male rat and sheep is made here. The male rat brain was found to possess a higher esterifying capacity than all other tissues examined, with 5.32 +/- 2.46 pmol dehydroepiandrosterone fatty acid esters synthesized/mg protein/h and 1.89 +/- 0.42 pmol pregnenolone fatty acid esters synthesized/mg protein/h. Sheep adrenal and sheep liver homogenates were found to have higher esterifying capacities than sheep brain. Within the brain of both species. delta 5-3 beta-hydroxy steroid acyl transferase enzyme(s) were concentrated in the microsomal fraction. The addition of a lecithin:cholesterol acyl transferase inhibitor to incubation assays resulted in a loss in enzyme activity. This effect was more significant in sheep brain microsomes than rat brain microsomes. It is likely that lecithin:cholesterol acyl transferase is more active in the sheep brain than the rat brain. Results from this study indicate that the steroid acyl transferase enzymes that are active in the mammalian brain are substrate specific.

Acyltransferases↗
PubMed1976

Mechanistic studies of glutamine synthetase from Escherichia coli. An integrated mechanism for biosynthesis, transferase, ATPase reaction.

The mechanism of biosynthetic, transferase, ATPase, and transphosphorylation reactions catalyzed by unadenylylated glutamine synthetase from E. coli was studied. Activation complex(es) involved in the biosynthetic reaction are produced in the presence of either Mg2+ or Mn2+ ; however, with the Mn2+-enzyme inhibition by the product, ADP, is so great that the overall forward biosynthetic reaction cannot be detected with the known assay methods. Binding studies show that substrates (except for NH3 and NH2OH which are not reported here) can bind to the enzyme in a random manner and that binding of the ATP-glutamate, ADP-Pi or ADP-arsenate pairs is strongly synergistic. Inhibition and binding studies show that the same binding site is utilized for glutamate and glutamine in biosynthetic and transferase reactions, respectively, and that a common nucleotide binding site is used for all reactions studied. Studies of the reverse biosynthetic reaction and results of fluorescent titration experiments suggest that both arsenate and orthophosphate bind at a site which overlaps the gamma-phosphate site of nucleoside triphosphate. In the reverse biosynthetic and transferase reactions, ATP serves as a substrate for the Mn2+-enzyme but not for the Mg2+-enzyme. The ATP supported transferase activity of Mn2+-enzyme is probably facilitated by the generation of ADP through ATP hydrolysis. When AMP was the only nucleotide substrate added, it was converted to ATP with concomitant formation of two equivalents of glutamate, under the reverse biosynthetic reaction conditions, and no ADP was detected. The reversibility of 180 transfer between orthophosphate and gamma-acyl group of glutamate was confirmed. ATPase activity of Mg2+ and Mn2+ unadenylylated enzymes is about the same. Both enzymes forms catalyze transphosphorylation reactions between various purine nucleoside triphosphates and nucleoside diphosphates under biosynthetic reaction conditions. The data are consistent with the hypothesis that a single active center is utilized for all reactions studied. Two stepwise mecanisms that could explain the results are discussed.

Adenosine Diphosphate↗
PubMed1998

Peptidyl-transferase ribozymes: trans reactions, structural characterization and ribosomal RNA-like features.

BACKGROUND: One of the most significant questions in understanding the origin of life concerns the order of appearance of DNA, RNA and protein during early biological evolution. If an 'RNA world' was a precursor to extant life, RNA must be able not only to catalyze RNA replication but also to direct peptide synthesis. Iterative RNA selection previously identified catalytic RNAs (ribozymes) that form amide bonds between RNA and an amino acid or between two amino acids. RESULTS: We characterized peptidyl-transferase reactions catalyzed by two different families of ribozymes that use substrates that mimic A site and P site tRNAs. The family II ribozyme secondary structure was modeled using chemical modification, enzymatic digestion and mutational analysis. Two regions resemble the peptidyl-transferase region of 23S ribosomal RNA in sequence and structural context; these regions are important for peptide-bond formation. A shortened form of this ribozyme was engineered to catalyze intermolecular ('trans') peptide-bond formation, with the two amino-acid substrates binding through an attached AMP or oligonucleotide moiety. CONCLUSIONS: An in vitro-selected ribozyme can catalyze the same type of peptide-bond formation as a ribosome; the ribozyme resembles the ribosome because a very specific RNA structure is required for substrate binding and catalysis, and both amino acids are attached to nucleotides. It is intriguing that, although there are many different possible peptidyl-transferase ribozymes, the sequence and secondary structure of one is strikingly similar to the 'helical wheel' portion of 23S rRNA implicated in ribosomal peptidyl-transferase activity.

Adenosine Monophosphate↗
PubMed1995

Binding of phenylarsenoxide to Arg-tRNA protein transferase is independent of vicinal thiols.

Reversible enzyme inhibition by phenylarsenoxides is generally taken to indicate the presence of functionally important vicinal thiol groups. Arginyl aminoacyl-tRNA transferase from eukaryotes is potently inhibited by phenylarsenoxides and possesses one or more essential sulfhydryl groups (Li, J., & Pickart C. M. (1995) Biochemistry 34, 139-147]. To map the putative Cys residues that mediate arsenoxide binding to the transferase from Saccharomyces cerevisiae, we systematically mutagenized the 15 Cys residues of the transferase, singly and in combination, to Ala (13 Cys) or Ser (2 Cys). Six mutant enzymes, encompassing all 15 Cys residues of the transferase, were characterized in detail. The results revealed that Cys-20, Cys-23, and Cys-94 and/or Cys-95 were important for activity, since mutations at these positions reduced activity by 100-fold (Cys-94 and Cys-95 were mutated simultaneously). Surprisingly, however, all of the mutant enzymes retained the ability to bind a radioiodinated phenylarsenoxide derivative, with undiminished stoichiometry and affinity. All of the mutant enzymes also remained susceptible to irreversible reaction with a bifunctional phenylarsenoxide bearing a paraalkyl halide substituent. Prior reaction of the enzyme with the bifunctional reagent blocked subsequent binding of the radiolabeled phenylarsenoxide, indicating that these two reagents bind at a single common site. These results indicate that high-affinity binding of trivalent arsenicals can occur by a thiol-independent mechanism.

Acyltransferases↗
PubMed1989

Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain.

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the nucleotide binding site by the substrate photoaffinity analogue [gamma-32P]-8-azido-dATP. The alpha and beta polypeptides of photolabeled terminal transferase were resolved by high-performance liquid chromatography. The beta polypeptide was digested with trypsin and fractionated by reverse-phase chromatography. Two 32P-containing fractions were isolated and subjected to amino acid sequence analysis. Peptides were identified as Ile209-Lys232 (B26) and Val233-Lys239 (B27). Peptide B26 was further resolved into two overlapping species; one contained an additional lysine residue at the N-terminus which resulted from tryptic cleavage between Lys207 and Lys208. In order to ensure that the sequenced peptides corresponded to the photolabeled species, we devised an anion-exchange procedure to isolate photolabeled peptides from the mixture. Analysis of photolabeled peptides from terminal transferase alpha beta using DEAE-cellulose chromatography followed by reverse-phase HPLC confirmed that the photolabeled species were peptides B26 and B27. Peptide B26, the major photolabeled species, contained a conserved octapeptide region found in several eucaryotic DNA polymerases. In addition, peptide B27 was flanked by a sequence that has been implicated in triphosphate binding in other proteins. Structure predictions, based on sequence data, place the two peptides identified by photolabeling in spatial proximity consistent with the participation of both in the nucleotide binding domain.

Adenosine Triphosphate↗
PubMed1998

Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosaminyl transferase from swine trachea epithelium.

UDP-GalNac: polypeptide N-acetylgalactosaminyltransferase from swine trachea epithelium was purified to homogeneity by procedures which included affinity chromatography on Sepharose 4B columns containing bound deglycosylated Cowper's gland mucin. The enzyme, purified 12,000-fold from microsomes with a yield of 40%, showed only a single band on dodecyl sulfate polyacrylamide gel electrophoresis. The homogenous enzyme has an apparent molecular mass of 70,000 Da, as determined by gel electrophoresis or gel filtration. The transferase has a broad pH optimum between 6.7-7.8 with maximal activity at pH 7.2, and required Mn2+ for activity with maximal activity at 5-7.5 mM. Higher concentrations of Mn2+, inhibited the enzyme. The purified transferase was specific for UDPGalNAc and glycosylated both threonine and serine residues in tryptic peptides prepared from deglycosylated Cowper's gland and swine and human trachea mucins. The apparent Km of the transferase for UDPGalNAc was 6.3 microM, and the Km values for deglycosylated Cowper's gland and human and swine trachea mucins were 0.83, 1.12 and 0.94 mg/ml, respectively. The Vmax of the purified enzyme was 2.1 micromol/min/mg with deglycosylated Cowper's gland mucin, as the glycosyl acceptor. However, the activities with peptides prepared from deglycosylated mucins by limited acid hydrolysis were 20-fold greater than the intact glycoprotein under identical conditions. The deglycosylated mucins and larger peptides aggregated with time of storage and precipitated from solution. Aggregation was accompanied by a corresponding loss of enzymatic activity even after dispersion of the aggregate by sonication. The deglycosylated mucins which were prepared by chemical treatment and periodate oxidation still contained about 20% of the N-acetylgalactosamine present in the intact mucin. When this residual amino sugar was removed by periodate oxidation the completely deglycosylated mucins became very poor substrates for the purified transferase. Data obtained in the current study indicate that the accessibility of serine and threonine in the polypeptide chains of mucin glycoproteins significantly influences the rate of glycosylation of these amino acids. The best substrates and affinity ligand for the enzyme were fragments of incompletely deglycosylated mucin polypeptide chains.

Amino Acid Sequence↗