Sepiapterin reductase in human amniotic and skin fibroblasts, chorionic villi, and various blood fractions

doi:10.1016/0009-8981(88)90053-8

Clinica Chimica Acta

Volume 174, Issue 3, 15 June 1988, Pages 271-282

https://doi.org/10.1016/0009-8981(88)90053-8 Get rights and content

Abstract

Sepiapterin reductase activity has been measured in amniotic fibroblasts by two procedures: one photometric and the other HPLC-fluorimetric. Both can be used for quantitative measurements, but the latter has considerable advantages including smaller standard deviation, much lower detection limit, and less volume of sample required. Sepiapterin reductase activity was also assayed in skin fibroblasts, chorionic villi and various blood fractions including stimulated mononuclear blood cells. Red blood cells have a low specific activity compared to unstimulated mononuclear blood cells, although the latter have a mean value with a high standard deviation. When the mononuclear blood cells were cultured for 5 days, the mean specific activity increased and the range became tighter. Enzyme stability and N-acetylserotonin inhibition were also studied.

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Cited by (24)

Sepiapterin reductase mediates chemical redox cycling in lung epithelial cells
2013, Journal of Biological Chemistry
Citation Excerpt :
To confirm that sepiapterin reductase possesses redox cycling activity, the human and mouse genes were cloned into a hexahistidine-tagged vector, expressed in E. coli, and purified by nickel affinity chromatography (Fig. 2, panel D, inset, and data not shown). The enzymes, which appeared as single bands on SDS-polyacrylamide gels (Mr ≈30,000), reduced sepiapterin to BH2, as measured by decreases in absorption of sepiapterin at 420 nm (25) and by the formation of BH2 by HPLC (26). Similar enzyme activity was detected in both assays (Figs. 2, panel D, and 3, panels A and B, and data not shown).
In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury.
Background: Enzymes mediating chemical redox cycling in the lung are poorly defined.
Results: Sepiapterin reductase was identified as a key mediator of redox cycling and was analyzed using inhibitors and site-directed mutagenesis.
Conclusion: Sepiapterin reductase generates reactive oxygen species during redox cycling in a mechanism distinct from sepiapterin reduction.
Significance: This is the first report demonstrating that sepiapterin reductase mediates chemical redox cycling.
Improvement of endothelial nitric oxide synthase activity retards the progression of diabetic nephropathy in db/db mice
2012, Kidney International
Impaired endothelial nitric oxide synthase (eNOS) activity may be involved in the pathogenesis of diabetic nephropathy. To test this, we used the type 2 diabetic db/db mouse (BKS background) model and found impaired eNOS dimerization and phosphorylation along with moderate glomerular mesangial expansion and increased glomerular basement membrane (GBM) thickness at 34 weeks of age. Cultured murine glomerular endothelial cells exposed to high glucose had similar alterations in eNOS dimerization and phosphorylation. Treatment with sepiapterin, a stable precursor of the eNOS cofactor tetrahydrobiopterin, or the nitric oxide precursor l-arginine corrected changes in eNOS dimerization and phosphorylation, corrected permeability defects, and reduced apoptosis. Sepiapterin or l-arginine, administered to db/db mice from weeks 26 to 34, did not significantly alter hyperfiltration or affect mesangial expansion, but reduced albuminuria and GBM thickness, and decreased urinary isoprostane and nitrotyrosine excretion (markers of oxidative stress). Although there was no change in glomerular eNOS monomer expression, both sepiapterin and l-arginine partially reversed the defect in eNOS dimerization and phosphorylation. Hence, our results support an important role for eNOS dysfunction in diabetes and suggest that sepiapterin supplementation might have therapeutic potential in diabetic nephropathy.
Nuclear localization of tetrahydrobiopterin biosynthetic enzymes
2004, Biochimica et Biophysica Acta - General Subjects
Biosynthesis of the tetrahydrobiopterin (BH₄) 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 β-galactosidase in the nucleus of neurons. In contrast, PTPS-β-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-β-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 BH₄-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 ∼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 BH₄-biosynthetic proteins with yet unknown biological function.
Localization of sepiapterin reductase in the human brain
2002, Brain Research
Citation Excerpt :
The enzyme activities were measured by using 10 μg of the brain homogenate, which was prepared as described above. SPR activity was assayed as described previously with a slight modification [11]. The incubation mixture (total volume of 50 μl) contained 100 mM potassium phosphate buffer (pH 6.4), 0.1 mM NADPH, and 0.15 mM sepiapterin as substrate.
Sepiapterin reductase (SPR) is the enzyme that catalyzes the final step of the synthesis of tetrahydrobiopterin (BH4), the cofactor for phenylalanine hydroxylase, tyrosine hydroxylase (TH), tryptophan hydroxylase, and nitric oxide synthase (NOS). Although SPR is essential for synthesizing BH4, the distribution of SPR in the human brain has not yet been clarified. In the present study, we purified recombinant human SPR from cDNA, raised an antibody against human SPR (hSPR), and examined the localization of SPR protein and SPR activity. Human brain homogenates from the substantia nigra (SN), caudate nucleus (CN), gray and white matters of the cerebral cortex (CTX), and dorsal and ventral parts of the medulla oblongata (MO) were subjected to Western blot analysis with anti-hSPR antibody or with anti-TH antibody. Whereas TH protein showed a restricted localization, being mainly detected in the SN and CN, SPR protein was detected in all brain regions examined. SPR activity was relatively high compared with the activity of GTP cyclohydrolase I (GCH), the rate-limiting biosynthetic enzyme of BH4, and was more widely distributed than GCH activity. Immunohistochemistry revealed SPR immunoreactivity in pyramidal neurons in the cerebral CTX, in a small number of striatal neurons, and in neurons of the hypothalamic and brain stem monoaminergic fields and olivary nucleus. Double-staining immunohistochemistry showed that TH and SPR were colocalized in the SN dopamine neurons. Localization of SPR immunoreactive neurons corresponded to monoamine or NOS neuronal fields, and also to the areas where no monoamine or NOS neurons were located. The results indicate that there might be a BH4 biosynthetic pathway where GCH is not involved and that SPR might have some yet unidentified function(s) in addition to BH4 biosynthesis.
Enhanced expression of GTP cyclohydrolase I in V-1-overexpressing PC12D cells
2002, Biochemical and Biophysical Research Communications
Citation Excerpt :
Measurement of SPR activity. SPR activity was assayed as described previously with a slight modification [13]. The incubation mixture (total volume of 50 μl) contained 100 mM potassium phosphate buffer (pH 6.4), 0.1 mM NADPH, and 0.15 mM sepiapterin as substrate.
Three of the catecholamine-synthesizing enzymes, i.e., tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase, and dopamine β-hydroxylase, were earlier shown to be up-regulated in cloned PC12D cells overexpressing V-1, a cdc10/SWI6 motif-containing protein. GTP cyclohydrolase I (GCH) is the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH₄), known as an essential cofactor for TH; and here we found the increased expression of GCH in V-1-overexpressing clones. Both GCH activity and total biopterin content were highly increased in the V-1 clones; whereas the activity of sepiapterin reductase, enzyme in the final step of the BH₄ biosynthesis, was not altered. Biochemical analyses revealed increased levels of GCH protein, mRNA, and transcription in the V-1 clones. Promoter analysis showed increased reporter activity in the construct with 150 bp of the promoter region of the human GCH gene, suggesting the involvement of cAMP-responsive element-mediated transcriptional regulation.
Mutations in the sepiapterin reductase gene cause a novel tetrahydrobiopterin-dependent monoamine-neurotransmitter deficiency without hyperphenylalaninemia
2001, American Journal of Human Genetics
Citation Excerpt :
For fibroblast cultures, the appropriate informed consent was obtained from all subjects. SR activity in red blood cells was assayed according to the method described by Ferre and Naylor (1988), with sepiapterin as a substrate and under assay conditions essentially the same as those for the fibroblasts (see the preceding paragraph). The assay monitors the conversion of sepiapterin to dihydrobiopterin, which is then measured as the oxidized product, biopterin.
Classic tetrahydrobiopterin (BH₄) deficiencies are characterized by hyperphenylalaninemia and deficiency of monoamine neurotransmitters. In this article, we report two patients with progressive psychomotor retardation, dystonia, severe dopamine and serotonin deficiencies (low levels of 5-hydroxyindoleacetic and homovanillic acids), and abnormal pterin pattern (high levels of biopterin and dihydrobiopterin) in cerebrospinal fluid. Furthermore, they presented with normal urinary pterins and without hyperphenylalaninemia. Investigation of skin fibroblasts revealed inactive sepiapterin reductase (SR), the enzyme catalyzing the final two-step reaction in the biosynthesis of BH₄. Mutations in the SPR gene were detected in both patients and their family members. One patient was homozygous for a TC→CT dinucleotide exchange, predicting a truncated SR (Q119X). The other patient was a compound heterozygote for a genomic 5-bp deletion (1397–1401delAGAAC) resulting in abolished SPR-gene expression and an A→G transition leading to an R150G amino acid substitution and to inactive SR as confirmed by recombinant expression. The absence of hyperphenylalaninemia and the presence of normal urinary pterin metabolites and of normal SR-like activity in red blood cells may be explained by alternative pathways for the final two-step reaction of BH₄ biosynthesis in peripheral and neuronal tissues. We propose that, for the biosynthesis of BH₄ in peripheral tissues, SR activity may be substituted by aldose reductase (AR), carbonyl reductase (CR), and dihydrofolate reductase, whereas, in the brain, only AR and CR are fully present. Thus, autosomal recessive SR deficiency leads to BH₄ and to neurotransmitter deficiencies without hyperphenylalaninemia and may not be detected by neonatal screening for phenylketonuria.

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Sepiapterin reductase in human amniotic and skin fibroblasts, chorionic villi, and various blood fractions

Abstract

Biochem Biophys Res Commun

Biochem Biophys Res Commun

Anal Biochem

Biochim Biophys Acta

Clin Chim Acta

J Chromatogr

Arch Biochem Biophys

Anal Biochem

Biochem Biophys Res Commun

Biochim Biophys Acta

Biochim Biophys Acta

Biochim Biophys Acta

Biosynthesis and metabolism of tetrahydrobiopterin and molyb-dopterin

Ann Rev Biochem