• BMB Reports
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• v.28 no.1
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• pp.72-78
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• 1995

Time-dependent inactivation of E. coli GTP cyclohydrolase I with a 2',3'-dialdehyde derivative of GTP (oGTP) was directed to the active site of the enzyme, and was dependent on the concentration of oGTP. The kinetics of inactivation were biphasic with a rapid reaction occurring immediately upon exposure of the enzyme to oGTP followed by a slow rate of inactivation. The $K_i$ value of oGTP for the enzyme was 0.25 mM. Inactivation was prevented by preincubation of the enzyme with GTP, the substrate of the enzyme. At 100% inactivation, 2.3 mol of [8.5'-$^3H$]oGTP were bound per each enzyme subunit, which consists of two identical polypeptides. The active site residue which reacted with the affinity label was lysine. oGTP interacted selectively with the ${\varepsilon}$-amino group of lysine in the GTP-binding site to form a morpholine-like structure which was stable without sodium borohydride treatment. However, triphosphate group was eliminated during the hydrolysis step. To identify the active site of the enzyme, [8.5'-$^3H$]oGTP-labeled enzyme was cleaved by endoproteinase Lys-C, and the $^3H$-labeled peptide was purified by HPLC. The amino acid sequence of the active site peptide was Pro-Ser-Leu-Ser-Lys, which corresponds to the aminoterminal sequence of the enzyme.

• Korean Journal of Microbiology
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• v.34 no.4
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• pp.243-247
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• 1998

Comparative analyses of intracellular biopterin contents and its biosynthetic enzymes were performed in Allomyces macrogynus. Biopterin content in fresh weight was 14-fold higher in mycelium than in zoospore. Enzyme activities of GTP cyclohydrolase I and 6-pyruvoyltetrahydropterin synthase in ammonium sulfate fractions were approximately 2-fold higher in mycelium. On the other hand, sepiapterin reductase (SR) activity was 10 fold higher in zoospore. Northern blot assay also demonstrated that SR transcript was abundant in zoospore. These results suggest a possible involvement of tetrahydrobiopterin in cellular differentiation of Allomyces macrogyllus as well as provide an experimental basis to elucidate the physiological function of SR in this organism.

• Proceedings of the Korea Crystallographic Association Conference
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• 2007.11a
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• pp.24-24
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• 2007

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.2
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• pp.129-134
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• 2014

It has been suggested that transition metal ions such as iron can produce an oxidative injuries to nigrostriatal dopaminergic neurons, like Parkinson's disease (PD) and subsequent compensative increase of tetrahydrobiopterin ($BH_4$) during the disease progression induces the aggravation of dopaminergic neurodegeneration in striatum. It had been established that the direct administration of $BH_4$ into neuron would induce the neuronal toxicity in vitro. To elucidate a role of $BH_4$ in pathogenesis in the PD in vivo, we assessed the changes of dopamine (DA) and $BH_4$ at striatum in unilateral intranigral iron infused PD rat model. The ipsistriatal DA and $BH_4$ levels were significantly increased at 0.5 to 1 d and were continually depleting during 2 to 7 d after intranigral iron infusion. The turnover rate of $BH_4$ was higher than that of DA in early phase. However, the expression level of GTP-cyclohydrolase I mRNA in striatum was steadily increased after iron administration. These results suggest that the accumulation of intranigral iron leads to generation of oxidative stress which damage to dopaminergic neurons and causes increased release of $BH_4$ in the dopaminergic neuron. The degenerating dopaminergic neurons decrease the synthesis and release of both $BH_4$ and DA in vivo that are relevance to the progression of PD. Based on these data, we propose that the increase of $BH_4$ can deteriorate the disease progression in early phase of PD, and the inhibition of $BH_4$ increase could be a strategy for PD treatment.

• Korean Journal of Microbiology
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• v.24 no.2
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• pp.119-126
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• 1986

A periplasmic endoprotease, named protease Pi, was purified to homogeneity from Escherkchia coli by conventional procedure with insulin as substrate. This enzyme degrades insulin and glucagon to trichloroacetic acid-soluble meterials, but shows little or no hydrolysis of bovine serum albumin, casein or globin. Its molecular weight was 110, 000 when determined by gel filtration on Sephacryl S-300 and was 105, 000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, it appears to be single polypeptide. This snzyme is metalloprotease, since it is completely inhibited by o-phenanthroline and can be activated by addition of divalent metal cations, such as $Mg^{2+}\;and\;Co^{2+}$. It is destinct from protease Ci, a cytoplasmic insulin degrading enzyme, since protease Pi is localized to the periplasm. Since protease Pi selectively degrades GTP cyclohydrolase I, it appears to play a role in the regulation of pteridine biosynthesis.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.74-74
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• 2003

Embryonic stem cells have several characteristics suitable for cell replacement therapy. To investigate a possibility of using human embryonic stem cell (hESC) as a carrier of therapeutic gene(s), hESC (MB03) was co-transfected with cDNAS coding for tyrosine hydroxylase (TH) and GTP cyclohydrolase Ⅰ (GTPCH Ⅰ) and bulk-selected using neomycin and hygromycin-B. Successful transfection was confirmed by western immunoblotting and RT-PCR. The genetically modified hESC (bk-THGC) relieved apomorphine-induced asymmetric motor behavior by approximately 54% when grafted into striatum of 6-OHDA-denervated rat brain. The number of rotation, however, increased up to 176+18% in 6 weeks when sham-grafted compared with number of rotation before graft. Immunohistochemical staining revealed that the grafted hESC survived and expressed TH for at least 6 weeks while the experiment was continued.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.101-101
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• 2003

• Journal of Genetic Medicine
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• v.17 no.1
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• pp.34-38
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• 2020

The term dopa-responsive dystonia (DRD) is used to describe a group of neurometabolic disorders, which are characterized by dystonia, and are typically associated with diurnal fluctuations and respond to levodopa treatment. Autosomal dominant DRD (DYT5a, MIM# 128230) is caused by a heterozygous mutation in the GTP cyclohydrolase 1 (GCH1) gene (MIM# 600225). GCH1 encodes an enzyme, which is involved in the biosynthesis of tetrahydrobiopterin, an essential co-factor for tyrosine hydroxylase. Herein, we report the case of a 16-year-old girl who was diagnosed with DYT5a. She exhibited additional unusual clinical features, including intellectual disability, depression, multiple skeletal anomalies, and short stature, which are not commonly observed in patients with DYT5a. The patient harbored a heterozygous missense variant, c.539A>C, p.Gln180Pro, in the GCH1 gene, which was identified by targeted gene panel analysis using next-generation sequencing.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.101-101
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• 2003

Main strategy for a treatment of Parkinson's disease (PD), due to a progressive degeneration of dopaminergic neurons, is a pharmaceutical supplement of dopamine derivatives or ceil replacement therapy. Both of these protocols have pros and cons; former exhibiting a dramatic relief but causing a severe side effects on long-term prescription and latter also having a proven effectiveness but having availability and ethical problems Embryonic stem (ES) cells have several characteristics suitable for this purpose. To investigate a possibility of using ES cells as a carrier of therapeutic gene(s), human ES (hES, MB03) cells were transfected with cDNAs coding for tyrosine hydroxylase (TH) in pcDNA3.1 (+) and the transfectants were selected using neomycin (250 $\mu /ml$). Expression of TH being confirmed, two of the positive clone (MBTH2 & 8) were second transfected with GTP cyclohydrolase 1 (GTPCH 1) in pcDNA3.1 (+)-hyg followed by selection with hygromycin-B (150 $\mu /ml$) and RT-PCR confirmation. By immune-cytochemistry, these genetically modified but undifferentiated dual drug-resistant cells were found to express few of the neuronal markers, such as NF200, $\beta$-tubulin, and MAP2 as well as astroglial marker GFAP. This results suggest that over-production of BH4 by ectopically expressed GTPCH I may be involved in the induction of those markers. Transplantation of the cells into striatum of 6-OHDA- denervated PD animal model relieved symptomatic rotational behaviors of the animals. Immunohistochemical analyses showed the presence of human cells within the striatum of the recipients. These results suggest a possibility of using hES cells as a carrier of therapeutic gene(s).

• Journal of Acupuncture Research
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• v.20 no.4
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• pp.85-92
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• 2003

목적 : 허혈시 발생되는 저산소중 상태에서는 세포독성을 유발한다고 알려져 있으나 정확한 기전은 아직 규명되지 않았다. 본 연구에서는 뇌허혈로 인한 세포독성의 기전을 유전자 발현을 통하여 살펴보고자 하였다. 방법 : 본 실험에서는 BV-2 microglia 세포주에 12시간 동안의 저산소 상태에서의 유전자 발현을 분석하기 위하여 마이크로에레이를 시행하였다. 결과 : 저산소 상태에서는 정상에 비하여 cathepsin F, growth factor independent 1, calcitonin/calcitonin-related poly, leucine-rich repeat LGI family membrane, dublecortin, cyclohydrolase 1, Ia-associated invariant chain, carbohydrate kinase-like과 erythrocyte protein band 4.1-like 3 등의 유전자 발현이 3배 이상 증가하였다. 한편 neuronal guanine nucleotide exchange factor, Bcl-2-related ovarian killer protein, chemokine (C-X-C motif) ligand 5, RNA binding motif protein 3, interleukin 2 receptor, alpha chain, crystallin zeta, cytochrome P450 subfamily IV B, asparagine synthetase과 moesin 등의 유전자 발현은 0.2배 이하로 감소하였다. 결론 : 이상의 결과는 저산소중에 관여하는 유전자 및 저산소중과 관련된 뇌경색 등의 질환의 기전을 밝히는데 기초적 자료로 이용될 수 있을 것이다.