Coupled-Enzyme System for Measuring Viral Neuraminidase Activity

A coupled-enzyme assay for determining viral neuraminidase activity is described. All reactants-viral neuraminidase, the initial substrate (fetuin), N-acetylneuraminic acid aldolase, lactic acid dehydrogenase, and reduced nicotinamide adenine dinucleotide-are combined in a single cuvette. Thus, in a single coupled system neuraminidase releases N-acetylneuraminic acid, which is cleaved to N-acetyl-D-mannosamine and pyruvic acid; finally, pyruvate is reduced to lactate as reduced nicotinamide adenine dinucleotide is oxidized. The rate of change of absorbance at 340 nm, as reduced nicotinamide adenine dinucleotide is oxidized, is a measure of the rate of reaction of the coupled system. This procedure, which measures the rate of release of N-acetylneuraminic acid by neuraminidase, is an alternate method for those procedures which require multistep, colorimetric determinations.     

Ligand-incorporation site in 5-methylcytosine-detection probe modulating the site of osmium complexation with the target DNA

ICON Probes, short DNA strands containing an adenine linked to a bipyridine ligand, formed an interstrand cross-link with 5-methylcytosine located opposite the modified adenine in the presence of an osmium oxidant. The location of a bipyridine-tethered adenine in the probes varied the selectivity of the reactive base. An ICON probe where the modified adenine was located at the probe center showed a 5-methylcytosine-selective osmium complexation, whereas an ICON probe with the modified adenine at the strand end exhibited high reactivity towards thymine as well as 5-methylcytosine. The modulation of reactive bases by the incorporation of a bipyridine-tethered adenine site made facilitates design of ICON probes for the fluorometric detection of 5-methylcytosine.     

Effects of functional group changes in the EcoRV recognition site on the cleavage reaction catalyzed by the endonuclease

Oligodeoxynucleotides have been prepared which contain changes in the functional group pattern present in the EcoRV recognition site d(GATATC). These modifications involve the deletion of specific functional groups or the reversal of the relative positions of functional groups within the canonical six base pair recognition site. The duplex stability of these modified oligodeoxynucleotides has been assessed by determining the thermodynamic parameters characterizing helix formation. Steady-state kinetic parameters have been used to characterize the interaction of the modified oligodeoxynucleotides with the EcoRV endonuclease. The enzyme is very sensitive to the deletion of either of the adenine amino or thymine methyl groups, or the reversal of the relative positions of the adenine amino group and thymine carboxy group which form an interstrand hydrogen bond in the major groove of the B-DNA helix. Conversely, deletion of the guanine amino group had only minimal effects upon the measured kinetic parameters. Deletion of the exocyclic amino group from the "inner" dA-dT base pair resulted in the fragment which interacted with the enzyme on the basis of observed inhibition experiments but was not cleaved. The results suggest that the endonuclease interacts with its recognition sequence via contacts in the major groove of the B-DNA helix and that both hydrogen bonding to the adenine amino groups and also hydrophobic interactions with the thymine methyl groups are involved.     

Ischemia decreases the content of the adenine nucleotide translocator in mitochondria of rat kidney

The activity of the adenine nucleotide translocator is decreased at ischemia. Studies were undertaken to elucidate changes in the adenine nucleotide translocator by determining its content in mitochondria of ischemic rat kidney. After 60 min of ischemia, the content of the adenine nucleotide translocator amounted only to about 55%, of that measured in control mitochondria. At the same time, the flux control coefficient was increased. These changes paralled the well-known effects of ischemia: the decrease in oxidative phosphorylation and in adenine nucleotides. It is supposed that the decrease in the adenine nucleotide translocatar content accounts, at least partially, for the ischemia-induced impairment of mitochondria.     

Unique modification of adenine in genomic DNA of the marine cyanobacterium Trichodesmium sp. strain NIBB 1067.

The genomic DNA of the marine nonheterocystous nitrogen-fixing cyanobacterium Trichodesmium sp. strain NIBB 1067 was found to be highly resistant to DNA restriction endonucleases. The DNA was digested extensively by the restriction enzyme DpnI, which requires adenine methylation for activity. The DNA composition, determined by high-performance liquid chromatography (HPLC), was found to be 69% AT. Surprisingly, it was found that a modified adenine which was not methylated at the usual N6 position was present and made up 4.7 mol% of the nucleosides in Trichodesmium DNA (15 mol% of deoxyadenosine). In order for adenine residues to be modified at this many positions, there must be many modifying enzymes or at least one of the modifying enzymes must have a degenerate recognition site. The reason(s) for this extensive methylation has not yet been determined but may have implications for the ecological success of this microorganism in nature.     

Synthesis and hybridization properties of oligonucleotides containing 6-membered azasugar nucleotides

A modified nucleoside was synthesized with adenine and a 6-membered azasugar, and it was converted to the phosphoramidite which was used for the incorporation into oligonucleotides. The hybridization properties of the modified oligonucleotides with DNA and RNA were studied.     

Decrease in mitochondrial levels of adenine nucleotides and concomitant mitochondrial dysfunction in ischemic rat liver

The process of mitochondrial dysfunction in ischemic rat liver was studied. A close correlation was found between decrease in the mitochondrial adenine nucleotide content and deterioration of oxidative phosphorylation capacity. The level of total adenine nucleotides, which was 15--20 nmol/mg protein in mitochondria isolated from normal liver, fell to 1--2 nmol/mg protein with concomitant loss of oxidative phosphorylation capacity after anoxic incubation in vitro or in vivo for 120 min. However, neither the permeability barrier to adenine nucleotides nor matrix enzymes were affected under these conditions. The loss of adenine nucleotides was ascribed to degradation of AMP to adenosine and then leakage of the latter. Conventional procedures for maintenance of oxidative phosphorylation capacity of isolated mitochondria, preservation in the cold and addition of ATP or a respiratory substrate under aerobic conditions, were very effective in maintaining the intramitochondrial levels of adenine nucleotides. Of the three species of adenine nucleotides, only AMP was ineffective in maintaining mitochondrial function; mitochondria containing more than 5 nmol of ATP plus ADP/mg protein exhibited normal activity of oxidative phosphorylation, but with less than 2 nmol they showed no activity.     

Determination of adenine nucleotides by thin-layer chromatography combined with the dansylation reaction

A new method was developed for the qualitative and quantitative determining of adenine nucleotides, based on thin-layer chromatography and dansilation. Chromatography mixtures 1,4-dioxan-water-ammonia (40:20:8) or 1,4-dioxan-water-propanol 2-ammonia (40:20:10:8) were used for separation of dansilated derivatives of ATP. ADP, AMP. The intensity of luminescence and the areas of spots served as measures of nucleotide quantities and were estimated using a microdensitometer. The method was applied to estimate the adenine nucleotide pool in E. coli K-12 cells.     

Suppression of human lymphocyte DNA and protein synthesis in vitro by adenosine and eight modified adenine nucleosides in the presence or in the absence of adenosine deaminase inhibitors, 2′-deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA)

Adenosine and eight modified adenine nucleosides in the presence or in the absence of adenosine deaminase (ADA) inhibitors, 2′-deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl) adenine, were investigated with regard to their effects on phytohemagglutinin-stimulated human lymphocytes in vitro. Protein and DNA synthesis were inhibited depending upon the specific nucleoside, presence or absence of ADA inhibitor, and time of addition of nucleoside to cultures. A possible role for the modified adenine nucleosides in causing in vivo immunodeficiency is proposed.     

A simple fluorimetric microassay for adenine compounds in platelets and plasma and its application to studies on the platelet release reaction

1. The formation of a stable fluorescent product between chloroacetaldehyde and adenine or its derivatives provides the basis of a rapid simple assay for total adenine compounds in blood platelets and in plasma. The assay will measure down to 200pmol of adenine nucleotides. An evaluation of the method established the optimum conditions for the production of maximum fluorescence. 2. Values obtained for total adenine compounds in platelets were 12.9nmol/10(8) cells in man and 7.8nmol/10(8) cells in rat. These closely agree with previous values for total platelet adenine nucleotides found by using a firefly luciferase assay, or a recycled NAD-linked photometric assay. This supports the concept that the chloroacetaldehyde reaction measures total adenine nucleotides in platelets. 3. Platelet aggregation induced by collagen was studied photometrically in 0.1ml volumes of citrated platelet-rich plasma, and total adenine nucleotides were assayed in platelets and plasma before and after aggregation. During aggregation 58% of adenine nucleotides were released from human platelets, and 36% from rat platelets. 4. The chloroacetaldehyde assay is no substitute for more sophisticated procedures, but is a simple sensitive means of monitoring the release of adenine nucleotides from blood platelets and is particularly valuable when small plasma samples must be used.     

A new ternary complex between horse liver alcohol dehydrogenase, NAD+, and acetone

Acetone was found to form a dead-end ternary complex with horse liver alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD⁺) when the reactants were incubated for a long time at relatively high concentrations. The complex formation was demonstrated by measuring the increase in absorbance at 320 nm, the quenching of protein fluorescence, and the loss of enzyme activity. Since acetone is a substrate of liver alcohol dehydrogenase, and the presence of acetaldehyde or pyrazole prevents acetone from forming the dead-end complex with liver alcohol dehydrogenase and NAD⁺, the acetone molecule in the complex may be bound to the substrate binding site of liver alcohol dehydrogenase. The dissociation of the complex was demonstrated by prolonged dialysis or by addition of reduced nicotinamide adenine dinucleotide (NADH) and iso-butyramide. A modified nicotinamide adenine dinucleotide was obtained as a main product from the dead-end complex after dissociation of the complex or denaturation of the apoenzyme. The modified nicotinamide adenine dinucleotide was found to exhibit an absorption spectrum similar to that of NADH; however, it was not oxidizable by liver alcohol dehydrogenase in the presence of acetaldehyde and exhibited no fluorescence.     

Motor Nerve Terminal Staining Combined with Catecholamine Histofluorescence or Immunocytochemistry

A number of excellent techniques are available to stain and characterize different types of neurons and nerve terminals. However, because these different techniques are frequently not compatible, their usefulness in determining the relationships between specific axons and neuromuscular junctions is often limited. The goal was to develop specific procedures for simultaneous visualization of different types of unmyelinated axons and motor nerve terminals in the same preparation. First we modified the formal-dehyde/glutaraldehyde staining solutions of the aqueous aldehyde fluorescence technique (Faglu) to observe catecholamine containing axons in whole mount amphibian skeletal muscle. The compatibility of this modified staining solution with other histological procedures made it possible to stain both motor nerve terminals with tetrazolium salts and, in the same preparation, to observe unmyelinated axons with aldehyde-induced catecholamine histofluorescence. This same general formaldehyde/glutaraldehyde staining procedure was also used with immunocytochemical techniques to visualize fluorescent antibody stained nerves and motor nerve terminals in the same whole mount preparation.     

Determination of hydrogen peroxide generated by reduced nicotinamide adenine dinucleotide oxidase

Hydrogen peroxide can be conveniently determined using horseradish peroxidase (HRP) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). However, interference occurs among assay components in the presence of reduced nicotinamide adenine dinucleotide (NADH) that is also a substrate of NADH oxidase. So, depletion of NADH is required before using the HRP method. Here, we report simple and rapid procedures to accurately determine hydrogen peroxide generated by NADH oxidase. All procedures developed were based on the extreme acid lability of NADH and the stability of hydrogen peroxide, because NADH was decomposed at pH 2.0 or 3.0 for 10 min, while hydrogen peroxide was stable at pH 2.0 or 3.0 for at least 60 min. Acidification and neutralization were carried out by adjusting sample containing NADH up to 30 microM to pH 2.0 for 10 min before neutralizing it back to pH 7.0. Then, hydrogen peroxide in the sample was measured using the HRP method and its determination limit was found to be about 0.3 microM. Alternatively, hydrogen peroxide in samples containing NADH up to 100 microM could be quantitated using a modified HRP method that required an acidification step only, which was found to have a determination limit of about 3 microM hydrogen peroxide in original samples.     

Heart phosphofructokinase. Allosteric kinetics following affinity labeling modification of the enzyme by 8-[m-(m-fluorosulfonylbenzamido) benzylthio] adenine.

An adenine analog 8-[m-(m-fluorosulfonylbenzamido)benzylthio]adenine (FSB-adenine) reacts covalently with sheep heart phosphofructokinase. Under conditions optimal for allosteric kinetics the modified enzyme is less sensitive to inhibition by ATP and insensitive to activation by AMP, cyclic AMP, and ADP. The concentration of fructose-6-P necessary for half-maximal activity is markedly decreased, while the cooperativity to the same substrate is not changed under the same conditions. The modified enzyme is more stable at pH 6.5 when compared with the native enzyme. Changes in the allosteric kinetics of the enzyme are proportional to the extent of modification reaching maximal effect when 3.2 mol of the reagent were bound/mol of tetrameric enzyme. Affinity labeling of the enzyme by the adenine derivative does not affect significantly the catalytic site. This is evidenced by the demonstration that under assay conditions optimal for Michaelian kinetics neither the Km for ATP nor for fructose-6-P is significantly changed following chemical modification. Maximal activity of the modified enzyme was 60% of the native enzyme. ADP gives the best protection, while AMP gives less protection against modification by the reagent. ATP slows the rate of the reaction and causes a slight decrease in maximum binding of the reagent to the enzyme. Modification of the enzyme caused a marked reduction of AMP and ADP binding. The evidence indicates that the modified site is a nucleotide mono- and diphosphate activation site.     

Determination of the major tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA adduct by 1H and 15N NMR studies

(+)-CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of the drug are thought to be due to the formation of a covalent adduct with DNA through N3 of adenine. Although the covalent linkage sites between (+)-CC-1065 and DNA have been determined, the tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA duplex adduct was not defined. A [6-15N]deoxyadenosine-labeled 12 base pair non-self-complementary oligomer, d(GGCGGAGTT*AGG).d(CCTAACTCCGCC) (asterisk indicates 15N-labeled base), containing the (+)-CC-1065 most preferred binding sequence 5'AGTTA, was synthesized and modified with (+)-CC-1065. This [6-15N]deoxyadenosine-labeled 12-mer duplex adduct was then studied by 1H and 15N NMR. One-dimensional NOE difference and two-dimensional NOESY 1H NMR experiments on the nonisotopically labeled 12-mer duplex adduct demonstrate that the 6-amino protons of the covalently modified adenine exhibit two signals at 9.19 and 9.08 ppm. Proton NMR experiments on the [6-15N]deoxyadenosine-labeled 12-mer duplex adduct show that the two resonance signals for adenine H6 observed on the nonisotopically labeled duplex adduct were split into doublets by the 15N nucleus with coupling constants of 91.3 Hz for non-hydrogen-bonded and 86.8 Hz for hydrogen-bonded amino protons. Parallel 15N NMR experiments on the [6-15N]deoxyadenosine-labeled (+)-CC-1065-12-mer duplex adduct show a triplet-like signal around -276.9 ppm and coupling constants of 91.5 and 85.6 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced cleavage by sodium hydroxide of methyladenine in DNA sequencing.

When the Maxam and Gilbert DNA sequencing method which is modified by Bencini et al. (Biotechniques Jan/Feb pp4-5, 1984) is applied to DNA containing methylated adenine in a GATC sequence, the cleavage reaction by sodium hydroxide is found to be greatly reduced in comparison to that of non-methylated adenine. Thus, a faint band in A greater than C reaction suggests a methyl adenine and can be used for its detection. That the faint band corresponds to a methyladenine was confirmed by Sanger sequencing of the same fragment and further by Maxam and Gilbert sequencing of the complementary strand of DNA, which was replicated in an E. coli strain either having or lacking methylation enzymes.     

Localization of oxalacetate keto-enol-tautomerase.

The intracellular location of oxalacetate keto-enol-tautomerase (oxaloacetate keto-enolisomerase) (EC 5.3.2.2) has been determined in two types of animal cells, rat liver and pig kidney. Two fractionation procedures were adopted and modified to suit each type of tissue. One fractionation procedure gave the soluble phase, microsomal and mitochondrial fractions, while the other isolated the nuclear fraction. The tautomerase is distributed among the soluble phase, microsomes and mitochondria in both tissues. Fractionation efficiency was checked by determining percentage recoveries of enzymic activity and total protein after each step, by microscopy studies and by determining the distribution of several marker enzymes.     

Synthesis and properties of nucleic acid methylene carboxamide mimics derived from morpholine nucleosides

Uracyl and adenine containing oligomers derived from carboxymethyl derivatives of morpholine nucleoside analogues (MorGly) were synthesized using the methods of peptide chemistry. Capillary electrophoresis conditions were found for the analysis of the homogeneity of the nucleic acid mimics protonated at physiological pH. The thermal stability of complementary complexes formed by the MorGly oligomers was shown to depend dramatically on the heterocyclic base structure (uracil or adenine). Based on the study of tandem complexes it was demonstrated that the impact on the thermal stability of cooperative interactions at oligomer junctions was higher for modified oligomers than for native oligodeoxyriboadenylates. Adenine containing MorGly oligomers formed more stable complexes with poly(U) than native oligodeoxyriboadenylates of the same length. Complexes formed by modified oligomers with polyribonucleotides were more stable if compared with polydeoxyribonucleotides.     

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.     

The Increasing Adenine Nucleotide Concentration and the Maturation of Rat Liver Mitochondria during Neonatal Development

The levels of adenosine triphosphate, diphosphate, and monophosphate in liver and isolated liver mitochondria were examined in foetal, neonatal, and suckling rats. It was shown that while the total adenine nucleotide level in liver varied only slightly during development, there was a steady increase in the concentration of mitochondrial adenine nucleotides. This increase was most dramatic just after birth. Experiments using pups obtained by caesarean section one day prior to normal birth and kept in a humidicrib for up to two hours, showed that the mitochondrial adenine nucleotide level doubles during this period. This increase is associated with the maturation of the mitochondrial inner membrane as measured by the enhancement of respiratory control.
The results indicate that in addition to the adenine nucleotide translocator—which effects the exchange of adenosine triphosphate and diphosphate—there must be a second transport mechanism present, at least in perinatal mitochondria, which is responsible for the net uptake of adenine nucleotides.
The adenine nucleotides in this study were measured, using a modified luciferin—luciferase assay. In this method the preincubation of sample with appropriate enzymes to convert adenosine monophosphate and diphosphate to adenosine triphosphate, was carried out in the same scintillation vial as the final assay. This eliminated a second sampling step and thereby increased the convenience, speed, and accuracy of this very sensitive method.