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PubMed1996

Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography.

An assay for human plasma xanthine oxidase activity was developed with pterin as the substrate and the separation of product (isoxanthopterin) by high-performance liquid chromatography with a fluorescence detector. The reaction mixture consists of 60 microliters of plasma and 240 microliters of 0.2 M Tris-HCl buffer (pH 9.0) containing 113 microM pterin. With this assay, the activity of plasma xanthine oxidase could be easily determined despite its low activity. As a result, it could be demonstrated that the intravenous administration of heparin or the oral administration of ethanol did not increase plasma xanthine oxidase activity in normal subjects, and also that plasma xanthine oxidase activity was higher in patients with hepatitis C virus infection than in healthy subjects or patients with gout. In addition, a single patient with von Gierke's disease showed a marked increase in the plasma activity of this enzyme, relative to that apparent in normal subjects.

Chromatography, High Pressure Liquid↗
PubMed1997

High performance liquid chromatographic determination of folic acid and its photodegradation products in the presence of riboflavin.

A high performance liquid chromatographic procedure was developed to determine folic acid and its photodegradation products, p-aminobenzoic acid, pterine-6-carboxylic acid, p-aminobenzoyl-L-glutamic acid, and pteroic acid in the presence of riboflavin. The method involves reversed phase, paired-ion chromatography on mu-BondaPak C18 column using a UV detector (254 nm), and isocratic solvent system (at ambient temperature) comprising 0.017 M monobasic potassium phosphate, tetrabutyl ammonium hydroxide solution (20%, aqueous) and methanol (870:15:250, v/v). The range of quantitation for the individual compounds was found to be: p-aminobenzoic acid, 0.01 - 1.25 x 10(-5) M; pterine-6-carboxylic acid, 0.01-2.0 x 10(-5) M; p-aminobenzoyl-L-glutamic acid, 0.02-2.0 x 10(-5) M; pteroic acid, 0.02-2.5 x 10(-5) M; folic acid, 1.0-5.0 x 10(-5) M; riboflavin, 1.0-5.0 x 10(-5) M. Linear regression analysis of the data demonstrates adequate performance of the method in terms of accuracy and precision (R. S. D. 3%). The method is specific, rapid and convenient and has been applied to photodegradation studies of folic acid in the presence and absence of riboflavin.

4-Aminobenzoic Acid↗
PubMed2000

A kinetic and conformational study on the interaction of tetrahydropteridines with tyrosine hydroxylase.

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).

Animals↗
PubMed2006

Reduction and oxidation of the active site iron in tyrosine hydroxylase: kinetics and specificity.

Tyrosine hydroxylase (TyrH) is a pterin-dependent enzyme that catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine. The oxidation state of the active site iron atom plays a central role in the regulation of the enzyme. The kinetics of reduction of ferric TyrH by several reductants were determined by anaerobic stopped-flow spectroscopy. Anaerobic rapid freeze-quench EPR confirmed that the change in the near-UV absorbance of TyrH upon adding reductant corresponded to iron reduction. Tetrahydrobiopterin reduces wild-type TyrH following a simple second-order mechanism with a rate constant of 2.8 +/- 0.1 mM(-)(1) s(-)(1). 6-Methyltetrahydropterin reduces the ferric enzyme with a second-order rate constant of 6.1 +/- 0.1 mM(-)(1) s(-)(1) and exhibits saturation kinetics. No EPR signal for a radical intermediate was detected. Ascorbate, glutathione, and 1,4-benzoquinone all reduce ferric TyrH, but much more slowly than tetrahydrobiopterin, suggesting that the pterin is a physiological reductant. E332A TyrH, which has an elevated K(m) for tetrahydropterin in the catalytic reaction, is reduced by tetrahydropterins with the same kinetic parameters as those of the wild-type enzyme, suggesting that BH(4) does not bind in the catalytic conformation during the reduction. Oxidation of ferrous TyrH by molecular oxygen can be described as a single-step second-order reaction, with a rate constant of 210 mM(-)(1) s(-)(1). S40E TyrH, which mimics the phosphorylated state of the enzyme, has oxidation and reduction kinetics similar to those of the wild-type enzyme, suggesting that phosphorylation does not directly regulate the interconversion of the ferric and ferrous forms.

Anaerobiosis↗
PubMed2004

Thermodynamic characterization of the binding of tetrahydropterins to phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) is the key enzyme in the catabolism of L-Phe. The natural cofactor of PAH, 6R-tetrahydrobiopterin (BH4), negatively regulates the enzyme activity in addition to being an essential cosubstrate for catalysis. The analogue 6-methyltetrahydropterin (6M-PH4) is effective in catalysis but does not regulate PAH. Here, the thermodynamics of binding of BH4 and 6M-PH4 to human PAH have been studied by isothermal titration calorimetry. At neutral pH and 25 degrees C, BH4 binds to PAH with higher affinity (Kd = 0.75 +/- 0.18 microM) than 6M-PH4 (Kd = 16.5 +/- 2.7 microM). While BH4 binding is a strongly exothermic process (DeltaH = -11.8 +/- 0.4 kcal/mol) accompanied by an entropic penalty (-TDeltaS = 3.4 +/- 0.4 kcal/mol), 6M-PH4 binding is both enthalpically (DeltaH = -3.3 +/- 0.3 kcal/mol) and entropically (-TDeltaS = -3.2 kcal/mol) driven. No significant changes in binding affinity were observed in the 5-35 degrees C temperature range for both pterins at neutral pH, but the enthalpic contribution increased with temperature rendering a heat capacity change (DeltaCp) of -357 +/- 26 cal/mol/K for BH4 and -63 +/- 12 cal/mol/K for 6M-PH4. Protons do not seem to be taken up or released upon pterin binding. Structure-based energetics calculations applied on the molecular dynamics simulated structures of the complexes suggest that in the case of BH4 binding, the conformational rearrangement of the N-terminal tail of PAH contribute with favorable enthalpic and unfavorable entropic contributions to the intrinsic thermodynamic parameters of binding. The entropic penalty is most probably associated to the reduction of conformational flexibility at the protein level and disappears for the L-Phe activated enzyme. The calculated energetic parameters aid to elucidate the molecular mechanism for cofactor recognition and the regulation of PAH by the dihydroxypropyl side chain of BH4.

Biopterins↗
PubMed2001

Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency in Dutch neonates.

Four neonates with a positive phenylalanine screening test (Phe concentrations between 258 and 1250 micromol/L) were investigated further to differentiate between phenylalanine hydroxylase (PAH) deficiency and variant hyperphenylalaninaemia (HPA) forms. In patients 1 and 2 a tetrahydrobiopterin (BH4) load caused a significant decrease of the plasma Phe levels. A combined phenylalanine/BH4 loading test was performed in patients 2, 3 and 4. In the latter two patients, plasma Phe concentrations completely normalized within 8 h after the BH4 load (20 mg/kg). Basal urinary pterins were normal in all four patients. The activity of dihydropteridine reductase (DHPR) was normal in patients 1, 2 and 3 and 50% of control values in patient 4 (not in the range of DHPR-deficient patients). In patient 3 a subsequent phenylalanine loading test with concomitant analysis of plasma biopterins revealed a normal increase of biopterin, excluding a BH4 biosynthesis defect. Pterins and neurotransmitter metabolites in CSF of patients 1, 3 and 4 were normal. DNA mutations detected in the PAH gene of patients 1-4 were A313T, and L367fsinsC; V190A and R243X; A300S and A403V; R241C and A403V. The results are suggestive for mutant PAH enzymes with decreased affinity for the cofactor BH4.

Biopterins↗
PubMed1981

Folate catabolism in tumour-bearing rats and rats treated with methotrexate.

The metabolism of (2-14C) + (3',5',7,9-3H) folic acid was studied in normal rats, tumour-bearing rats and rats treated with methotrexate (MTX). The experiments were designed to investigate changes in the catabolism and folate. The breakdown of folate to scission products was again demonstrated to be a normal phenomenon. Catabolites excreted included p-acetamidobenzoate, p-acetamidobenzoyl-L-glutamate, 3H2O, urea and a number of pterins. The catabolic process was decreased in the presence of a tumour and increased by the administration of MTX. MTX also led to the excretion of 4 additional radioactive pterins not found in normal urine. The possible mechanisms of folate breakdown are discussed with reference to the point of action of MTX.

4-Aminobenzoic Acid↗
PubMed1994

Tetrahydrobiopterin is a limiting factor of nitric oxide generation in interleukin 1 beta-stimulated rat glomerular mesangial cells.

Treatment of mesangial cells with recombinant human interleukin 1 beta (IL-1 beta) triggers the expression of a macrophage-type of nitric oxide (NO) synthase and the subsequent increase of cellular concentration of cGMP and nitrite production. Tetrahydrobiopterin (BH4) is an essential cofactor for NO synthase, and in the present study we investigated its impact on inducible NO synthesis in mesangial cells. Inhibition of GTP-cyclohydrolase I, the rate-limiting enzyme for BH4 synthesis, with 2,4-diamino-6-hydroxy-pyrimidine (DAHP) potently suppresses IL-1 beta-induced nitrite production and elevation of cellular cGMP levels. This inhibitory effect of DAHP is reversed by sepiapterin, which provides BH4 via the pterin salvage pathway. Most importantly, sepiapterin dose-dependently augments IL-1 beta-stimulated NO synthesis, indicating that the availability of BH4 limits the production of NO in cytokine-induced mesangial cells. N-acetylserotonin, an inhibitor of the BH4 synthetic enzyme sepiapterin reductase, completely abolishes IL-1 beta-stimulated nitrite production, whereas methotrexate, which inhibits the pterin salvage pathway, displays only a moderate inhibitory effect, thus suggesting that mesangial cells predominantly synthesize BH4 by de novo synthesis from GTP. In conclusion, these data demonstrate that BH4 synthesis is an absolute requirement for, and limits IL-1 beta induction of NO synthesis in mesangial cells. Inhibition of BH4 synthesis may provide new therapeutic approaches to the treatment of pathological conditions involving increased NO formation.

Amino Acid Oxidoreductases↗
PubMed2004

Synthesis of N3- and 2-NH2-substituted 6,7-diphenylpterins and their use as intermediates for the preparation of oligonucleotide conjugates designed to target photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene.

Two 17-mer oligodeoxynucleotide-5'-linked-(6,7-diphenylpterin) conjugates, 2 and 3, were prepared as photosensitisers for targeting photooxidative damage to a 34-mer DNA oligodeoxynucleotide (ODN) fragment 1 representing the chimeric bcr-abl gene that is implicated in the pathogenesis of chronic myeloid leukaemia (CML). The base sequence in the 17-mer was 3'G G T A G T T A T T C C T T C T T5'. In the first of these ODN conjugates (2) the pterin was attached at its N3 atom, via a -(CH2)3OPO(OH)- linker, to the 5'-OH group of the ODN. Conjugate 2 was prepared from 2-amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone 10, using phosphoramidite methodology. Starting material 10 was prepared from 5-amino-7-methylthiofurazano[3,4-d]pyrimidine 4 via an unusual highly resonance stabilised cation 8, incorporating the rare 2H,6H-pyrimido[6,1-b][1,3]oxazine ring system. In the characterisation of 10 two pteridine phosphazenes, 15 and 29, were obtained, as well as new products containing two uncommon tricyclic ring systems, namely pyrimido[2,1-b]pteridine (20 and 24) and pyrimido[1,2-c]pteridine (27). In the second ODN conjugate the linker was -(CH2)5CONH(CH2)6OPO(OH)- and was attached to the 2-amino group of the pterin. In the preparation of 3, the N-hydroxysuccinimide ester 37 of 2-(5-carboxypentylamino)-6,7-diphenyl-4(3H)-pteridinone was condensed with the hexylamino-modified 17-mer. Excitation of 36 with near UV light in the presence of the single-stranded target 34-mer, 5'T G A C C A T C A A T A A G14 G A A G18 A A G21 C C C T T C A G C G G C C3' 1 caused oxidative damage at guanine bases, leading to alkali-labile sites which were monitored by polyacrylamide gel electrophoresis. Cleavage was observed at all guanine sites with a marked preference for cleavage at G14. In contrast, excitation of ODN-pteridine conjugate 2 in the presence of 1 caused oxidation of the latter predominantly at G18, with a smaller extent of cleavage at G15 and G14 (in the double-stranded portion) and G21. These results contrast with our previous observation of specific cleavage at G21 with ruthenium polypyridyl sensitisers, and suggest that a different mechanism, probably one involving Type 1 photochemical electron transfer, is operative. Much lower yields were found with the ODN-pteridine conjugate 3, perhaps as a consequence of the longer linker between the ODN and the pteridine in this case.

Base Pairing↗
PubMed1983

Km and kcat. values for [6,6,7,7-2H]7,8(6H)-dihydropterin and 2,6-diamino-5-iminopyrimidin-4-one with dihydropteridine reductase.

The Km and kcat. values for [6,6,7,7-2H]7,8(6H)-dihydropterin and 2,6-diamino-5-iminopyrimidin-4-one were determined for dihydropteridine reductase (EC 1.6.99.10) from two sources. The parameters of the pterin are of the same order as those of the most effective substrates of dihydropteridine reductase. The Km values of the pterin are one order of magnitude smaller than those of the pyrimidinone, although the kcat. values are of the same order.

Animals↗
PubMed1997

The role of mutants in the search for the photoreceptor for phototropism in higher plants.

Early attempts to identify the chromophore of the photoreceptor for phototropism are reviewed. Carotenoids and flavins were the principal candidates, but studies with grass coleoptiles devoid of carotenoids suggest that at least in these organs carotenoids are most unlikely to play that role. The status of characterization of a gene for a putative photoreceptor protein is also reviewed. As the action spectrum for phototropism resembles the absorption spectrum of a flavoprotein, flavoproteins are attractive candidates at present, especially since the CRY1 photoreceptor in Arabidopsis thaliana that mediates blue light-dependent hypocotyl growth suppression has flavin adenine dinucleotide as one of its two chromophores. As the second chromophore appears to be pterin, pterins should not be ruled out as candidate chromophores for the photoreceptor for phototropism.

Arabidopsis↗
PubMed1999

Tyrosine hydroxylase binds tetrahydrobiopterin cofactor with negative cooperativity, as shown by kinetic analyses and surface plasmon resonance detection.

Kinetic studies of tetrameric recombinant human tyrosine hydroxylase isoform 1 (hTH1) have revealed properties so far not reported for this enzyme. Firstly, with the natural cofactor (6R)-Lerythro-5,6,7, 8-tetrahydrobiopterin (H4biopterin) a time-dependent change (burst) in enzyme activity was observed, with a half-time of about 20 s for the kinetic transient. Secondly, nonhyperbolic saturation behaviour was found for H4biopterin with a pronounced negative cooperativity (0.39 < h < 0.58; [S]0.5 = 24 +/- 4 microM). On phosphorylation of Ser40 by protein kinase A, the affinity for H4biopterin increased ([S]0.5 = 11 +/- 2 microM) and the negative cooperativity was amplified (h = 0.27 +/- 0.03). The dimeric C-terminal deletion mutant (Delta473-528) of hTH1 also showed negative cooperativity of H4biopterin binding (h = 0.4). Cooperativity was not observed with the cofactor analogues 6-methyl-5,6,7,8-tetrahydropterin (h = 0.9 +/- 0.1; Km = 62.7 +/- 5.7 microM) and 3-methyl-5,6,7, 8-tetrahydropterin (H43-methyl-pterin)(h = 1.0 +/- 0.1; Km = 687 +/- 50 microM). In the presence of 1 mM H43-methyl-pterin, used as a competitive cofactor analogue to BH4, hyperbolic saturation curves were also found for H4biopterin (h = 1.0), thus confirming the genuine nature of the kinetic negative cooperativity. This cooperativity was confirmed by real-time biospecific interaction analysis by surface plasmon resonance detection. The equilibrium binding of H4biopterin to the immobilized iron-free apoenzyme results in a saturable positive resonance unit (DeltaRU) response with negative cooperativity (h = 0.52-0.56). Infrared spectroscopic studies revealed a reduced thermal stability both of the apo-and the holo-hTH1 on binding of H4biopterin and Lerythro-dihydrobiopterin (H2biopterin). Moreover, the ligand-bound forms of the enzyme also showed a decreased resistance to limited tryptic proteolysis. These findings indicate that the binding of H4biopterin at the active site induces a destabilizing conformational change in the enzyme which could be related to the observed negative cooperativity. Thus, our studies provide new insight into the regulation of TH by the concentration of H4biopterin which may have significant implications for the physiological regulation of catecholamine biosynthesis in neuroendocrine cells.

Animals↗
PubMed1980

Tyrosine hydroxylase: a substrate of cyclic AMP-dependent protein kinase.

Data demonstrating the direct phosphorylation of tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] purified from rat pheochromocytoma by ATP, Mg2+ and cyclic AMP-dependent protein kinase catalytic subunit are presented. The incorporation of phosphate is highly correlated with the activation of the hydroxylase when either the time of preincubation or the amount of protein kinase subunit is varied. The rate of phosphorylation of tyrosine hydroylase compares favorably with that of H1 histone, a known substrate of protein kinase. Lineweaver-Burk analysis of crude or purified rat pheochromocytoma tyrosine hydroxylase activity, as a function of pterin cofactor concentration, in the absence of ATP, Mg2+, and protein kinase catalytic subunit, yields a curvilinear relationship which can be resolved into two lines, suggesting two enzyme forms with different affinities for pterin cofactor. A fraction of the hydroxylase present in the tumor exists in the activated state, presumably due to the presence of ATP and endogenous protein kinase activity. When the solubl enzyme is activated by cyclic AMP, ATP, Mg2+, and protein kinase, virtually all of the enzyme is converted to the low Km state. We conclude that tyrosine hydroxylase is a substrate of cyclic AMP-dependent protein kinase in vitro and, presumably, in vivo.

Adenosine Triphosphate↗
PubMed1988

Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains.

The sequence of nitrate reductase (EC 1.6.6.1) mRNA from the plant Arabidopsis thaliana has been determined. A 3.0-kilobase-long cDNA was isolated from a lambda gt10 cDNA library of Arabidopsis leaf poly(A)+ RNA. The cDNA hybridized to a 3.2-kilobase mRNA whose level increased 15-fold in response to treatment of the plant with nitrate. An open reading frame encoding a 917 amino acid protein was found in the sequence. This protein is very similar to tobacco nitrate reductase, being greater than 80% identical within a section of 450 amino acids. By comparing the Arabidopsis protein sequence with other protein sequences, three functional domains were deduced: (i) a molybdenum-pterin-binding domain that is similar to the molybdenum-pterin-binding domain of rat liver sulfite oxidase, (ii) a heme-binding domain that is similar to proteins in the cytochrome b5 superfamily, and (iii) an FAD-binding domain that is similar to NADH-cytochrome b5 reductase.

Amino Acid Sequence↗
PubMed1996

Role of tetrahydrobiopterin availability in the regulation of nitric-oxide synthase expression in human mesangial cells.

Human mesangial cells express an inducible form of nitric-oxide synthase (iNOS) after treatment with cytokines. Tetrahydrobiopterin (BH4), an essential cofactor for NOS, is required for cytokine-induced NO generation. We report here that BH4 is necessary not only for the activity but also for the expression of iNOS in human mesangial cells. Inhibition of de novo BH4 synthesis with 2,4-diamino-6-hydroxypyrimidine (DAHP) significantly attenuated iNOS activity as well as mRNA and protein expression in response to interleukin 1beta plus tumor necrosis factor alpha (IL-1beta/TNF-alpha). In contrast, sepiapterin, which provides BH4 through the pterin salvage pathway, strongly potentiated IL-1beta/TNF-alpha-induced iNOS expression and abrogated the inhibitory effect of DAHP. Inhibition of the pterin salvage pathway with methotrexate abolished sepiapterin potentiation of iNOS induction but did not alter the effect of IL-1beta/TNF-alpha. Determination of intracellular pteridines confirmed that sepiapterin markedly raised BH4 content, an effect that was blocked by methotrexate. These results suggest that BH4 availability plays an important role in the regulation of iNOS expression. The effect of BH4 appears to be mediated, at least in part, by an increase in mRNA stability, as indicated by the observation that DAHP shortened, whereas sepiapterin prolonged the half-life of IL-1beta/TNF-alpha-induced iNOS mRNA. Taken together, our results suggest that the biosynthesis of BH4 contributes to cytokine induction of iNOS expression in human mesangial cells through the stabilization of iNOS mRNA.

Arginine↗
PubMed2000

Purification and properties of a folate-catabolizing enzyme.

We have identified and purified to homogeneity an enzyme from rat liver that catalyzes the oxidative catabolism of 5-formyltetrahydrofolate to p-aminobenzoylglutamate and a pterin derivative. Purification of the enzyme utilized six column matrices, including a pterin-6-carboxylic acid affinity column. Treatment of crude rat liver extracts with EDTA or heat decreased the specific activity of the enzyme by up to 85%. Peptides generated from the purified protein were sequenced and found to be identical to primary sequences present within rat light chain or heavy chain ferritin. Commercial rat ferritin did not display catabolic activity, but activity could be acquired with iron loading. The purified enzyme contained 2000 atoms of iron/ferritin 24-mer and displayed similar electrophoretic properties as commercial rat liver ferritin. The ferritin-catalyzed reaction displayed burst kinetics, and the enzyme catalyzed only a single turnover in vitro. Expression of rat heavy chain ferritin cDNA resulted in increased rates of folate turnover in cultured Chinese hamster ovary cells and human mammary carcinoma cells and reduced intracellular folate concentrations in Chinese hamster ovary cells. These results indicate that ferritin catalyzes folate turnover in vitro and in vivo and may be an important factor in regulating intracellular folate concentrations.

Amino Acid Sequence↗
PubMed1990

Cloning and molecular characterization of a metabolic gene with development functions in Drosophila. I. Analysis of the head function of Punch.

In an effort to understand the functions of pterins throughout development we have been studying Punch (Pu), the structural gene for the enzyme GTP cyclohydrolase in Drosophila melanogaster. This enzyme catalyzes the first step in the pterin biosynthetic pathway. The Pu gene product is required for vital functions at two distinct stages in embryogenesis, and a pigmentation function in the eye of the young adult. We have localized the Pu region to 29 kb of DNA through the analysis of lesions present in Pu mutants. Since all of the mutations that were mapped affect the eye pigmentation function of Pu, and since this function is the best defined biochemically, we have concentrated on identifying and characterizing Pu products required for eye pigmentation in our initial examination of the cloned region. Four different transcripts from this region are expressed in the adult head. We show that one of these transcripts, the 1.7-kb species, is responsible for the pigmentation function through the analysis of mutant transcripts and the use of an in vitro translation assay. A 2-kb region lying within the locus is specifically required for this eye pigmentation function.

Animals↗
PubMed1978

Stoichiometric studies on the oxidation of tetrahydropterin with ferri-cytochrome c.

The oxidation of tetrahydropterin with ferri-cytochrome c was studied using a tetrahydropterin-generating system composed of dihydropteridine reductase [EC 1.6.99.7] and NADH. Under aerobic conditions, 1.5 to 1.8 mol of cytochrome c was reduced per mol of NADH, whereas 2 mol of cytochrome c was reduced under anaerobic conditions. When superoxide dismutase [EC 1.15.1.1] was added to the system under aerobic conditions, only 1 mol of cytochrome c was reduced per mol of NADH, while the pterin oxidation was scarcely affected. Based on these results, we propose that the oxidation of tetrahydropterin to quinonoid dihydropterin proceeds via two steps: tetrahydropterin is first oxidized by ferri-cytochrome c to give a pterin intermediate, which has lost one electron, then in turn this reduces O2 to form O2-.

Cytochrome c Group↗