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2.
Anal Biochem ; 298(2): 231-40, 2001 Nov 15.
Article in English | MEDLINE | ID: mdl-11700977

ABSTRACT

Two fluorescent adenosine analogs, 4-amino-6-methyl-8-(2-deoxy-beta-d-ribofuranosyl)-7(8H)-pteridone (6MAP) and 4-amino-2,6-dimethyl-8-(2'-deoxy-beta-d-ribofuranosyl)-7(8H)-pteridone (DMAP), have been synthesized as phosphoramidites. These probes are site-selectively incorporated into oligonucleotides using automated DNA synthesis. Relative quantum yields are 0.39 for 6MAP and 0.48 for DMAP as monomers and range from >0.01 to 0.11 in oligonucleotides. Excitation maxima are 310 (6MAP) and 330 nm (DMAP) and the emission maximum for each is 430 nm. Fluorescence decay curves of each are monoexponential exhibiting lifetimes of 3.8 and 4.8 ns for 6MAP and DMAP, respectively. When these probes are incorporated into oligonucleotides they display quenching of fluorescence intensity, increases in the complexity of decay curves, and decreases in mean lifetimes. Because these changes are apparently mediated by interactions with neighboring bases, spectral changes that occur as probe-containing oligonucleotides meet and react with other molecules provide a means of monitoring these interactions in real time. These probes are minimally disruptive to DNA structure as evidenced by melting temperatures of probe-containing oligonucleotides that are very similar to those of controls. Digestion of probe-containing oligonucleotides with P1 nuclease confirms probe stability as fluorescence levels are restored to those expected for each monomer. These adenosine analog probes are capable of providing information on DNA structure as it responds to binding or catalysis through interaction with other molecules.


Subject(s)
Adenosine/chemical synthesis , Pteridines/chemical synthesis , Adenosine/analogs & derivatives , Adenosine/chemistry , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , Molecular Structure , Organophosphorus Compounds/chemistry , Pteridines/chemistry , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Spectrometry, Fluorescence
3.
J Biol Chem ; 276(52): 49133-41, 2001 Dec 28.
Article in English | MEDLINE | ID: mdl-11590164

ABSTRACT

Pathological nitric oxide (NO) generation in sepsis, inflammation, and stroke may be therapeutically controlled by inhibiting NO synthases (NOS). Here we targeted the (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)Bip)-binding site of NOS, which, upon cofactor binding, maximally increases enzyme activity and NO production from substrate l-arginine. The first generation of H(4)Bip-based NOS inhibitors employed a 4-amino pharmacophore of H(4)Bip analogous to antifolates such as methotrexate. We developed a novel series of 4-oxo-pteridine derivatives that were screened for inhibition against neuronal NOS (NOS-I) and a structure-activity relationship was determined. To understand the structural basis for pterin antagonism, selected derivatives were docked into the NOS pterin binding cavity. Using a reduced 4-oxo-pteridine scaffold, derivatives with certain modifications such as electron-rich aromatic phenyl or benzoyl groups at the 5- and 6-positions, were discovered to markedly inhibit NOS-I, possibly due to hydrophobic and electrostatic interactions with Phe(462) and Ser(104), respectively, within the pterin binding pocket. One of the most effective 4-oxo compounds and, for comparisons an active 4-amino derivative, were then co-crystallized with the endothelial NOS (NOS-III) oxygenase domain and this structure solved to confirm the hypothetical binding modes. Collectively, these findings suggest (i) that, unlike the antifolate principle, the 4-amino substituent is not essential for developing pterin-based NOS inhibitors and (ii), provide a steric and electrostatic basis for their rational design.


Subject(s)
Biopterins/analogs & derivatives , Biopterins/chemistry , Biopterins/metabolism , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase/metabolism , Animals , Antioxidants/chemistry , Antioxidants/metabolism , Binding Sites , Cerebellum/enzymology , Humans , Isoenzymes/chemistry , Isoenzymes/metabolism , Models, Molecular , Molecular Structure , Nitric Oxide Synthase/antagonists & inhibitors , Protein Binding , Protein Structure, Tertiary , Structure-Activity Relationship , Swine
4.
Biochem J ; 356(Pt 1): 43-51, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11336634

ABSTRACT

Besides oxidizing L-arginine, neuronal NO synthase (NOS) NADPH-dependently reduces various electron acceptors, including cytochrome c and tetrazolium salts. The latter NADPH diaphorase reaction is used as a NOS-specific histochemical stain. Both reductase activities have been utilized to analyse electron transfer mechanisms within NOS. Basal L-arginine turnover by homodimeric NOS is enhanced by exogenous tetrahydrobiopterin, and the intra-subunit electron flow may include intermediate trihydrobiopterin. In the present work we have investigated the possible role of the tetrahydrobiopterin binding site of NOS in its reductase activities by examining the effects of anti-pterin type (PHS) NOS inhibitors. Although the type I anti-pterin, PHS-32, which does not affect basal dimeric NOS activity, also had no effect on either reductase activity, the type II anti-pterin, PHS-72, which inhibits basal NOS activity, inhibited both reductase activities and the NADPH diaphorase histochemical stain. Pterin-free NOS monomers catalysed both cytochrome c and tetrazolium salt reduction. Our data suggest that both NOS reductase activities are independent of tetrahydrobiopterin. However, occupation of an exosite near the pterin site in NOS by type II anti-pterins may interfere with the electron flow within the active centre, suggesting that steric perturbation of the pterin binding pocket or reductase interaction contribute to the mechanism of inhibition by this class of NOS inhibitors.


Subject(s)
Biopterins/analogs & derivatives , Biopterins/pharmacology , NADPH Dehydrogenase/drug effects , Nitric Oxide Synthase/drug effects , Nitric Oxide/metabolism , Calcium/pharmacology , Calmodulin/pharmacology , Catalase/metabolism , Electron Transport , NADH Dehydrogenase/metabolism , Nitric Oxide Synthase Type II , Oxidation-Reduction , Oxidoreductases , Pterins/antagonists & inhibitors , Superoxide Dismutase/metabolism , Superoxides/metabolism , Tetrazolium Salts/metabolism
5.
Biochemistry ; 39(45): 13676-86, 2000 Nov 14.
Article in English | MEDLINE | ID: mdl-11076506

ABSTRACT

Tetrahydropterins are obligatory cofactors for tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis. A series of synthetic analogues of 6(R)-L-erythro-5,6,7, 8-tetrahydrobiopterin (BH(4)) with different substituents in positions C2, N3, C4, N5, C6, C7, and N8 on the ring were used as active site probes for recombinant human TH. The enzyme tolerates rather bulky substituents at C6, as seen by the catalytic efficiency (V(max)/K(m)) and the coupling efficiency (mol of L-DOPA produced/mol of tetrahydropterin oxidized) of the cofactors. Substitutions at C2, C4, N5, and N8 abolish the cofactor activity of the pterin analogues. Molecular docking of BH(4) into the crystal structure of the catalytic domain of ligand-free rat TH results in complexes in which the pteridine ring pi-stacks with Phe300 and the N3 and the amino group at C2 hydrogen bonds with Glu332. The pteridine ring also establishes interactions with Leu294 and Gln310. The distance between C4a in the pteridines and the active site iron was 4.2 +/- 0.5 A for the ensemble of docked conformers. Docking of BH(4) analogues into TH also shows that the most bulky substituents at C6 can be well-accommodated within the large hydrophobic pocket surrounded by Ala297, Ser368, Tyr371, and Trp372, without altering the positioning of the ring. The pterin ring of 7-BH(4) shows proper stacking with Phe300, but the distance between the C4a and the active site iron is 0.6 A longer than for bound BH(4), a finding that may be related to the high degree of uncoupling observed for 7-BH(4).


Subject(s)
Pterins/chemistry , Tyrosine 3-Monooxygenase/chemistry , Animals , Binding Sites , Binding, Competitive , Catalysis , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Kinetics , Protein Conformation , Pterins/chemical synthesis , Pterins/metabolism , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Structure-Activity Relationship , Tyrosine 3-Monooxygenase/antagonists & inhibitors , Tyrosine 3-Monooxygenase/genetics , Tyrosine 3-Monooxygenase/metabolism
6.
Biochem J ; 346 Pt 3: 767-76, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10698705

ABSTRACT

The underlying mechanisms regulating the activity of the family of homodimeric nitric oxide synthases (NOSs) and, in particular, the requirement for (6R)-5,6,7,8-tetrahydro-L-biopterin (H(4)Bip) are not fully understood. Here we have investigated possible allosteric and stabilizing effects of H(4)Bip on neuronal NOS (NOS-I) during the conversion of substrate, L-arginine, into L-citrulline and nitric oxide. Indeed, in kinetic studies dual allosteric interactions between L-arginine and H(4)Bip activated recombinant human NOS-I to increase L-arginine turnover. Consistent with this was the observation that H(4)Bip, but not the pterin-based NOS inhibitor 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydrooxazolo[1, 2-f]-pteridine (PHS-32), caused an L-arginine-dependent increase in the haem Soret band, indicating an increase in substrate binding to recombinant human NOS-I. Conversely, L-arginine was observed to increase in a concentration-dependent manner H(4)Bip binding to pig brain NOS-I. Secondly, we investigated the stabilization of NOS quaternary structure by H(4)Bip in relation to uncoupled catalysis. Under catalytic assay conditions and in the absence of H(4)Bip, dimeric recombinant human NOS-I dissociated into inactive monomers. Monomerization was related to the uncoupling of reductive oxygen activation, because it was inhibited by both superoxide dismutase and the inhibitor N(omega)-nitro-L-arginine. Importantly, H(4)Bip was found to react chemically with superoxide (O(2)(-.)) and enzyme-bound H(4)Bip was consumed under O(2)(-.)-generating conditions in the absence of substrate. These results suggest that H(4)Bip allosterically activates NOS-I and stabilizes quaternary structure by a novel mechanism involving the direct interception of auto-damaging O(2)(-.).


Subject(s)
Biopterins/analogs & derivatives , Nitric Oxide Synthase/metabolism , Superoxides/antagonists & inhibitors , Allosteric Regulation , Arginine/pharmacology , Binding Sites , Biopterins/metabolism , Biopterins/pharmacology , Catalysis , Cell Line , Enzyme Activation , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Indicators and Reagents , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase Type I , Protein Structure, Quaternary , Superoxides/metabolism
7.
Nucleosides Nucleotides Nucleic Acids ; 19(10-12): 1765-77, 2000.
Article in English | MEDLINE | ID: mdl-11200271

ABSTRACT

2'-O-Methyloligoribonucleotides, deoxyoligonucleotides and 2'-O-methyl/deoxy gapmers were synthesized using solid phase phosphoramidite chemistry employing the 2-(4-nitrophenyl)ethyl (npe) protection strategy. Melting temperatures of the synthesized oligonucleotides as well as their stability against degradation by several different nucleases were determined. 2'-O-Methyloligoribonucleotides showed the highest melting temperatures (Tm's) whereas 2'-O-methyl/deoxy gapmers revealed either slightly higher or surprizingly no thermal stabilities compared with their deoxy analogs when using self-complementary sequences. Gapmers with four 2'-O-methyl nucleotides on both ends showed about the same stability as all 2'-O-methyloligoribonucleotides against micrococal nuclease, nuclease S1, and snake venom phosphodiesterase.


Subject(s)
Micrococcal Nuclease/metabolism , Oligonucleotides/chemical synthesis , Phosphoric Diester Hydrolases/metabolism , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Base Sequence , Chromatography, High Pressure Liquid , Hydrolysis , Nucleic Acid Hybridization , Oligonucleotides/metabolism , Temperature
8.
J Med Chem ; 42(20): 4108-21, 1999 Oct 07.
Article in English | MEDLINE | ID: mdl-10514281

ABSTRACT

The family of nitric oxide synthases (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO), an important cellular messenger molecule which has been implicated in the pathophysiology of septic shock and inflammatory and neurodegenerative disease states. NOS can be maximally activated by the ubiquitous cofactor, (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip), and antagonists of H(4)Bip may be of therapeutic importance to inhibit pathologically high NO formation. The 4-amino substituted analogue of H(4)Bip was reported to be a potent NOS inhibitor. Therefore, we developed a series of novel 4-amino pteridine derivatives, anti-pterins, to pharmacologically target the neuronal isoform of nitric oxide synthase (NOS-I). To functionally characterize the pterin/anti-pterin interaction and establish a structure-activity relationship (SAR), we systematically altered the substituents in the 2-, 4-, 5-, 6-, and 7-position of the pteridine nucleus. Varying the substitution pattern in the 2-, 5-, and 7-position resulted in no significant inhibitory effect on enzyme activity. In contrast, bulky substituents in the 6-position, such as phenyl, markedly increased the inhibitory potency of the reduced 4-amino-5,6,7,8-tetrahydropteridines, possibly as a consequence of hydrophobic interactions within NOS-I. However, this was not the case for the aromatic 4-amino pteridines. Interestingly, chemical modification of the 4-amino substituent by dialkyl/diaralkylation together with 6-arylation of the aromatic 2,4-diamino pteridine resulted in potent and efficacious inhibitors of NOS-I, suggesting possible hydrophilic and hydrophobic interactions within NOS-I. This SAR agrees with (a) the recently published crystal structure of the oxygenase domain of the inducible NOS isoform (NOS-II) and (b) the comparative molecular field analysis of selected NOS-I inhibitors, which resulted in a 3D-QSAR model of the pterin binding site interactions. Further optimization should be possible when the full length structure of NOS-I becomes available.


Subject(s)
Biopterins/analogs & derivatives , Enzyme Inhibitors/chemical synthesis , Neurons/enzymology , Nitric Oxide Synthase/antagonists & inhibitors , Pteridines/chemical synthesis , Animals , Biopterins/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Isoenzymes/antagonists & inhibitors , Pteridines/chemistry , Structure-Activity Relationship , Swine
9.
Nucleosides Nucleotides ; 18(6-7): 1219-20, 1999.
Article in English | MEDLINE | ID: mdl-10474216

ABSTRACT

The introduction of the base-labile 9-fluorenylmethoxycarbonyl (Fmoc) group into the exocyclic amino function of 2'-deoxynucleosides and their dimethoxytritylation and phosphitylation is described. The resulting key intermediates were investigated in the built-up of different oligodeoxyribonucleoside phosphate and thiophosphate chains which were deprotected under mild basic conditions leading to crude oligomers of high purity.


Subject(s)
Fluorenes/chemistry , Oligonucleotides/chemical synthesis , Base Sequence , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel
11.
J Biol Chem ; 274(35): 24921-9, 1999 Aug 27.
Article in English | MEDLINE | ID: mdl-10455167

ABSTRACT

The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO synthases (NOS). For unknown reasons, all NOS co-purify with substoichiometric amounts of (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) and require additional H(4)Bip for maximal activity. We examined the effects of H(4)Bip and pterin-derived inhibitors (anti-pterins) on purified neuronal NOS-I quaternary structure and H(4)Bip content. During L-arginine turnover, NOS-I dimers time dependently dissociated into inactive monomers, paralleled by a loss of enzyme-associated pterin. Dimer dissociation was inhibited when saturating levels of H(4)Bip were added during catalysis. Similar results were obtained with pterin-free NOS-I expressed in Escherichia coli. This stabilizing effect of H(4)Bip was mimicked by the anti-pterin 2-amino-4,6-dioxo-3,4,5,6,8,8a,9, 10-octahydro-oxazolo[1,2f]-pteridine (PHS-32), which also displaced NOS-associated H(4)Bip in a competitive manner. Surprisingly, H(4)Bip not only dissociated from NOS during catalysis, but was only partially recovered in the solute (50.0 +/- 16.5% of control at 20 min). NOS-associated H(4)Bip appeared to react with a NOS catalysis product to a derivative distinct from dihydrobiopterin or biopterin. Under identical conditions, reagent H(4)Bip was chemically stable and fully recovered (95.5 +/- 3.4% of control). A similar loss of both reagent and enzyme-bound H(4)Bip and dimer content was observed by NO generated from spermine NONOate. In conclusion, we propose a role for H(4)Bip as a dimer-stabilizing factor of neuronal NOS during catalysis, possibly by interfering with enzyme destabilizing products.


Subject(s)
Biopterins/analogs & derivatives , Nitric Oxide Synthase/chemistry , Arginine/metabolism , Biopterins/metabolism , Biopterins/pharmacology , Dimerization , Enzyme Stability/drug effects , Escherichia coli , Humans , Kinetics , Nitric Oxide/metabolism , Nitric Oxide Synthase Type I , Protein Conformation , Pteridines/pharmacology , Recombinant Proteins/chemistry , Spectrophotometry , Spermine/analogs & derivatives
12.
Biochim Biophys Acta ; 1410(1): 61-70, 1999 Jan 27.
Article in English | MEDLINE | ID: mdl-10076015

ABSTRACT

A new pteridine glycoside, called cyanopterin, was isolated from Synechocystis sp. PCC 6803 and its structure was elucidated as 6-[1-(4-O-methyl-(alpha-d-glucuronyl)-(1, 6)-(beta-d-galactosyloxy]methylpterin by chemical degradation and 1H- and 13C-NMR spectroscopic means. Cyanopterin is constitutively synthesized at a relatively high intracellular concentration that is comparable to that of chlorophyll a in a molar ratio of approximately 1 to 1.6. The in vivo oxidation state of cyanopterin is primarily the fully reduced 5,6,7,8-tetrahydro form. The cellular function is unknown at present. The findings have established a model system, using Synechocystis sp. PCC 6803, for studies of the physiological functions of unconjugated pteridine glycosides found mostly in cyanobacteria.


Subject(s)
Cyanobacteria/chemistry , Glycosides/chemistry , Pteridines/chemistry , Chromatography, High Pressure Liquid , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Structure , Pterins/chemistry
13.
J Biol Chem ; 273(50): 33142-9, 1998 Dec 11.
Article in English | MEDLINE | ID: mdl-9837881

ABSTRACT

Nitric oxide synthases (NOS) are homodimeric enzymes that NADPH-dependently convert L-arginine to nitric oxide and L-citrulline. Interestingly, all NOS also require (6R)-5,6,7, 8-tetrahydro-L-biopterin (H4Bip) for maximal activity although the mechanism is not fully understood. Basal NOS activity, i.e. that in the absence of exogenous H4Bip, has been attributed to enzyme-associated H4Bip. To elucidate further H4Bip function in purified NOS, we developed two types of pterin-based NOS inhibitors, termed anti-pterins. In contrast to type II anti-pterins, type I anti-pterins specifically displaced enzyme-associated H4Bip and inhibited H4Bip-stimulated NOS activity in a fully competitive manner but, surprisingly, had no effect on basal NOS activity. Moreover, for a number of different NOS preparations basal activity (percent of Vmax) was frequently higher than the percentage of pterin saturation and was not affected by preincubation of enzyme with H4Bip. Thus, basal NOS activity appeared to be independent of enzyme-associated H4Bip. The lack of intrinsic 4a-pterincarbinolamine dehydratase activity argued against classical H4Bip redox cycling in NOS. Rather, H4Bip was required for both maximal activity and stability of NOS by binding to the oxygenase/dimerization domain and preventing monomerization and inactivation during L-arginine turnover. Since anti-pterins were also effective in intact cells, they may become useful in modulating states of pathologically high nitric oxide formation.


Subject(s)
Biopterins/analogs & derivatives , Nitric Oxide Synthase/metabolism , Animals , Biopterins/antagonists & inhibitors , Biopterins/metabolism , Catalysis , Cerebellum/enzymology , Humans , Kinetics , Recombinant Proteins/metabolism , Swine
14.
Biophys J ; 73(6): 3277-86, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9414238

ABSTRACT

The fluorescence properties of 3-methyl-isoxanthopterin (3-MI) incorporated into different oligonucleotides have been determined. This highly fluorescent guanosine analog has its absorption and fluorescence spectra well resolved from those of the normal nucleotides and the aromatic amino acids. The small shifts observed in absorption and fluorescence emission spectra upon incorporation of 3-MI into these oligonucleotides are consistent with a general solvent effect and do not suggest any contribution from the position of the probe from the 5' end, the sequence of nucleotides immediately 5' or 3' to the probe, or the single- or double-stranded nature of the oligomer. However, steady-state and time-resolved fluorescence studies indicate that the presence of a purine immediately 5' or 3' to the probe results in some dynamic but mostly static quenching in the single-stranded oligomer. Furthermore, a 3' purine is more effective than a 5' purine, and an adenine appears to be more effective than a guanine for these static quenching interactions. Formation of the double-stranded oligomer leads to an additional loss of quantum yield, which can also be ascribed primarily to static quenching. These results show that this new class of spectrally enhanced fluorescent purine analogs will be able to provide useful information concerning the perturbation of nucleic acid structures.


Subject(s)
Deoxyguanosine/analogs & derivatives , Fluorescent Dyes/chemistry , Oligonucleotides/chemistry , Xanthopterin/analogs & derivatives , Base Sequence , Biophysical Phenomena , Biophysics , Deoxyguanosine/chemistry , Fluorescence Polarization , Polydeoxyribonucleotides/chemistry , Quantum Theory , Spectrometry, Fluorescence , Spectrophotometry , Xanthopterin/chemistry
15.
J Interferon Cytokine Res ; 17(7): 377-85, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9243369

ABSTRACT

Previous studies from this laboratory have demonstrated a statistically significant dysregulation in several key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNase L and PKR antiviral pathways in chronic fatigue syndrome (CFS) (Suhadolnik et al. Clin Infect Dis 18, S96-104, 1994; Suhadolnik et al. In Vivo 8, 599-604, 1994). Two methodologies have been developed to further examine the upregulated RNase L activity in CFS. First, photoaffinity labeling of extracts of peripheral blood mononuclear cells (PBMC) with the azido 2-5A photoaffinity probe, [32P]pApAp(8-azidoA), followed by immunoprecipitation with a polyclonal antibody against recombinant, human 80-kDa RNase L and analysis under denaturing conditions. A subset of individuals with CFS was identified with only one 2-5A binding protein at 37 kDa, whereas in extracts of PBMC from a second subset of CFS PBMC and from healthy controls, photolabeled/immunoreactive 2-5A binding proteins were detected at 80, 42, and 37 kDa. Second, analytic gel permeation HPLC was completed under native conditions. Extracts of healthy control PBMC revealed 2-5A binding and 2-5A-dependent RNase L enzyme activity at 80 and 42 kDa as determined by hydrolysis of poly(U)-3'-[32P]pCp. A subset of CFS PBMC contained 2-5A binding proteins with 2-5A-dependent RNase L enzyme activity at 80, 42, and 30 kDa. However, a second subset of CFS PBMC contained 2-5A binding and 2-5A-dependent RNase L enzyme activity only at 30 kDa. Evidence is provided indicating that the RNase L enzyme dysfunction in CFS is more complex than previously reported.


Subject(s)
2',5'-Oligoadenylate Synthetase/metabolism , Endoribonucleases/metabolism , Fatigue Syndrome, Chronic/enzymology , Adult , Antibody Specificity , Carrier Proteins , Case-Control Studies , Endoribonucleases/immunology , Female , Humans , Hydrolysis , Leukocytes, Mononuclear/enzymology , Male , Middle Aged , Molecular Weight , Precipitin Tests , Recombinant Proteins/immunology , Up-Regulation
16.
Anal Biochem ; 244(1): 86-95, 1997 Jan 01.
Article in English | MEDLINE | ID: mdl-9025913

ABSTRACT

Eighteen fluorescent pteridine-based nucleoside analogs have been prepared that are suitable for synthesis as phosphormidites and site-specific incorporation into oligonucleotides. Their quantum yields ranged from < or = 0.03 to 0.88. The maximum excitation and emission wavelenghts of seven selected probes with quantum yields > 0.15 ranged from 334 to 358 and 400 to 444 nm, respectively. Fluorescence decay curves of the seven probes were biexponential, and the mean intensity-weighted lifetimes ranged from 0.87 to 6.54 ns. Incorporation of probes 4 and 17 (3-methylisoxanthopterin and 6-methylisoxanthopterin) into oligonucleotides significantly quenched their fluorescence signal, and the degree of quench correlated with the number and proximity of purines in the oligonucleotide. Incorporation also resulted in a shift in absorbance-, emission-, and decay-associated spectra for 6-methylisoxanthopterin. An increase in the complexity of the decay curve and a decrease in the mean lifetime occurred for both probes. Formation of double-stranded oligonucleotides did not substantially increase the degree of quenching but generally increased the complexity of decay curves and decreased the mean lifetimes. Melting temperature, Tm, depression equivalent to that of a single base pair mismatch was observed in 3-methylisoxanthopterin-containing double-stranded oligonucleotides, while the Tm of 6-methylisoxanthopterin-containing double-stranded oligonucleotides were unperturbed, e.g., equivalent to unlabeled double-stranded oligonucleotides. This new class of fluorophore yields promising probes for the study of protein/DNA interactions.


Subject(s)
Nucleosides/chemistry , Oligonucleotides/chemistry , Pteridines/chemistry , Fluorescent Dyes , Spectrometry, Fluorescence
17.
Free Radic Biol Med ; 21(4): 449-56, 1996.
Article in English | MEDLINE | ID: mdl-8886794

ABSTRACT

Neopterin, 6-D-erythro-1',2',3'-trihydroxypropyl-pterin, and its dihydroform, 7,8-dihydro-neopterin, are synthesized by human monocytes/macrophages upon stimulation by interferon-gamma. In the presence of iron chelator complexes neopterin enhances hydrogen peroxide-induced luminol chemiluminescence at neutral or slightly alkaline pH (7.5). In contrast, 7,8-dihydroneopterin scavenges chemiluminescence independently from the pH value and iron. In this study, we explored in more detail the mechanism possibly involved: analysis of the reaction products shows that 7,8-dihydroneopterin is oxidized and degraded to 7,8-dihydroxanthopterin and xanthopterin, whereas the neopterin molecule is not chemically altered during the chemiluminescence reaction. Investigations of the neopterin-induced effect show that mannitol, a scavenger of hydroxyl radicals, does not alter the enhancing effect of neopterin. L-histidine, which scavenges singlet oxygen almost as effective as hydroxyl radicals, reduces the enhancing effect of neopterin. However, singlet oxygen was not detectable during the reaction by measuring monomol light emission (1270 nm). When replacing hydrogen peroxide by 3-morpholinosydnonimine, a generator of hydroxyl radicals, or naphthalene-endoperoxide, a generator of singlet oxygen, in the luminol chemiluminescence assay, neopterin shows no enhancing effect irrespective of the presence of iron-(III)-EDTA. The data suggest that neopterin enhances hydrogen peroxide-induced luminol chemiluminescence in the presence of iron-(III)-EDTA by formation of a catalytic complex that seems to favor the formation of oxygen intermediates which derive from hydrogen peroxide and react with luminol.


Subject(s)
Biopterins/analogs & derivatives , Biopterins/pharmacology , Hydrogen Peroxide , Luminol , Biopterins/biosynthesis , Chromatography, High Pressure Liquid , Edetic Acid , Ferric Compounds , Free Radical Scavengers , Histidine , Humans , Interferon-gamma/pharmacology , Iron Chelating Agents , Kinetics , Luminescent Measurements , Macrophages/drug effects , Macrophages/physiology , Monocytes/drug effects , Monocytes/physiology , Neopterin , Oxygen/analysis , Pteridines/analysis , Singlet Oxygen
18.
Acta Biochim Pol ; 43(1): 37-44, 1996.
Article in English | MEDLINE | ID: mdl-8790710

ABSTRACT

New blocking group combinations for the machine-aided oligoribonucleotide synthesis on solid phase material have been developed and tested regarding their general application. An acetal function for 2'-OH protection offers a series of advantages in the synthetic approach but special conditions have to be fulfilled in order to guarantee a selective cleavage of the temporary 5'-OH blocking group such as the dansylethoxycarbonyl or even the acid-labile dimethoxytrityl group in the chain elongation process. The final removal of the 2'-O-acetal function in the partially deblocked oligomer proceeds unexpectedly well under weak acidic conditions due to a supposed intramolecular acid catalysis.


Subject(s)
Oligonucleotides/chemical synthesis , Acetals , Dansyl Compounds , Indicators and Reagents , Molecular Structure , Oligodeoxyribonucleotides/chemical synthesis , Oligoribonucleotides/chemical synthesis
19.
Nucleic Acids Res ; 23(15): 2872-80, 1995 Aug 11.
Article in English | MEDLINE | ID: mdl-7659509

ABSTRACT

We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3-Mi) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-phosphodiester linkage using an automated DNA synthesizer. Fluorescence is partially quenched within an oligonucleotide and the degree of quench is a function of the fluorophore's proximity to purines and its position in the oligonucleotide. As an example of the potential utility of this class of fluorophores, we developed a continuous assay for HIV-1 integrase 3'-processing reaction by incorporating 3-MI at the cleavage site in a double-stranded oligonucleotide identical to the U5 terminal sequence of the HIV genome. Integrase cleaves the 3'-terminal dinucleotide containing the fluorophore, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. Substitution of the fluorophore for guanosine at the cleavage site does not inhibit integrase activity. This assay is specific for the 3'-processing reaction. The change in fluorescence intensity is linear over time and proportional to the rate of the reaction. This assay demonstrates the potential utility of this new class of fluorophore for continuous monitoring of protein/DNA interactions.


Subject(s)
DNA Nucleotidyltransferases/metabolism , Deoxyguanosine/analogs & derivatives , Fluorescent Dyes , Guanosine/analogs & derivatives , HIV-1/enzymology , Xanthopterin/analogs & derivatives , Base Sequence , Deoxyguanosine/chemical synthesis , Deoxyguanosine/metabolism , Deoxyribonucleotides/chemical synthesis , Deoxyribonucleotides/metabolism , Genome, Viral , Guanosine/metabolism , Humans , Integrases , Molecular Sequence Data , Sensitivity and Specificity , Xanthopterin/chemical synthesis , Xanthopterin/metabolism
20.
Eur J Biochem ; 231(2): 414-23, 1995 Jul 15.
Article in English | MEDLINE | ID: mdl-7635153

ABSTRACT

Pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor-1 alpha is a protein with two different functions. We have overexpressed and purified the human wild-type protein, and its Cys81Ser and Cys81Arg mutants. The Cys81Arg mutant has been proposed to be causative in a hyperphenylalaninaemic patient [Citron, B. A., Kaufman, S., Milstien, S., Naylor, E. W., Greene, C. L. & Davis, M. D. (1993) Am. J. Hum. Genet. 53, 768-774]. The dehydratase behaves as a tetramer on gel filtration, while cross-linking experiments showed mono-, di-, tri-, and tetrameric forms, irrespective of the presence of the single Cys81. Sulfhydryl-modifying reagents did not affect the activity, but rather showed that Cys81 is exposed. Various pterins bind and quench the tryptophan fluorescence suggesting the presence of a specific binding site. The fluorescence is destroyed upon light irradiation. Wild-type and the Cys81Ser protein enhance the rate of the phenylalanine hydroxylase assay approximately 10-fold, a value similar to that of native dehydratase from rat liver; the Cys81Arg mutant, in contrast, has significantly lower activity. This is compatible with the hypothesis that the dehydratase is a rate-limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spontaneous dehydration of the synthetic substrate 6,6-dimethyl-7,8-dihydropterin-4a-carbinolamine approximately 50-70-fold at 4 degrees C and pH 8.5. The results are discussed in view of the recently solved three-dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier, G. & Suck, D. (1995) EMBO J. 14, 2032-2042].


Subject(s)
Hydro-Lyases/chemistry , Hydro-Lyases/metabolism , Liver/enzymology , Pterins/metabolism , Transcription Factors/metabolism , Base Sequence , Biopterins/analogs & derivatives , Biopterins/pharmacology , Catalysis , Cysteine/chemistry , Cysteine/metabolism , Humans , Hydro-Lyases/genetics , Hydro-Lyases/isolation & purification , Hydrogen-Ion Concentration , Hydroxylation , Kinetics , Molecular Sequence Data , Mutagenesis , Phenylalanine/metabolism , Phenylalanine Hydroxylase/metabolism , Protein Binding , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Spectrometry, Fluorescence , Temperature , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/isolation & purification , Ultraviolet Rays
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