Sepiapterin Reductase Research Articles

Global analysis of proteins synthesized by Mycobacterium smegmatis provides direct evidence for physiological heterogeneity in stationary-phase cultures.

We have characterized the induction kinetics of approximately 1,700 proteins during entry into and survival in carbon-starved stationary phase by Mycobacterium smegmatis. Strikingly, among the patterns of expression observed were a group of proteins that were expressed in exponential-phase cultures and severely repressed in 48-h stationary-phase cultures (Spr or stationary-phase-repressed proteins) but were synthesized again at high levels in > or =128-day stationary-phase cultures (Spr(128) proteins). A number of Spr(128) proteins were identified, and they included the heat shock protein DnaK, the tricarboxylic acid cycle enzyme succinyl coenzyme A synthase, a FixA-like flavoprotein, a single-stranded DNA binding protein, and elongation factor Tu (EF-Tu). The identification of EF-Tu as an Spr(128) protein is significant, as ribosomal components are known to be expressed in a growth rate-dependent way. We interpreted these data in terms of a model whereby stationary-phase mycobacteria comprise populations of cells that differ in both their growth status and gene expression patterns. To investigate this further, we constructed gene fusions between the rpsL gene promoter (which heads the Mycobacterium smegmatis operon encoding the tuf gene encoding EF-Tu) or the rrnA promoter gene and an unstable variant of green fluorescent protein. While the majority of cells in old stationary-phase cultures had low levels of fluorescence and so rpsL expression, a small but consistently observed population of approximately 1 in 1,000 cells was highly fluorescent. This indicates that a small fraction of the cells was expressing rpsL at high levels, and we argue that this represents the growing subpopulation of cells in stationary-phase cultures.

Antioxidant Effects of N‐Acetylserotonin

Abstract: This paper will review our recent data relevant to the antioxidant effects of N‐acetylserotonin (NAS), the immediate precursor of melatonin, the pineal gland indole. Mechanisms of the antioxidant effects of NAS might involve interaction with melatonin type 3 receptors and nonreceptor mechanisms such as stimulation of glutathione peroxidase, an antioxidant enzyme; inhibition of lipid peroxidation; suppression of phospholipase A2 activation; attenuation of tumor necrosis factor‐α production; prevention of pathological opening of the mitochondrial permeability transition pores; and inhibition of sepiapterin reductase, the key enzyme of biosynthesis of tetrahydrobiopterin, the essential cofactor of nitric oxide synthase. NAS actions on some of these enzymes might be receptor‐mediated. Protective effects of NAS against oxidative damage are independent from the effect of melatonin and, depending on the model, are 5 to 20 times stronger than that of melatonin. Antioxidant effect of NAS might underpin its cognition‐enhancing, antiaging, antidepressant, antihypertensive, and antitumor effects. NAS and its derivatives might be useful in protection against oxidative stress‐related disorders (cell death, mutagenesis, aging) and diseases (sepsis, cancer, postischemic trauma, Alzheimer's disease, parkinsonism).

N‐Acetyldopamine Inhibits Rat Brain Lipid Peroxidation Induced by Lipopolysaccharide

The effects of N-acetyldopamine, a sepiapterin reductase inhibitor, on lipopolysaccharide-induced lipid peroxidation were examined in rat brain homogenates in vitro. Lipid peroxidation in the form of malondialdehyde (MDA) was evaluated by the measurement of thiobarbituric acid (TBA) reactive substances. N-Acetyldopamine inhibited the formation of MDA in a concentration-dependent manner. The effect was similar to that of N-acetylserotonin, but stronger than that of the endogenous antioxidant agent, melatonin. Possible clinical applications of N-acetyldopamine and its derivatives are discussed.

Antioxidant Effects of N-Acetylserotonin: Possible Mechanisms and Clinical Implications

This paper will review our recent data relevant to the antioxidant effects of N-acetylserotonin (NAS), the immediate precursor of melatonin, the pineal gland indole. Mechanisms of the antioxidant effects of NAS might involve interaction with melatonin type 3 receptors and nonreceptor mechanisms such as stimulation of glutathione peroxidase, an antioxidant enzyme; inhibition of lipid peroxidation; suppression of phospholipase A2 activation; attenuation of tumor necrosis factor-alpha production; prevention of pathological opening of the mitochondrial permeability transition pores; and inhibition of sepiapterin reductase, the key enzyme of biosynthesis of tetrahydrobiopterin, the essential cofactor of nitric oxide synthase. NAS actions on some of these enzymes might be receptor-mediated. Protective effects of NAS against oxidative damage are independent from the effect of melatonin and, depending on the model, are 5 to 20 times stronger than that of melatonin. Antioxidant effect of NAS might underpin its cognition-enhancing, antiaging, antidepressant, antihypertensive, and antitumor effects. NAS and its derivatives might be useful in protection against oxidative stress-related disorders (cell death, mutagenesis, aging) and diseases (sepsis, cancer, postischemic trauma, Alzheimer's disease, parkinsonism).

Sepiapterin reductase deficiency: a congenital dopa-responsive motor and cognitive disorder

This study presents the clinical findings on seven children from Malta (population 385,000). All of them had early motor delay and a significant degree of cognitive impairment. Diurnal variation of the motor impairments was clear in six out of seven of the subjects and oculogyric crises occurred from an early stage also in six out of the seven. Five out of seven had clear evidence of dystonia but the early picture was dominated by hypotonia in five. Two had early Parkinsonian tremor and chorea was seen in four, although in two this was attributable to the use of L-dopa. Three had early bulbar involvement. In all, although minor motor problems persisted, the response to L-dopa was dramatic and there was a need to balance improvement in dystonia against aggravation of chorea. The majority were not able to walk until they were treated. Increased doses of L-dopa were required in hot weather, to which they were sensitive. Despite a good response of improved motor ability and abolition of oculogyric crises, there was no obvious change in cognitive function with learning remaining in the moderate impairment range. This report widens the phenotype of dopa-responsive motor disorders and the range of young children with primary motor delay (cerebral palsy) who need a clinical trial of L-dopa. All of the subjects had the same novel mutation in the tetrahydrobiopterin pathway involving sepiapterin reductase, and no abnormality in the gene encoding guanosine triphosphate cyclohydrolase 1. Clinically and molecularly the condition shows autosomal recessive inheritance.

D-threo-tetrahydrobiopterin is synthesized via 1′-oxo-2′- d-hydroxypropyl-tetrahydropterin in Dictyostelium discoideum Ax2

The biosynthesis of d-threo-tetrahydrobiopterin (DH4, tetrahydrodictyopterin) in Dictyostelium discoideum Ax2 was investigated through the mutant disrupted in the gene encoding sepiapterin reductase (SR) by insertional inactivation. The mutant cells, being completely devoid of SR protein, showed 18.1% of l-erythro-tetrahydrobiopterin (BH4) and 0.6% of DH4 productions in the wild type cells. The mutant cells were also identified to excrete d- and l-sepiapterin, which were presumed to originate from intracellular 1′-oxo-2′- d-hydroxypropyl- and 1′-oxo-2′- l-hydroxypropyl-tetrahydropterin (H4-pterin), respectively. Furthermore, in a coupled assay with Dictyostelium SR, the mutant cell extract exhibited a novel enzyme activity converting 6-pyruvoyltetrahydropterin to 1′-oxo-2′- d-hydroxypropyl-H4-pterin. These results are clear demonstration of the in vivo synthesis of DH4 via 1′-oxo-2′- d-hydroxypropyl-H4-pterin as well as an alternative synthesis of BH4 and DH4 in the complete absence of SR.

Characterization of a critical region in the hormone binding domain of sperm progesterone receptor

Our previous studies have demonstrated that the ligand binding characteristics of the non-genomic sperm progesterone receptor (sPR) are different from those of the conventional progesterone receptor (PR). Unlike PR, sPR does not bind efficiently to progesterone antagonists i.e. mifepristone (RU38486), onapristone (ZK98.299) etc. The present study was undertaken to determine the molecular composition of a region that plays a critical role in the interaction of the hormone-binding domain (HBD) of sPR with progesterone and its antagonists. Detection, cloning and sequencing of the HBD region of sPR revealed its complete sequence homology to the corresponding region in the conventional PR. No nucleotide substitution/mutation/deletion, which could account for the differential antihormone binding, was observed in the HBD of sPR. The critical codon (nucleotide 3216-3218) in all three clones for the HBD of sPR was found to encode for glycine. This ruled out the possibility of steric hindrance because of the placement of amino acids with side chains at a critical position in the HBD, which may interfere in binding of sPR with antiprogestins. It is likely that the post-transcriptional modifications contribute to the differential binding characteristics of sPR. This warrants future investigations to focus more on the characterization of the mature sPR protein.

Expression, purification, crystallization and preliminary X-ray analysis of sepiapterin reductase from Chlorobium tepidum.

Sepiapterin reductase from Chlorobium tepidum (CT-SR) produces L-threo-tetrahydrobiopterin, an isomer of tetrahydrobiopterin, in the last step of de novo synthesis initiating from GTP. Native CT-SR and a selenomethionine (SeMet) derivative of CT-SR have been crystallized by the hanging-drop vapour-diffusion method using PEG 400 as precipitant. CT-SR crystals belong to space group R32, with unit-cell parameters a = b = 201.142, c = 210.184 A, and contain four molecules in the asymmetric unit. Diffraction data were collected to 2.1 A resolution using synchrotron radiation. The structure of CT-SR has been determined using MAD phasing. There is one CT-SR tetramer in the asymmetric unit formed by two closely interacting CT-SR dimers. The solvent content is calculated to be about 67.2%.

Sepiapterin reductases from Chlorobium tepidum and Chlorobium limicola catalyze the synthesis of l-threo-tetrahydrobiopterin from 6-pyruvoyltetrahydropterin

The ORF sequences of the gene encoding sepiapterin reductase were cloned from the genomic DNAs of Chlorobium tepidum and Chlorobium limicola, which are known to produce l-threo- and l-erythro-tetrahydrobiopterin (BH4)- N-acetylglucosamine, respectively. The deduced amino acid sequence of C. limicola consists of 241 residues, while C. tepidum SR has three residues more at the C-terminal. The overall protein sequence identity was 87.7%. Both recombinant proteins generated from Escherichia coli were identified to catalyze reduction of diketo compound 6-pyruvoyltetrahydropterin to l-threo-BH4. This result suggests that C. limicola needs an additional enzyme for l-erythro-BH4 synthesis to yield its glycoside. The catalytic activity of Chlorobium SRs also supports the previously proposed mechanism of two consecutive reductions of C1′ carbonyl group of 6-pyruvoyltetrahydropterin via isomerization reaction.

Heterozygous mutation in 5'-untranslated region of sepiapterin reductase gene (SPR) in a patient with dopa-responsive dystonia.

The search for mutations in genes coding for components of the biopterin pathway other than GTPCH1 revealed a mutation in the gene coding for sepiapterin reductase (SPR) in 1 of 95 patients with GCH1-negative dopa-responsive dystonia (DRD). The mutation detected in SPR is a G-->A transition at position -13 of the untranslated region of the gene. This resulted in drastically reduced activity of sepiapterin reductase in the patient's fibroblasts. The findings indicate that haploinsufficiency of SPR can be a rare cause of DRD.

Functional tetrahydrobiopterin synthesis in human platelets.

Previous studies have provided evidence for the importance of platelet-derived nitric oxide (NO) for the regulation of hemostasis. Tetrahydrobiopterin (BH4) is an essential cofactor and regulator of NO synthase activity in the vasculature; however, it is as yet unknown whether platelets dispose over a functional BH4 synthesis. We quantified mRNA expression of genes involved in BH4 synthesis, measured enzymatic activities, and determined intraplatelet levels of pteridines in platelets from healthy volunteers and from patients treated for prolonged periods of time with glucocorticoids. Freshly isolated platelets from healthy volunteers show functional BH4 synthesis, as evidenced by the presence of mRNA species and enzymatic activity of GTP cyclohydrolase I (GTPCH), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase. Biopterin was the major intraplatelet pteridine, whereas no neopterin was found. mRNA expression and enzymatic activity of GTPCH were undetectably low in platelets that had been stored for 5 days, and no pteridines were found in these platelets. Freshly isolated platelets from patients treated with glucocorticoids had decreased mRNA expression and activity of GTPCH compared with platelets from healthy volunteers. Human platelets dispose over a functional de novo BH4 synthesis. Furthermore, our results indicate the potential of external factors, eg, prolonged storage or glucocorticoid therapy, to significantly affect BH4 synthesis within platelets. Together, these findings offer new insights into the biology and pathobiology of platelet function in humans.

Activity of synthetic enzymes of tetrahydrobiopterin in the human placenta

Tetrahydrobiopterin (BH4) is an essential co-factor for nitric oxide (NO) synthase (NOS) and regulates the production of NO, or endothelium-derived relaxation factor. Although NOS is highly expressed in the placenta and NO plays a critical role in the regulation of feto-placental circulation, the mechanism maintaining the level of BH4 is not known. To investigate the de novo synthesis of BH4 in the human placenta, the activity of guanosine triphosphate cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR) in the chorionic tissue during the first, second and third trimester of pregnancy was analyzed. GTPCH activity was the lowest of the three enzymes and became negligible after the second trimester. There was no significant change in PTPS activity throughout pregnancy. Although SR activity decreased significantly after the second trimester, the levels remained abundant throughout pregnancy. These results showed that GTPCH is a rate-limiting enzyme and the total activity of the de novo synthesis of BH4 is negligible in the mature placenta after the second trimester when fetal growth is accelerated. The present study suggests that the level of BH4 in the placenta depends principally on the system other than de novo synthesis. The salvage pathway is considered the most potent system, which is formed by the transfer of the substrates from the fetus and their enzymatic conversion to BH4 in the placenta.

A haplotype at the PARK3 locus influences onset age for Parkinson's disease: the GenePD study.

To identify a haplotype influencing onset age for Parkinson's disease (PD) in the PARK3 region on chromosome 2p13. Single nucleotide polymorphisms (SNP) spanning 2.2 Mb and located in or near potential candidate genes were used to fine map the PARK3 region in 527 patients with familial PD, from 264 families. TT homozygotes for rs1876487 (G/T) had a 7.4-year younger mean age at onset (p = 0.005) compared to patients with GT and GG genotypes. Furthermore, SNP flanking the sepiapterin reductase (7,8-dihydrobiopterin: NADP+ oxidoreductase) (SPR) gene, rs1876487 (p = 0.02) and rs1150500 (p = 0.04), were associated with younger onset age among persons who did not carry the 174 allele of D2S1394. The SPR gene is implicated in dopamine synthesis. Haplotype analysis of three SNP-rs2421095, rs1876487, rs1561244-revealed an association with onset age (p = 0.023) and a haplotype of A-T-G alleles was associated with younger onset for PD (p = 0.005). A haplotype at the PARK3 locus, harboring the SPR gene, is associated with onset age of PD. This may suggest a role for the SPR gene in modifying the age at onset of PD.

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.

Localization of sepiapterin reductase in pigment cells of Oryzias latipes.

Body colors of poikilothermal vertebrates are derived from three distinct types of pigment cells, melanophores, erythro/xanthophores and irido/leucophores. It is well known that melanin in melanophores is synthesized by tyrosinase within a specific organelle termed the melanosome. Although sepiapterin reductase (SPR) is an important enzyme involved in metabolizing biopterin and sepiapterin (a conspicuous pteridine as a coloring pigment in xanthophores) the distribution of SPR has not been shown in pigment cells. An antibody raised in rabbits against rat SPR was used to demonstrate the presence of SPR in pigment cells of Oryzias latipes. This study, which used immunohistochemistry with fluorescence or peroxidase/diaminobenzidine as markers, revealed that SPR could be detected readily in xanthophores, but only faintly in melanophores. These results suggest that sepiapterin is metabolized within xanthophores. Moreover, these experiments show that a protein sharing immunological cross-reactivity with rat SPR is located in teleost O. latipes xanthophores, which is significant considering the relationship of pteridine metabolism between poikilothermal vertebrates and mammals. Further progress in investigations of the roles of pteridines in vertebrates will be promoted by using these fish which can be bred in mass rather easily in the laboratory.

Genetic engineering of Escherichia coli for production of tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is an essential cofactor for various enzymes in mammals. In vivo, it is synthesized from GTP via the three-step pathway of GTP cyclohydrolase I (GCHI), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SPR). BH4 is a medicine used to treat atypical hyperphenylalaninemia. It is currently synthesized by chemical means, which consists of many steps, and requires costly materials and complicated procedures. To explore an alternative microbial method for BH4 production, we utilized recombinant DNA technology to construct recombinant Escherichia coli ( E. coli) strains carrying genes expressing GCHI, PTPS and SPR enzymes. These strains successfully produced BH4, which was detected as dihydrobiopterin and biopterin, oxidation products of BH4. In order to increase BH4 productivity we made further improvements. First, to increase the de novo GTP supply, an 8-azaguanine resistant mutant was isolated and an additional guaBA operon was introduced. Second, to augment the activity of GCHI, the folE gene from E. coli was replaced by the mtrA gene from Bacillus subtilis. These modifications provided us with a strain showing significantly higher productivity, up to 4.0 g of biopterin/L of culture broth. The results suggest the possibility of commercial BH4 production by our method.

Tetrahydrobiopterin is synthesized from 6-pyruvoyl-tetrahydropterin by the human aldo-keto reductase AKR1 family members

Tetrahydrobiopterin (BH 4) is a cofactor for aromatic amino acid hydroxylases and nitric oxide synthase. The biosynthesis includes two reduction steps catalyzed by sepiapterin reductase. An intermediate, 6-pyruvoyltetrahydropterin (PPH 4) is reduced to 1 ′-oxo-2 ′-hydroxypropyl-tetrahydropterin (1 ′-OXPH 4) or 1 ′-hydroxy-2 ′-oxopropyl-tetrahydropterin (2 ′-OXPH 4), which is further converted to BH 4. However, patients with sepiapterin reductase deficiency show normal urinary excretion of pterins without hyperphenylalaninemia, suggesting that other enzymes catalyze the two reduction steps. In this study, the reductase activities for the tetrahydropterin intermediates were examined using several human recombinant enzymes belonging to the aldo-keto reductase (AKR) family and short-chain dehydrogenase/reductase (SDR) family. In the reduction of PPH 4 by AKR family enzymes, 2 ′-OXPH 4 was formed by 3α-hydroxysteroid dehydrogenase type 2, whereas 1 ′-OXPH 4 was produced by aldose reductase, aldehyde reductase, and 20α-hydroxysteroid dehydrogenase, and both 1 ′-OXPH 4 and 2 ′-OXPH 4 were detected as the major and minor products by 3α-hydroxysteroid dehydrogenases (types 1 and 3). The activities of aldose reductase and 3α-hydroxysteroid dehydrogenase type 2 (106 and 35 nmol/mg/min, respectively) were higher than those of the other enzymes (0.2–4.0 nmol/mg/min). Among the SDR family enzymes, monomeric carbonyl reductase exhibited low 1 ′-OXPH 4-forming activity of 5.0 nmol/mg/min, but l-xylulose reductase and peroxisomal tetrameric carbonyl reductase did not form any reduced product from PPH 4. Aldose reductase reduced 2 ′-OXPH 4 to BH 4, but the other enzymes were inactive towards both 2 ′-OXPH 4 and 1 ′-OXPH 4. These results indicate that the tetrahydropterin intermediates are natural substrates of the human AKR family enzymes and suggest a novel alternative pathway from PPH 4 to BH 4, in which 3α-hydroxysteroid dehydrogenase type 2 and aldose reductase work in concert.

Two suppressors of sel-12 encode C2H2 zinc-finger proteins that regulate presenilin transcription in Caenorhabditis elegans.

Mutations in presenilin genes are associated with familial Alzheimer's disease in humans and affect LIN-12/Notch signaling in all organisms tested so far. Loss of sel-12 presenilin activity in Caenorhabditis elegans results in a completely penetrant egg-laying defect. In screens for extragenic suppressors of the sel-12 egg-laying defect, we have isolated mutations in at least five genes. We report the cloning and characterization of spr-3 and spr-4, which encode large basic C(2)H(2) zinc-finger proteins. Suppression of sel-12 by spr-3 and spr-4 requires the activity of the second presenilin gene, hop-1. Mutations in both spr-3 and spr-4 de-repress hop-1 transcription in the early larval stages when hop-1 expression is normally nearly undetectable. As sel-12 and hop-1 are functionally redundant, this suggests that mutations in spr-3 and spr-4 bypass the need for one presenilin by stage-specifically de-repressing the transcription of the other. Both spr-3 and spr-4 code for proteins similar to the human REST/NRSF (Re1 silencing transcription factor/neural-restrictive silencing factor) transcriptional repressors. As other Spr genes encode proteins homologous to components of the CoREST co-repressor complex that interacts with REST, and the INHAT (inhibitor of acetyltransferase) co-repressor complex, our data suggest that all Spr genes may function through the same mechanism that involves transcriptional repression of the hop-1 locus.

Co-ordinate transcriptional regulation of dopamine synthesis genes by alpha-synuclein in human neuroblastoma cell lines.

Abnormal accumulation of alpha-synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) alpha-synuclein using microarrays, with confirmation of selected genes by quantitative RT-PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis-inducing molecules, transcription factors and membrane-bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co-ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild-type but not mutant alpha-synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co-ordinate regulation of dopamine synthesis is regulated through this transcription factor.

Effect of antisense oligodeoxynucleotide for sepiapterin reductase on the viability of PC12 cells in the presence of exogenous carbonyl compounds

Sepiapterin reductase (SPR) is known as an essential enzyme for the biosynthesis of tetrahydrobiopterin. SPR belongs to the short-chain dehydrogenase/reductase (SDR) family and also reduces various exogenous carbonyl compounds including phenylpropanedione. We found in the present study that phenylpropanedione decreased the rate of proliferation of PC12 cells and that this rate was further diminished by the transfection of the cells with antisense oligodeoxynucleotide for SPR mRNA. When the cells were treated with N-acetylserotonin, a specific inhibitor of SPR, in the presence of phenylpropanedione, the cell number decreased to almost the same level as when the cells were transfected with the antisense oligodeoxynucleotide. Thus, the SDR activity of SPR in PC12 cells may serve for detoxification of exogenous carbonyl compounds besides functioning as a specific enzyme for the formation of tetrahydrobiopterin.