Enzymatic polymerisation involving 2'-amino-LNA nucleotides

The triphosphate of the thymine derivative of 2′-amino-LNA (2′-amino-LNA-TTP) was synthesised and found to be a good substrate for Phusion® HF DNA polymerase, allowing enzymatic synthesis of modified DNA encoded by an unmodified template. To complement this, 2′-amino-LNA-T phosphoramidites were incorporated into DNA oligonucleotides which were used as templates for enzymatic synthesis of unmodified DNA using either KOD, KOD XL or Phusion polymerases. 2′-Amino-LNA-T in the template and 2′-amino-LNA-TTP as a substrate both decreased reaction rate and yield compared to unmodified DNA, especially for sequences with multiple 2′-amino-LNA-T nucleotides.     

Enzymatic properties of Escherichia coli and human 7,8-dihydro-8-oxoguanine DNA glycosylases.

Oxidative damage to DNA generates aberrant guanine bases such as 2,6-diamino-4-hydroxy-formamido-pyrimidine (Fapy) and 7,8-dihydro-8-oxoguanine (8-oxoG). Although synthetic oligonucleotides containing a single 8-oxoG have been widely used to study enzymatic processing of this lesion, the synthesis of oligonucleotides containing Fapy as a unique lesion has not been achieved to date. In this study, an oligonucleotide containing a single 2,6-diamino-4-hydroxy-5-(N-methyl)formamido-pyrimidine (me-Fapy, a methylated derivative of Fapy) was prepared by a DNA polymerase reaction and the subsequent alkali treatment. The repair activity of Fpg and hOGG1 proteins were compared using oligonucleotide substrates containing me-Fapy and 8-oxoG.     

Enzymatic incorporation in DNA of 1,5-anhydrohexitol nucleotides

The ability of several DNA polymerases to catalyze the template-directed synthesis of duplex oligonucleotides containing a base pair between a nucleotide with anhydrohexitol ring and its natural complement has been investigated. All DNA polymerases were able to accept the chemically synthesized anhydrohexitol triphosphate as substrate and to catalyze the incorporation of one anhydrohexitol nucleotide. However, only family B DNA polymerases succeeded in elongating the primer after the incorporation of an anhydrohexitol nucleotide. In this family, Vent (exo(-)) DNA polymerase is the most successful one and was therefore selected for further investigation. Results revealed that at high enzyme concentrations six hATPs could be incorporated; however, a selective incorporation proved only feasible under experimental conditions where no more than two analogues could be inserted. Also the synthesis of a mixed HNA-DNA sequence was examined. Kinetic parameters for incorporation of one anhydrohexitol adenine nucleoside were similar to those of its natural analogue.     

Enzymatic properties of human hemalbumin.

The binding of hemin to the primary site of human serum albumin (HSA) has been reinvestigated using UV-Vis, CD and NMR techniques. The major fraction of bound hemin contains a five-coordinated high-spin iron(III) center, but a minor fraction of the metal appears to be in a six-coordinated, low-spin state, where a 'distal' residue, possibly a second histidine residue, completes the coordination sphere. The reduced, iron(II) form of the adduct contains six-coordinated low-spin heme. The distal residue hinders the access to the iron(III) center of hemin-HSA to small anionic ligands like azide and cyanide and destabilizes the binding of neutral diatomics like dioxygen and carbon monoxide to the iron(II) form. In spite of these limitations, the hemin-HSA complex promotes hydrogen peroxide activation processes that bear the characteristics of enzymatic reactions and may have biological relevance. The complex is in fact capable of catalyzing peroxidative reactions on phenolic compounds related to tyrosine and hydrogen peroxide dismutation. Kinetic and mechanistic studies confirm that the low efficiency with which peroxidative processes occur depends on the limited rate of the reaction between hydrogen peroxide and the iron(III) center, to form the active species, and by the competitive peroxide degradation reaction.     

Synthesis and properties of novel acyclic nucleotides.

Acyclic adenosine and thymidine analogs derived from L- and D-threonie were synthesised and incorporated into oligonucleotides by automated protocols using a standard phosphoramidite method. UV melting experiments with thus obtained oligonucleotides showed that incorporation of those acyclic nucleosides did not destabilize the hybrid duplexes and that the stabilities of them are influenced by the stereochemical structures of acyclic analogs. Modification of 3'-end of oligonucleotide with acyclic analogs protected the oligonucleotide against 3'-exonuclease.     

Enzymatic analysis of guanine nucleotides in tissues and cells

GTP and GDP concentrations can be determined by a simple and specific spectrophotometric assay that uses commercially available enzymes. The conversion of GTP to GDP catalyzed by nucleosidediphosphate kinase in the presence of ADP enables the subsequent use of guanylate kinase which is coupled with hexokinase and glucose-6-phosphate dehydrogenase as indicator enzymes. Guanylate kinase which is highly specific for GDP and 5′-GMP (Miech, R. P., and Parks, R. E., Jr. (1965) J. Biol. Chem.240, 351–357) is also used for the determination of 5′-GMP and of the sum of all acid-soluble guanine 5′-nucleotides. The latter are hydrolyzed by snake venom phosphodiesterase and assayed as 5′-GMP. The assays are highly reproducible with standard deviations of less than 2% when performed in the optimal range between 2 and 100 nmol of guanine nucleotide per cuvette. The sensitivity can be increased by use of dual wavelength measurements of fluorimetry or by following the generation of ATP with the luciferase-catalyzed luminescence. Contents of guanine nucleotides and of total nucleoside 5′-triphosphates were measured in liver, kidney, brain, and skeletal muscle of the rat. The effect of guanosine and of inhibitors of inosinate dehydrogenase (virazole and mycophenolate) on the level of GTP and GDP was examined in ascites hepatoma cells in suspension.     

Enzymatic properties of rat myelencephalon-specific protease.

Myelencephalon-specific protease (MSP), first identified in the rat and now known to have a human homologue (human kallikrein 6), is preferentially expressed in the central nervous system (CNS), compared with nonneural tissues. MSP has been postulated to have trypsin-like activity, is upregulated in response to glutamate receptor-mediated excitotoxic injury in the CNS, and is downregulated in the brain of Alzheimer's patients. The preferential expression of this enzyme by oligodendrocytes in CNS white matter points to a role in myelin homeostasis. To further characterize the activity and substrate specificity of this newly identified enzyme, we have heterologously expressed MSP in a baculovirus/insect cell line system. We demonstrate that recombinant MSP exhibits a broad specificity for cleavage after arginine but not lysine residues, with kinetic characteristics intermediate between trypsin and pancreatic kallikrein. We show that the pro form of MSP does not self-activate but, rather, requires cleavage after lysine, indicating that mature active MSP is regulated by a distinct protease. MSP may be regulated in part by autolysis, since the active protein is readily inactivated through autolysis at specific internal arginine positions. Additionally, we show that MSP is abundantly expressed in inflammatory cells at sites of demyelination in the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis (MS). In conjunction with data demonstrating the ability of MSP to degrade myelin-associated as well as several extracellular matrix proteins, these findings delineate MSP as a broad-specificity arginine-specific protease with the potential to play a key role in immune-mediated demyelination.     

Enzymatic properties of Escherichia coli peptide deformylase.

Since its discovery in crude extracts in the late sixties, Escherichia coli peptide deformylase activity could not be further characterized because of an apparent extreme instability. We show that this behavior was caused by an inadequate activity assay, involving substrate concentration inhibition and substrate precipitation in crude extracts. The homogeneous protein, as it was previously purified (T. Meinnel and S. Blanquet J. Bacteriol. 175:7737-7740, 1993), had actually retained its initial activity. The influence on the deformylation reaction of several factors was studied and used to improve the activity assay. Pure peptide deformylase proves to act only on peptide substrates with an N-formylmethionyl moiety. In agreement with the occurrence of zinc in the enzyme, peptide deformylase activity is inhibited by 1,10-phenanthroline.     

Enzymatic properties of neuraminidases from Arthrobacter ureafaciens.

Neuraminidase I and neuraminidase II from Arthrobacter ureafaciens were characterized. As determined by gel filtration on Ultrogel AcA 44, the molecular weights of neuraminidases I and II were 51,000 and 39,000, respectively. Neuraminidases I and II were similar to each other in their enzymatic properties except for the substrate specificities towards gangliosides and erythrocyte stroma. Their optimal pHs were between 5.0 and 5.5 with N-acetylneuraminosyl-lactose or bovine submaxillary mucin as substrates, but with colominic acid as a substrate, the pH optimum was between 4.3 and 4.5. They were most active around 53 degrees C, were stable between pH 6.0 and 9.0, and were thermostable up to 50 degrees C. They did not require Ca2+ for activity and were not inhibited by EDTA. They were inhibited only slightly or not at all by p-chloromercuribenzoic acid of Hg2+. Both neuraminidases I and II were able to hydrolyze the alpha-ketosidic linkage of N-glycolylneuraminic acid as well as that of N-acetylneuraminic acid, and were able to liberate substantially all of the sialic acid from various kinds of substrates. However, they cleaved only about 50% of the sialic acid from bovine submaxillary mucin. The saponification of bovine submaxillary mucin by mild alkali treatment, on the other hand, resulted in an increased susceptibility to the neuraminidases and brought about the complete liberation of sialic acid. Remarkable differences were observed between neuraminidases I and II as regards substrate specificities on gangliosides; the initial rate of hydrolysis by neuraminidase I was 74 times, and its maximum velocity constant was 91 times those of neuraminidase II. The addition of sodium cholate markedly stimulated the enzymatic hydrolysis of gangliosides, and increased the maximum velocity constant of neuraminidase I twofold and that of neuraminidase II 143-fold. Although neuraminidases I and II were able to hydrolyze (alpha,2-3), (alpha,2-6), and (alpha,2-8) linkages, the initial rate of hydrolysis of N-acetylneuraminosyl-alpha,2-6-lactose was greater than that of the alpha,2-3-isomer.     

Single-channel properties of ionic channels gated by cyclic nucleotides.

This paper presents an extensive analysis of single-channel properties of cyclic nucleotide gated (CNG) channels, obtained by injecting into Xenopus laevis oocytes the mRNA encoding for the alpha and beta subunits from bovine rods. When the alpha and beta subunits of the CNG channel are coexpressed, at least three types of channels with different properties are observed. One type of channel has well-resolved, multiple conductive levels at negative voltages, but not at positive voltages. The other two types of channel are characterized by flickering openings, but are distinguished because they have a low and a high conductance. The alpha subunit of CNG channels has a well-defined conductance of about 28 pS, but multiple conductive levels are observed in mutant channels E363D and T364M. The conductance of these open states is modulated by protons and the membrane voltage, and has an activation energy around 44 kJ/mol. The relative probability of occupying any of these open states is independent of the cGMP concentration, but depends on extracellular protons. The open probability in the presence of saturating cGMP was 0.78, 0.47, 0.5, and 0.007 in the w.t. and mutants E363D, T364M, and E363G, and its dependence on temperature indicates that the thermodynamics of the transition between the closed and open state is also affected by mutations in the pore region. These results suggest that CNG channels have different conductive levels, leading to the existence of multiple open states in homomeric channels and to the flickering behavior in heteromeric channels, and that the pore is an essential part of the gating of CNG channels.     

Structural and enzymatic properties of adenine 1-oxide nucleotides.

We decribed the preparation of adenine 1-oxide nucleotides by oxidation of the natural compounds with monopermaleic acid in aqueous solutions at neutral pH, with an overall yield after chromatographic purification between 75 and 80%. If irradiated, the adenine 1-oxide nucleotides undergo a photochemical rearrangement reaction, the main photoproducts in aqueous solution at alkaline pH being the corresponding isoguanine nucleotides. The modified ring vibration pattern of the 1-oxide analogues as well as the 13C chemical shift indicate a loss of aromaticity as compared to the natural compounds. Coupling constant measurements show that the dihedral angle between the 31POC and OC13C planes is around 180degree, i.e., trans, as in the natural adenine nucleotides. The modified adenine nucleotides were tested as potential substrates and/or inhibitors of mitochondrial processes, as substrates of varous phosphotransferases from mitochondria or cytosol, and as allosteric effectors in the reactions catalyzed by glutamate dehydrogenase and phosphofructokinase. Although the adenine 1-oxide nucleotides are not recognized by the translocase system of the inner mitochondrial membrane, they are good substrates for mitochondrial phosphotransferases located in the intermembrane space. Similarly, they participate in the phosphoryl group transfer reactions catalyzed by pyruvate kinase, phosphofructokinase, and hexokinase. As allosteric effectors, the modified nucleotides are less active than the natural compounds, probably because of a lower binding capacity to the allosteric sites of the regulatory enzymes.