Affinity of (alpha-P-borano)-NTP analogs to rabbit muscle pyruvate kinase

The binding affinity of (alpha-P-borano) and other NTP analogs to rabbit muscle pyruvate kinase (PK) was investigated using a fluorescence quenching approach to obtain structure-activity relationships for substrate specificity of nucleotide analogs.     

SYNTHESIS AND PROPERTIES OF NOVEL TRIPHOSPHATE ANALOGUES: RIBONUCLEOSIDE AND DEOXYRIBONUCLEOSIDE (α-P-BORANO, α-P-THIO)TRIPHOSPHATES

The first ribo- and deoxyribo-nucleoside (α-P-borano, α-P-thio)triphosphates have been synthesized. The chemical and biochemical properties of adenosine (α-P-borano, α-P-thio)triphosphate and thymidine (α-P-borano, α-P-thio) triphosphate have been investigated.     

Thermodynamic properties of enzyme-catalyzed reactions involving cytosine, uracil, thymine, and their nucleosides and nucleotides

The standard Gibbs energies of formation of species in the cytidine triphosphate series, uridine triphosphate series, and thymidine triphosphate series have been calculated on the basis of the convention that Delta(f)G=0 for the neutral form of cytidine in aqueous solution at 298.15 K at zero ionic strength. This makes it possible to calculate apparent equilibrium constants for a number of reactions for which apparent equilibrium constants have not been measured or cannot be measured because they are too large. This paper adds fifteen reactants to the database BasicBiochemData3 at MathSource that includes 199 reactants. The standard transformed Gibbs energies of formation of these fifteen reactants are used to calculate apparent equilibrium constants at 298.15 K, ionic strength 0.25 M, and pHs 5, 6, 7, 8, and 9 for thirty two reactions. The pKs, standard Gibbs energies of hydrolysis, and standard Gibbs energies of deamination are given for these fifteen reactants.     

Partial purification of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate synthetase from chicken liver.

An enzyme that catalyzes the formation of 6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate (D-erythrodihydroneopterin triphosphate) and formic acid from GTP has been purified about 3700-fold from homogenates of chicken liver. The molecular weight of the enzyme, D-erythrodihydroneopterin triphosphate synthetase (GTP cyclohydrolase), has been estimated to be 125,000 by gel filtration on Ultrogel AcA-34. The enzyme functions optimally between pH 8.0 and 9.2 and is considerably heat-stable. No cofactors or metal ions have been demonstrated to be required for activity; however, the reaction is strongly inhibited by Cu2+ and Hg2+. GTP is the most efficient substrate, with GDP being 1/17 as active and guanosine, GMP, and ATP being inactive. The Km for GTP has been found to be 14 micrometer. Although the overall reaction catalyzed by D-erythrodihydroneopterin triphosphate synthetase from chicken liver is identical with that from Escherichia coli GTP cyclohydrolase, immunological studies show no apparent homology between the two enzymes.     

Structural and functional analysis of biopolymers and their complexes: Enzymatic synthesis of high-modified DNA

Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology.     

Biosynthesis of tetrahydrobiopterin. Purification and characterization of 6-pyruvoyl-tetrahydropterin synthase from human liver

6-Pyruvoyl-tetrahydropterin synthase, which catalyzes the first step in the conversion of 7,8-dihydroneopterin triphosphate to tetrahydrobiopterin, was purified approximately 140,000-fold to apparent homogeneity from human liver. The molecular mass of the enzyme is estimated to be 83 kDa. 7,8-Dihydroneopterin triphosphate was a substrate of the enzyme in the presence of Mg2+, and the pH optimum of the reaction was 7.5 in Tris HCl buffer. The Km value for 7,8-dihydroneopterin triphosphate was 10 microM. The product of this enzymatic reaction was the presumed intermediate 6-pyruvoyl-tetrahydropterin. This latter compound was converted to tetrahydrobiopterin in the presence of NADPH and partially purified sepiapterin reductase from human liver. The conditions and the effect of N-acetylserotonin on this reaction, and on the formation of the intermediates 6-(1'-hydroxy-2'-oxopropyl)-tetrahydropterin and 6-(1' oxo-2'-hydroxypropyl)-tetrahydropterin have been studied.     

Differential recognition of Toxoplasma gondii recombinant nucleoside triphosphate hydrolase isoforms by naturally infected human sera

Toxoplasma gondii possesses a highly active nucleoside triphosphate hydrolase, which has been shown to be an immunodominant antigen in mice and humans. Two isoforms (I and II) which exhibit different activities with respect to hydrolysis of ATP exist. Past studies suggest that all strains of T. gondii contain the less active nucleoside triphosphate hydrolase II, whilst only virulent strains contain the nucleoside triphosphate hydrolase I isoform. In order to further investigate the correlation between nucleoside triphosphate hydrolase isoform and biological significance, we cloned and expressed as glutathione S-transferase fusion proteins the full-length nucleoside triphosphate hydrolase I and II isoforms and two truncations of the nucleoside triphosphate hydrolase I isoform in Escherichia coli. We then used ELISAs with the full-length recombinant nucleoside triphosphate hydrolases as antigens to examine 188 naturally infected T. gondii-positive sera and 83 T. gondii-negative sera for antibody reactivity. All positive sera reacted to T. gondii whole tachyzoite lysate antigen, 31 sera reacted to both nucleoside triphosphate hydrolase isoforms, three sera reacted specifically to nucleoside triphosphate hydrolase I and two sera reacted to only nucleoside triphosphate hydrolase II. Immunoblot analysis of the five sera reacting to either nucleoside triphosphate hydrolase I or II revealed both quantitative and qualitative differences in reactivity to the two isoforms. Comparative immunoblot analysis using the truncations of the nucleoside triphosphate hydrolase I isoform, and one of these positive sera identified a presumptive differential epitope between the nucleoside triphosphate hydrolase I and II isoforms within an 81-aa region (aa 445–526) at the C-terminus of the nucleoside triphosphate hydrolase I isoform. This differential reactivity was further localised to the 12-residue region of greatest variability between the two isoforms (residues 488–499) using synthetic peptides. This is the first report where naturally infected human sera have been used to identify a differential epitope. Because this region is essential for substrate binding, an antibody response to this region may play some role in inhibition of this highly active enzyme.     

Incorporation of biotin-labeled deoxyuridine triphosphate into DNA during excision repair and electron microscopic visualization of repair patches

Biotin-labeled deoxyuridine triphosphate (BiodUTP) has the potential to be a useful affinity probe for studies on DNA repair, if it can be incorporated into DNA repair patches and does not inhibit subsequent steps in the excision repair pathway. We have synthesized BiodUTP by an improved procedure and have used permeable normal human fibroblasts to determine the effect of substituting BiodUTP for thymidine triphosphate on several steps in the excision repair pathway: incision, polymerization, ligation, and nucleosome rearrangement. The results demonstrate that BiodUTP is efficiently incorporated into repair patches and has little or no effect on the repair process. The presence of BiodUMP in ligated repair patches has been used to visualize the repair patches by electron microscopy following incubation with ferritin-labeled avidin. This approach has been used to estimate the maximum size of repair patches induced by ultraviolet radiation.     

Improvements in the chain-termination method of DNA sequencing through the use of 7-deaza-2'-deoxyadenosine.

Significant improvements in the quality of DNA sequencing data have been shown when deoxyadenosine triphosphate (dATP) is replaced by 7-deaza-2'-deoxyadenosine triphosphate (c7dATP). The use of c7dATP in conjunction with 7-deaza-2'-deoxyguanosine triphosphate (c7dGTP) further decreases anomalies in electrophoretic mobility which are caused by compressions involving G and/or A residues. This effect is observed for both isotope-based and fluorescence-based sequencing approaches. Replacing dATP with c7dATP also results in a higher degree of uniformity in the frequency of chain termination reactions, when such terminations involve the incorporation of fluorescence-labeled dideoxynucleotides by T7 polymerase. These improvements in the gel-resolution and distribution of chain-terminated DNA products result in higher accuracy in both manual and automated base assignment.     

Dynamic affinity chromatography of heavy meromyosin subfragment-1.

Heavy meromyosin subfragment-1 and its trinitrophenylated derivative have been chromatographed on immobilized ATP, ADP and adenosine 5'-(geta, gamma-imino) triphosphate affinity chromatography columns, in the presence and in the absence of Ng-2+ or Ca-2+.ma-32-P] ATP columns. While the divalent cations had little effect on the chromatographic pattern in the case of the non-hydrolyzable ADP and adenosine 5' (beta, gamma-imino) triphosphate, they catalyzed splitting in the case of ATP and at the same time strongly increased the affinity of adsorption of the proteins. The protein-elution and the Pi-release patterns were different for the native and the modified proteins. These results have been interpreted in terms of protein binding to the various intermediates of the ATP hydrolysis reaction.     

Separation and partial characterization of two deoxyribonucleic acid polymerases from Spiroplasma citri.

The separation and partial characterization of two deoxyribonucleic acid polymerases from Spiroplasma citri have been achieved. The two enzymes had different elution properties on diethylaminoethyl (DEAE) cellulose and differed in their sensitivity to N-ethylmaleimide (NEM), preference for different template-primers, and sedimentation velocity in linear glycerol gradients. The first enzyme activity, ScA, was retained on DEAE-cellulose and was not inhibited by NEM. Activated deoxyribonucleic acid and poly(dA)-oligo(dT12) were the preferred template-primers. Arabinosyl-cytidine triphosphate had no effect. The sedimentation coefficient of ScA was 6.3s. The second activity, ScB, was not retained on DEAE-cellulose and was inhibited by NEM. Poly(dA)-oligo(dT12) was the preferred template-primer, whereas activated DNA was only poorly utilized. ScB was not affected by arabinosyl-cytidine triphosphate, and its sedimentation coefficient was 4.4s. The polymerization activities of the two enzymes were maximum at 37 to 40 degrees C.     

Dyspropterin, an intermediate formed from dihydroneopterin triphosphate in the biosynthetic pathway of tetrahydrobiopterin

The structure of dyspropterin, a new name given to an intermediate which is formed from dihydroneopterin triphosphate in the biosynthetic pathway of tetrahydrobiopterin, has been studied. Sepiapterin reductase (EC 1.1.1.153) was found to reduce dyspropterin to tetrahydrobiopterin in the presence of NADPH. Several lines of evidence showing the formation of tetrahydrobiopterin have been presented. Stoichiometric analysis revealed that there is a 1:2 relationship between the production of biopterin and the oxidation of NADPH during the reductase-catalyzed reduction of dyspropterin. The tetrahydrobiopterin production from dyspropterin was enhanced by dihydropteridine reductase (EC 1.6.99.7). Dyspropterin could also serve as a cofactor in phenylalanine hydroxylase (EC 1.14.16.1) system. These results are consistent with the view that dyspropterin is 6-(1,2-dioxopropyl)-5,6,7,8-tetrahydropterin. Based on our findings, the biosynthetic pathway of tetrahydrobiopterin from dihydroneopterin triphosphate has been discussed.     

The role of DNA polymerases alpha, beta and gamma in nuclear DNA synthesis.

The effects of the inhibitors 2'3' dideoxythymidine triphosphate (ddTTP) and 1-beta-D-arabinofuranosyl cytosine triphosphate (araCTP) on DNA synthesis in isolated S-phase HeLa S3 nuclei have been examined. These effects are compared with the effects of the same inhibitors in partially purified preparations of DNA polymerases alpha and beta. The effect of ddTTP on partially purified DNA polymerase gamma was also tested. DNA polymerases beta and gamma were very sensitive to ddTTP whereas DNA polymerase alpha and DNA synthesis in isolated nuclei were quite resistant. The synthesis and subsequent ligation of primary DNA pieces ('Okazaki fragments') were not affected by the presence of this inhibitor. DNA synthesis in isolated nuclei and DNA polymerase alpha activity were very sensitive to araCTP whereas DNA polymerase beta was almost totally resistant to the inhibitor. The results indicate a major role for DNA polymerase alpha in DNA replication.     

5-Hydroxypyrimidine deoxynucleoside triphosphates are more efficiently incorporated into DNA by exonuclease-free Klenow fragment than 8-oxopurine deoxynucleoside triphosphates.

Recent studies with 8-oxodeoxyguanosine triphosphate (8-oxodGTP) have suggested that incorporation of oxidized nucleotides from the precursor pool into DNA may have deleterious effects. Here we show that 5-hydroxydeoxycytosine triphosphate (5-OHdCTP) and 5-hydroxydeoxyuridine triphosphate (5-OHdUTP) are more efficient substrates than 8-oxodGTP for Escherichia coli DNA polymerase I Klenow fragment lacking proofreading activity, while 8-oxodeoxyadenosine triphosphate (8-oxodGTP, 5-OHdCTP can mispair with dA in DNA but with lower efficiency. Since the 5-hydroxypyrimidines are present in normal and oxidized cellular DNA in amounts similar to the 8-oxopurines, these data suggest that enzymatic mechanisms might exist for removing them from the DNA precursor pools.     

Biochemical studies on the mutagen, 6-N-hydroxylaminopurine. Synthesis of the deoxynucleoside triphosphate and its incorporation into DNA in vitro

The nucleotide analogue, 6-N-hydroxylaminopurine deoxynucleoside triphosphate (dHAPTP) has been synthesized from 6-chloropurine by a procedure involving both enzymatic and chemical reagents. In a series of experiments involving several different DNA polymerases including 3 procaryotic and 2 eucaryotic enzymes, it was shown that dHAPTP is ambiguous in its base-pairing characteristics, since it can replace both dATP and dGTP in DNA synthesis. It was also shown that different enzymes have different capacities to distinguish dHAPTP from the canonical deoxynucleoside triphosphates. These results are consistent with (but do not prove) the hypothesis that the mechanism of 6-N-hydroxylaminopurine mutagenesis seen in both eucaryotic and procaryotic organisms is due to its conversion, in vivo, to a deoxynucleoside triphosphate which is incorporated ambiguously for dATP and dGTP during DNA replication.     

A novel Raman spectrophotometric method for quantitative measurement of nucleoside triphosphate hydrolysis

A novel spectrophotometric method, based upon Raman spectroscopy, has been developed for accurate quantitative determination of nucleoside triphosphate phosphohydrolase (NTPase) activity. The method relies upon simultaneous measurement in real time of the intensities of Raman marker bands diagnostic of the triphosphate (1115 cm(-1)) and diphosphate (1085 cm(-1)) moieties of the NTPase substrate and product, respectively. The reliability of the method is demonstrated for the NTPase-active RNA-packaging enzyme (protein P4) of bacteriophage phi6, for which comparative NTPase activities have been estimated independently by radiolabeling assays. The Raman-determined rate for adenosine triphosphate substrate (8.6 +/- 1.3 micromol x mg(-1) x min(-1) at 40 degrees C) is in good agreement with previous estimates. The versatility of the Raman method is demonstrated by its applicability to a variety of nucleotide substrates of P4, including the natural ribonucleoside triphosphates (ATP, GTP) and dideoxynucleoside triphosphates (ddATP, ddGTP). Advantages of the present protocol include conservative sample requirements (approximately 10(-6) g enzyme/protocol) and relative ease of data collection and analysis. The latter conveniences are particularly advantageous for the measurement of activation energies of phosphohydrolase activity.