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PubMed1991

Human carbonyl and aldose reductases: new catalytic functions in tetrahydrobiopterin biosynthesis.

New catalytic functions of human carbonyl- and aldose reductase in tetrahydrobiopterin biosynthesis are proposed. 6-Pyruvoyl tetrahydropterin, an intermediate in the biosynthesis of tetrahydrobiopterin, was converted to 6-lactoyl tetrahydropterin and 1'-hydroxy-2'-oxopropyl tetrahydropterin by carbonyl reductase under anaerobic condition. 1'-Hydroxy-2'-oxopropyl tetrahydropterin was subsequently metabolized to tetrahydrobiopterin by aldose reductase. Based on these results alternative pathways for the synthesis of tetrahydrobiopterin in patients with genetic defects of sepiapterin reductase are suggested.

Alcohol Oxidoreductases↗
PubMed1996

Regulation of tyrosine hydroxylase and tetrahydrobiopterin biosynthetic enzymes in PC12 cells by NGF, EGF and IFN-gamma.

The regulation of catecholamine and tetrahydrobiopterin synthesis was investigated in cultured rat pheochromocytoma PC12 cells following treatments with nerve growth factor (NGF), epidermal growth factor (EGF) and interferon-gamma (IFN-gamma). NGF and EGF, but not IFN-gamma, caused an increase after 24 h in the levels of BH4 and catecholamines, and the activities of tyrosine hydroxylase and GTP cyclohydrolase, the rate-limiting enzymes in catecholamine and BH4 synthesis, respectively. Actinomycin D, a transcriptional inhibitor, blocked treatment-induced elevations in tyrosine hydroxylase and GTP cyclohydrolase activities. NGF, EGF or IFN-gamma did not affect the activity of sepiapterin reductase, the final enzyme in BH4 biosynthesis. Rp-cAMP, an inhibitor of cAMP-mediated responses, blocked the induction of tyrosine hydroxylase by NGF or EGF; inhibition of protein kinase C partially blocked the EGF effect, but not the NGF effect, NGF also induced GTP cyclohydrolase in a cAMP-dependent manner, while the EGF effect was not blocked by Rp-cAMP or protein kinase C inhibitors. Sphingosine induced GTP cyclohydrolase in a protein kinase C-independent manner without affecting tyrosine hydroxylase activity. Our results suggest that both tyrosine hydroxylase and GTP cyclohydrolase are induced in a coordinate and transcription-dependent manner by NGF and EGF, while conditions exist where the induction of tyrosine hydroxylase and GTP cyclohydrolase is not coordinately regulated.

Animals↗
PubMed1990

Synthesis of tetrahydrobiopterin in Friend erythroleukemia cells and its modulator effect on cell proliferation.

The induction of the enzymes in the tetrahydrobiopterin pathway by dimethyl sulfoxide (DMSO) was investigated in subclones F4N and B8/3 of the proerythroblastoid Friend erythroleukemia cell line (MEL). GTP-cyclohydrolase, the initial enzyme in the biosynthetic pathway, is virtually absent in both clones, but expression increases during 3 days of DMSO treatment. The final enzyme levels show 12-fold (subclone B8/3) and 40-fold (subclone F4N) increases compared to initial values. Enhancement of 6-pyruvoyl tetrahydropterin synthase activity is detectable 6 h after exposure to DMSO and continues to increase in the 3-day time period to 2.4-fold and 1.8-fold levels in subclones B8/3 and F4N, respectively. Sepiapterin reductase is present in unstimulated F4N cells and absent in B8/3 cells. The enzyme activity is not affected by DMSO treatment in either cell line. This explains why DMSO treatment causes accumulation of tetrahydrobiopterin in the MEL subclone F4N, but not in subclone B8/3. MEL cells are devoid of phenylalanine hydroxylase for which tetrahydrobiopterin serves as cofactor. In F4N, but not in B8/3, tetrahydrobiopterin modulates the rate of [3H]thymidine incorporation, thus being functionally linked with cell proliferation rather than with differentiation. In contrast to T lymphocytes, periods of tetrahydrobiopterin synthesis and of modulator function are uncoupled in MEL cells.

Alcohol Oxidoreductases↗
PubMed1986

Pigment cell differentiation: the relationship between pterin content, allopurinol treatment, and the melanoid gene in axolotls.

The effects of allopurinol (an inhibitor of the enzyme xanthine dehydrogenase (XDH] and the melanoid gene on pigment cell differentiation in the axolotl were examined by analyzing pigment components of the xanthophore (pterins). Pterin contents of skin extracts (70% ethanol) from wild type, allopurinol-treated and melanoid axolotls were determined by thin layer chromatography (TLC) and fluorometric scanning of TLC plates. Heights of peaks produced were used as a quantitative measure for pterin content. Results reveal that melanoid animals contain significantly reduced amounts of all seven pterins examined as compared with wild type animals. Allopurinol-treated animals have reduced levels of four pterins (xanthopterin, isoxanthopterin, biopterin and sepiapterin) as compared with the wild type. These findings suggest that the alterations in pterin biosynthetic pathways, either by drug-induced inhibition of XDH activity or by the melanoid gene, produce similar dramatic changes in pigment phenotype which are manifested by alterations in pigment cell differentiation.

Allopurinol↗
PubMed1987

Induction of xanthophores from non-pigmented dermal cells of xanthic goldfish in vitro.

To identify precursor cells of xanthophores (xanthoblasts), non-pigmented cells without any phenotypic traits as pigment cells were isolated from the dermal tissue of xanthic goldfish with an adult color pattern and cultured in a medium containing 1 mM db-cAMP or 0.25 U/ml ACTH and 10% carp serum. These non-pigmented cells differentiated into xanthophores which showed a dendritic morphology and contained a large quantity of fluorescent pteridines and numerous vesicular inclusions. Sepiapterin was the major component, and the vesicles contained fuzzy material in addition to small membranous elements. The fluorescent pattern and the morphological characteristics indicated that the differentiated pigment cells were xanthophores of larval type.

Adrenocorticotropic Hormone↗
PubMed1990

Purification and characterization of 6-pyruvoyl-tetrahydropterin synthase from Drosophila melanogaster.

The enzyme 6-pyruvoyl-tetrahydropterin synthase (PTP synthase), which catalyzes the conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, has been purified approx. 230-fold to apparent homogeneity from head extracts of Drosophila melanogaster. A partially purified 6-pyruvoyl-tetrahydropterin reductase (PTP reductase) was also prepared and in its presence, along with Mg2+ and NADPH, the purified PTP synthase converted 7,8-dihydroneopterin triphosphate to metastable 6-lactoyltetrahydropterin, which was autoxidized to sepiapterin under aerobic conditions. Purified PTP synthase had a specific activity of 3792 units per mg protein and migrated as a single protein band on both nondenaturing polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified active enzyme consisted of at least two identical subunits which had a molecular mass of 37.5 kDa on SDS-PAGE and NH2-Asx-Pro- as N-terminal amino acids. The native enzyme in crude extract was shown to be more complex, existing as higher multimeric forms.

Alcohol Oxidoreductases↗
PubMed1992

Structural requirements for the modulatory effect of 6-substituted pterins on interleukin 2 receptor binding.

(6R)-5,6,7,8-Tetrahydrobiopterin is produced by stimulated human T lymphocytes, and is known to affect various aspects of interleukin-2-directed T cell proliferation. Using an increased apparent affinity of interleukin 2 receptor to interleukin 2 as a measure of activity, this study explores whether other 6-substituted pterins might have the same effect, and what structural features are necessary for activity. Of the compounds tested, only the T-lymphocyte-derived (6R)-5,6,7,8-tetrahydrobiopterin was active. The diastereomeric (6S)-5,6,7,8-tetrahydrobiopterin was inactive, as were 7,8-dihydrobiopterin, sepiapterin, 5,6,7,8-tetrahydroneopterin, 6,7-dimethyl-5,6,7,8-tetrahydropterin and 6-hydroxymethylpterin. 7,8-Dihydroneopterin and neopterin were also found to be inactive. It follows that neither of these compounds participates in the feedback modulation of IL-2 receptor affinity, although both of them can be detected upon IFN-gamma stimulation of human monocytes/macrophages. A computer-based molecular modelling study of (6R)-5,6,7,8-tetrahydrobiopterin and (6R)-5,6,7,8-tetrahydroneopterin revealed substantial differences in overall shape between the two molecules, with certain features figuring prominently in the low-energy conformers of (6R)-5,6,7,8-tetrahydrobiopterin.

Biopterins↗
PubMed1994

2,4-Diamino-6-hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, suppresses nitric oxide production by chicken macrophages.

Biosynthesis of nitric oxide (.NO) from L-arginine by nitric oxide synthase (NOS) represents a major cytotoxic effector function of macrophages. It has been shown that most mammalian NOS requires tetrahydrobiopterin (BH4) as a cofactor and that inhibition of BH4 synthesis results in suppressed .NO production. Chicken L-arginine metabolism differs from that of mammals in that chickens cannot synthesize L-arginine de novo. Therefore, it is important to examine whether chicken macrophage .NO synthesis is also BH4-dependent. 2,4-diamino-6-hydroxypyrimidine (DAHP), a specific inhibitor for GTP cyclohydrolase I (GTP-CH; EC 3.5.4.16), the rate-limiting enzyme in de novo pterin synthesis, was used to block synthesis of BH4. Both chicken peritoneal macrophages (PECs) and the avian MC29 virus-transformed macrophage cell line, HD11, exhibited a dose-dependent reduction in .NO production (measured as nitrite accumulation) relative to DAHP concentration. Authentic BH4 and a substrate for pterin salvage pathway of BH4 synthesis, sepiapterin, were both capable of restoring the production of .NO in DAHP-treated PECs and HD11 macrophages. These results suggest that chicken macrophages require active synthesis of BH4 to produce .NO and that chemicals interfering with BH4 synthesis may result in suppressed .NO production and, hence, .NO-mediated immune function.

Animals↗
PubMed1985

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.

Alcohol Oxidoreductases↗
PubMed1986

The application of 8-aminoguanosine triphosphate, a new inhibitor of GTP cyclohydrolase I, to the purification of the enzyme from human liver.

GTP cyclohydrolase I from human liver and Escherichia coli is competitively inhibited by 8-aminoguanosine triphosphate with a dissociation constant (Ki) of 0.25 mumol/l. 8-Aminoguanosine triphosphate, prepared from GTP and hydroxylamine-O-sulfonic acid, was coupled to Sepharose 4B and used as an affinity adsorbent for a 309-fold purification of GTP cyclohydrolase I from human liver. GTP cyclohydrolase I from human liver is a relatively heat-stable enzyme with a half-life of 2 min at 80 degrees C, an isoelectric point (pI) of about 5.6, and a Km for GTP of 31 mumol/l. Addition of KCl (0.3 mol/l) increased the Km to 153 mumol/l. No cofactors were required for activity. L-erythro-5,6,7,8-Tetrahydrobiopterin, L-erythro-7,8-dihydrobiopterin, L-sepiapterin and 8-aminoguanosine triphosphate were strong inhibitors.

Aminohydrolases↗
PubMed1988

Inhibition of GTP cyclohydrolase I by pterins.

Pterins inhibit rat liver GTP cyclohydrolase I activity noncompetitively. Reduced pterins, such as 7,8-dihydro-D-neopterin, (6R,S)-5,6,7,8-tetrahydro-D-neopterin, 7,8-dihydro-L-biopterin, (6R)-5,6,7,8-tetrahydro-L-biopterin, L-sepiapterin, and DL-6-methyl-5,6,7,8-tetrahydropterin are approximately 12-times more potent as inhibitors than are oxidized pterins, such as D-neopterin, L-biopterin, and isoxanthopterin. They are also 12-times more potent than folates, such as folic acid, dihydrofolic acid, (+/-)-L-tetrahydrofolic acid, and aminopterin. The Ki values for 7,8-dihydro-D-neopterin, 7,8-dihydro-L-biopterin, and (6R)-5,6,7,8-tetrahydro-L-biopterin are 12.7 microM, 14.4 microM, and 15.7 microM, respectively. These results suggest that mammalian GTP cyclohydrolase I may be regulated by its metabolic end products.

Aminohydrolases↗
PubMed1991

6-Pyruvoyl tetrahydropterin synthase assay in extracts of cultured human cells using high-performance liquid chromatography with fluorescence detection of biopterin.

An assay for 6-pyruvoyl tetrahydropterin synthase, the second enzyme in the conversion of guanosine triphosphate into tetrahydrobiopterin, has been developed. Cell extracts were incubated with enzymatically prepared dihydroneopterin triphosphate (80 microM) in the presence of Mg2+ (12 mM), excess sepiapterin reductase (EC 1.1.1.153) (2 nmol/min) and NADPH (2 mM). 6-Pyruvoyl tetrahydropterin, the product of the reaction, was thus converted into tetrahydrobiopterin. After oxidation of the reduced biopterin derivatives in acidic iodine solution, biopterin was enriched and separated from the abundant neopterin phosphates by solid-phase extraction on a strong cation exchanger. Biopterin was then directly eluted on a reversed-phase liquid chromatographic column and detected fluorimetrically using excitation at 353 nm and emission at 438 nm. The biopterin concentrations formed by the coupled enzyme reaction increased linearly with incubation times up to 90 min. The assay allows the quantification of 6-pyruvoyl tetrahydropterin synthase in cultured human cells.

Alcohol Oxidoreductases↗
PubMed1996

High-performance liquid chromatographic methods for the quantification of tetrahydrobiopterin biosynthetic enzymes.

Tetrahydrobiopterin is a cofactor in hydroxylation reactions, including phenylalanine 4-monooxygenase, tyrosine 3-monooxygenase, tryptophan 5-monooxygenase, alkyl glycol ether monooxygenase and nitric oxide synthase. Determination of its biosynthesis is carried out to diagnose inherited diseases leading to partial defects in tetrahydrobiopterin synthesis. In addition, tetrahydrobiopterin synthesis is induced by proinflammatory cytokines, and intracellular levels of tetrahydro-biopterin in many cases limit the activity of tetrahydrobiopterin-dependent reactions, such as nitric oxide synthase in intact cells. Biosynthesis of tetrahydrobiopterin from guanosine 5'-triphosphate (GTP) requires the action of three enzymes, GTP-cyclohydrolase I (E.C. 3.5.4.16), 6-pyruvoyl tetrahydropterin synthase (EC, 4.6.1.10) and sepiapterin reductase (E.C. 1.1.1.153). Methods for quantification of biopterin and related pteridines in biological matrices by HPLC and application of these for determining the activity of the three tetrahydrobiopterin biosynthetic enzymes are reviewed in this article.

Alcohol Oxidoreductases↗
PubMed2004

Nuclear localization of tetrahydrobiopterin biosynthetic enzymes.

Biosynthesis of the tetrahydrobiopterin (BH(4)) cofactor, essential for catecholamines and serotonin production and nitric oxide synthase (NOS) activity, requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Upon studying the distribution of GTPCH and PTPS with polyclonal immune sera in cross sections of rat brain, prominent nuclear staining in many neurons was observed besides strong staining in peri-ventricular structures. Furthermore, localization studies in transgenic mice expressing a Pts-LacZ gene fusion containing the N-terminal 35 amino acids of PTPS revealed beta-galactosidase in the nucleus of neurons. In contrast, PTPS-beta-galactosidase was exclusively cytoplasmic in the convoluted kidney tubules but nuclear in other parts of the nephron, indicating again that nuclear targeting may occur only in specific cell categories. Furthermore, the N terminus of PTPS acts as a domain able to target the PTPS-beta-galactosidase fusion protein to the nucleus. In transiently transfected COS-1 cells, which do not express GTPCH and PTPS endogenously, we found cytoplasmic and nuclear staining for GTPCH and PTPS. To further investigate nuclear localization of all three BH(4)-biosynthetic enzymes, we expressed Flag-fusion proteins in transiently transfected COS-1 cells and analyzed the distribution by immunolocalization and sub-cellular fractionation using anti-Flag antibodies and enzymatic assays. Whereas 5-10% of total GTPCH and PTPS and approximately 1% of total SR were present in the nucleus, only GTPCH was confirmed to be an active enzyme in nuclear fractions. The in vitro studies together with the tissue staining corroborate specific nuclear localization of BH(4)-biosynthetic proteins with yet unknown biological function.

Alcohol Oxidoreductases↗
PubMed2004

Nitric oxide production and regulation of neuronal NOS in tyrosine hydroxylase containing neurons.

CAD cells are a murine CNS catecholaminergic (tyrosine hydroxylase-positive; TH+) neuronal cell line that undergoes morphological differentiation to resemble CNS catecholaminergic neurons upon serum deprivation. We show here that CAD cells also express neuronal nitric oxide synthase (nNOS) mRNA and protein and produce readily measurable levels of NO. Since both NO and catecholamines (L-DOPA; dopamine; norepinephrine) are redox active molecules, their production within the same cell may affect the cell's vulnerability to insult. Thus, we examined the regulation of NO production by CAD cells and the effect of NO on cell survival. NO is generated in a dose-dependent fashion by treatment with agents (ionomycin; A23817; KCl) known to increase calcium entry across the cell membrane. The NO level can be increased further by pretreatment with sepiapterin, a membrane permeable precursor for BH4 synthesis, suggesting that the BH4 levels or access required for nNOS activation is limited in CAD cells. Reducing mitochondrial Ca2+ uptake using ruthenium red (RuR) increased ionomycin-mediated NO production over ionomycin alone and indicates a critical role for mitochondria in nNOS regulation. Cell death was significantly increased by ionomycin treatment alone or in conjunction with reduced mitochondrial Ca2+ uptake. However, NO was not the primary mediator of cell death since NOS inhibitors rescued only less than 10% of the cells. These data suggest that endogenous NO production by nNOS is not a major factor in CAD cell death under these conditions.

Animals↗
PubMed2004

Homocysteine induces oxidative stress by uncoupling of NO synthase activity through reduction of tetrahydrobiopterin.

Hyperhomocysteinemia is a risk factor for cardiovascular diseases that induces endothelial dysfunction. Here, we examine the participation of endothelial NO synthase (eNOS) in the homocysteine-induced alterations of NO/O(2)(-) balance in endothelial cells from human umbilical cord vein. When cells were treated for 24 h, homocysteine dose-dependently inhibited thrombin-activated NO release without altering eNOS phosphorylation and independently of the endogenous NOS inhibitor, asymmetric dimethylarginine. The inhibitory effect of homocysteine on NO release was associated with increased production of reactive nitrogen and oxygen species (RNS/ROS) independent of extracellular superoxide anion (O(2)(-)) and was suppressed by the NOS inhibitor L-NAME. In unstimulated cells, L-NAME markedly decreased RNS/ROS formation and the ethidium red fluorescence induced by homocysteine. This eNOS-dependent O(2)(-) synthesis was associated with reduced intracellular levels of both total biopterins (-45%) and tetrahydrobiopterin (-80%) and increased release of 7,8-dihydrobiopterin and biopterin in the extracellular medium (+40%). In addition, homocysteine suppressed the activating effect of sepiapterin on NO release, but not that of ascorbate. The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.

Antioxidants↗
PubMed2004

Tetrahydrobiopterin rapidly reduces the SOD mimic Mn(III) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin.

Mn(III) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) effectively scavenges reactive oxygen and nitrogen species in vitro, and protects in vivo, in different rodent models of oxidative stress injuries. Further, Mn(III)TE-2-PyP(5+) was shown to be readily reduced by cellular reductants such as ascorbic acid and glutathione. We now show that tetrahydrobiopterin (BH(4)) is also able to reduce the metal center. Under anaerobic conditions, in phosphate-buffered saline (pH 7.4) at 25 +/- 0.1 degrees C, reduction of Mn(III)TE-2-PyP(5+) occurs through two reaction steps with rate constants k(1) = 1.0 x 10(4) M(-1) s(-1) and k(2) = 1.5 x 10(3) M(-1) s(-1). We ascribe these steps to the formation of tetrahydrobiopterin radical (BH(4)(.+)) (k(1)) that then undergoes oxidation to 6,7-dihydro-8H-biopterin (k(2)), which upon rearrangement gives rise to 7,8-dihydrobiopterin (7,8-BH(2)). Under aerobic conditions, Mn(III)TE-2-PyP(5+) catalytically oxidizes BH(4). This is also true for its longer chain alkyl analog, Mn(III) ortho-tetrakis(N-n-octylpyridinium-2-yl)porphyrin. The reduced Mn(II) porphyrin cannot be oxidized by 7,8-BH(2) or by l-sepiapterin. The data are discussed with regard to the possible impact of the interaction of Mn(III)TE-2-PyP(5+) with BH(4) on endothelial cell proliferation and hence on tumor antiangiogenesis via inhibition of nitric oxide synthase.

Biopterins↗
PubMed2004

Tetrahydropteridines suppress gene expression and induce apoptosis of activated RAW264.7 cells via formation of hydrogen peroxide.

Tetrahydrobiopterin, a redox-active cofactor, is essential for nitric oxide (NO) biosynthesis. Previous work showed that intracellular tetrahydrobiopterin levels modulate activity of nitric oxide synthases (NOSs). The 4-amino analog of tetrahydrobiopterin is an effective inhibitor of all three purified NOS isoforms that, in intact cells, preferentially targets the inducible isoenzyme. In vivo, 4-amino-tetrahydrobiopterin prolonged allograft survival and rescued rats from septic shock. Here we investigated the effects of tetrahydrobiopterin and its 4-amino analog on RAW264.7 murine macrophages activated with lipopolysaccharide. Surprisingly, both tetrahydropteridines inhibited NO formation. This was caused by downregulation of inducible NOS expression rather than by affecting enzyme activity. In addition, expression of tumor necrosis factor-alpha was impaired, and apoptosis, as characterized by quantifying DNA content and caspase-3 activation and being associated with the formation of a 33 kDa fragment of nuclear factor-kappaB p65, was induced. The effects of tetrahydropteridines were scavenged by catalase or glutathione but not by superoxide dismutase. Like tetrahydropteridines, hydrogen peroxide at concentrations comparable to those found in tetrahydropteridine-treated cultures affected gene expression and cell survival, whereas increasing intracellular tetrahydrobiopterin levels by sepiapterin did not. Thus, extracellular tetrahydropteridines suppress gene expression and induce apoptosis in RAW264.7 cells via hydrogen peroxide formed in the culture medium during autoxidation.

Animals↗