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Tae-cheon Kang - One of the best experts on this subject based on the ideXlab platform.
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tat mediated protein transduction of human brain Pyridoxal Kinase into pc12 cells
Biochimie, 2005Co-Authors: Dae Won Kim, Oh-shin Kwon, Tae-cheon Kang, Chung Kwon Kim, Soo Hyun Choi, Hee Soon Choi, So Young Kim, Seung Ree Lee, Sun Hwa Lee, Mooho WonAbstract:Abstract Pyridoxal Kinase (PK) catalyses the phosphorylation of vitamin B6 to Pyridoxal-5′-phosphate (PLP). A human brain PK gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat–PK fusion protein. The expressed and purified Tat–PK fusion proteins transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously in culture media. Once inside the cells, the transduced Tat–PK proteins showed catalytic activity and are stable for 48 h. The intracellular concentration of PLP, which is known as a biologically active form of vitamin B6, was increased by pre-treatment of Tat–PK to the PC12 cells. Those results suggest that the transduction of Tat–PK fusion protein can be one of the ways to regulate the PLP level and to replenish this enzyme in the various neurological disorders related to vitamin B6.
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vigabatrin inhibits pyridoxine 5 phosphate oxidase not Pyridoxal Kinase in the hippocampus of seizure prone gerbils
Neurochemistry International, 2004Co-Authors: Seung Kook Park, Oh-shin Kwon, Soo Young Choi, In Koo Hwang, Mooho Won, Tae-cheon KangAbstract:To identify the effects of vigabatrin (VGB) on the metabolism of Pyridoxal 5'-phosphate (PLP) in the seizure prone gerbil hippocampus, we conducted a chronological and comparative analysis of Pyridoxal Kinase (PLK) and pyridoxine-5'-phosphate oxidase (PNP oxidase) expression. In the VGB treated animals, PNP oxidase immunoreactivity was reduced, although the distribution and immunodensity of PLK were unaltered, as compared with control animals. In a Western blot study, the densities of PNP oxidase immunoreactivities in VGB treated animals were found to have decreased significantly. However, no differences in PLK immunoreactive bands were observed in controls or in VGB treated animals. By enzyme activity assay, and in contrast to PLK, the specific activity of PNP oxidase in the VGB treated gerbils was significantly reduced. In conclusion, the present data presents a piece of in vivo evidence that supports the anti-epileptic effects mediated by pyridoxamine-5'-phosphate (PMP) metabolism, and which may be helpful in the development of an anti-epileptic drug.
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Changes in Pyridoxal Kinase immunoreactivity in the gerbil hippocampus following spontaneous seizure.
Brain research, 2002Co-Authors: Tae-cheon Kang, Oh-shin Kwon, Soo Young Choi, Jae Hoon Bahn, Seung Kook Park, In Koo Hwang, Dae Won Kim, Nam-in Baek, Hyeon Yong LeeAbstract:To identify the roles of Pyridoxal Kinase (PLK) in epileptogenesis and the recovery mechanisms in spontaneous seizure, a chronological and comparative analysis of PLK expression in the gerbil hippocampus was conducted. PLK immunoreactivity in a pre-seizure group of seizure sensitive (SS) gerbils was more strongly detected than that in a seizure resistant (SR) group. The density of PLK immunoreactivity in a 30-min postictal group was significantly lower than that of a pre-seizure group. In a 12 h postictal group, PLK immunodensity recovered to pre-seizure level. The over-expression of PLK in the hippocampus of pre-seizure SS gerbils suggests that PLP play an important role in the modulation of GAD activity and GABA reuptake as mediated by membrane transporter via neurons.
Oh-shin Kwon - One of the best experts on this subject based on the ideXlab platform.
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tat mediated protein transduction of human brain Pyridoxal Kinase into pc12 cells
Biochimie, 2005Co-Authors: Dae Won Kim, Oh-shin Kwon, Tae-cheon Kang, Chung Kwon Kim, Soo Hyun Choi, Hee Soon Choi, So Young Kim, Seung Ree Lee, Sun Hwa Lee, Mooho WonAbstract:Abstract Pyridoxal Kinase (PK) catalyses the phosphorylation of vitamin B6 to Pyridoxal-5′-phosphate (PLP). A human brain PK gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat–PK fusion protein. The expressed and purified Tat–PK fusion proteins transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously in culture media. Once inside the cells, the transduced Tat–PK proteins showed catalytic activity and are stable for 48 h. The intracellular concentration of PLP, which is known as a biologically active form of vitamin B6, was increased by pre-treatment of Tat–PK to the PC12 cells. Those results suggest that the transduction of Tat–PK fusion protein can be one of the ways to regulate the PLP level and to replenish this enzyme in the various neurological disorders related to vitamin B6.
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vigabatrin inhibits pyridoxine 5 phosphate oxidase not Pyridoxal Kinase in the hippocampus of seizure prone gerbils
Neurochemistry International, 2004Co-Authors: Seung Kook Park, Oh-shin Kwon, Soo Young Choi, In Koo Hwang, Mooho Won, Tae-cheon KangAbstract:To identify the effects of vigabatrin (VGB) on the metabolism of Pyridoxal 5'-phosphate (PLP) in the seizure prone gerbil hippocampus, we conducted a chronological and comparative analysis of Pyridoxal Kinase (PLK) and pyridoxine-5'-phosphate oxidase (PNP oxidase) expression. In the VGB treated animals, PNP oxidase immunoreactivity was reduced, although the distribution and immunodensity of PLK were unaltered, as compared with control animals. In a Western blot study, the densities of PNP oxidase immunoreactivities in VGB treated animals were found to have decreased significantly. However, no differences in PLK immunoreactive bands were observed in controls or in VGB treated animals. By enzyme activity assay, and in contrast to PLK, the specific activity of PNP oxidase in the VGB treated gerbils was significantly reduced. In conclusion, the present data presents a piece of in vivo evidence that supports the anti-epileptic effects mediated by pyridoxamine-5'-phosphate (PMP) metabolism, and which may be helpful in the development of an anti-epileptic drug.
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Changes in Pyridoxal Kinase immunoreactivity in the gerbil hippocampus following spontaneous seizure.
Brain research, 2002Co-Authors: Tae-cheon Kang, Oh-shin Kwon, Soo Young Choi, Jae Hoon Bahn, Seung Kook Park, In Koo Hwang, Dae Won Kim, Nam-in Baek, Hyeon Yong LeeAbstract:To identify the roles of Pyridoxal Kinase (PLK) in epileptogenesis and the recovery mechanisms in spontaneous seizure, a chronological and comparative analysis of PLK expression in the gerbil hippocampus was conducted. PLK immunoreactivity in a pre-seizure group of seizure sensitive (SS) gerbils was more strongly detected than that in a seizure resistant (SR) group. The density of PLK immunoreactivity in a 30-min postictal group was significantly lower than that of a pre-seizure group. In a 12 h postictal group, PLK immunodensity recovered to pre-seizure level. The over-expression of PLK in the hippocampus of pre-seizure SS gerbils suggests that PLP play an important role in the modulation of GAD activity and GABA reuptake as mediated by membrane transporter via neurons.
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Human Pyridoxal Kinase: overexpression and properties of the recombinant enzyme.
Molecules and cells, 2000Co-Authors: Hyun-shik Lee, Sun-hye Choi, Oh-shin KwonAbstract:Pyridoxal Kinase catalyses the phosphorylation of the vitamin B6. A human brain Pyridoxal Kinase cDNA was isolated, and the recombinant enzyme was overexpressed in E. coli as a fusion protein with maltose binding protein (MBP). Pure Pyridoxal Kinase exhibits a molecular mass of about 40 kDa when examined by SDS-PAGE and FPLC gel filtration. The recombinant enzyme is a monomer endowed with catalytic activity, indicating that the native quaternary structure of Pyridoxal Kinase is not a prerequisite for catalytic function. Zn2+ is the most effective divalent cation in the phosphorylation of Pyridoxal, and the human enzyme has maximum catalytic activity in the narrow pH range of 5.5-6.0. The Km values for two substrates Pyridoxal and ATP are 97 microM and 12 microM, respectively. In addition, the unfolding processes of the recombinant enzyme were monitored by circular dichroism. The values of the free energy change of unfolding (AGo = 1.2 kcal x mol(-1) x K(-1)) and the midpoint transition (1 M) suggested that the enzyme is more stable than ovine Pyridoxal Kinase against denaturation by guanidine hydrochloride. Intrinsic fluorescence spectra of the human enzyme from red-edge excitation and fluorescence quenching experiments showed that the tryptophanyl residues are not completely exposed and more accessible to neutral acrylamide than to the negatively charged iodide. The first complete set of catalytic and structural properties of human Pyridoxal Kinase provide valuable information for further biochemical studies on this enzyme.
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Molecular Cloning and Catalytic Properties of Human Brain Pyridoxal Kinase
Biochemistry and Molecular Biology of Vitamin B6 and PQQ-dependent Proteins, 2000Co-Authors: Hyun-shik Lee, Soo Young Choi, Byung Jo Moon, Oh-shin KwonAbstract:cDNA fragments of ovine liver Pyridoxal Kinase were amplified by PCR using degenerate oligonucleotide primers based on partial amino acid sequence data. Using the PCR product as probes, we have isolated a full-length cDNA encoding the human brain Pyridoxal Kinase. The recombinant enzyme was overexpressed inE. coliand the recombinant enzyme has maximum catalytic activity in the narrow pH range of 5.5-6.0. The Km values for two substrates Pyridoxal and ATP are 97 µM and 12 µM, respectively. Zn2+ is the most effective divalent cation in the phosphorylation of Pyridoxal. Results from intrinsic fluorescence quenching measurements showed that the tryptophanyl residues in the recombinant protein are more accessible to the neutral acrylamide than to the negatively charged iodide.
Longquan Huang - One of the best experts on this subject based on the ideXlab platform.
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Direct and indirect effects of RNA interference against Pyridoxal Kinase and pyridoxine 5'-phosphate oxidase genes in Bombyx mori.
Gene, 2016Co-Authors: Shuohao Huang, Lili Yao, Jianyun Zhang, Longquan HuangAbstract:Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which Pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize Pyridoxal 5'-phosphate by Pyridoxal Kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how Pyridoxal 5'-phosphate biosynthesis is regulated, and Pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of Pyridoxal Kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of Pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of Pyridoxal Kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using Pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that Pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks.
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bombyx mori Pyridoxal Kinase cdna cloning and enzymatic characterization
Journal of Genetics and Genomics, 2007Co-Authors: Ruijun Shi, Jianyun Zhang, Changjun Jiang, Longquan HuangAbstract:Pyridoxal Kinase (PLK) (EC 2.7.1.35) catalyzes the ATP-dependent phosphorylation of Pyridoxal, generating Pyridoxal-5.-phosphate (PLP), an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method (GenBank accession number: DQ452397). The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 kDa. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.
Makoto Matsuda - One of the best experts on this subject based on the ideXlab platform.
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Pyridoxal Kinase immunoreactivity in rabbit brain
Neurochemical Research, 1994Co-Authors: Kivoshi Ohkawa, Tae Hirakawa-sakurai, Tadashi Asakura, Koji Takada, Makoto MatsudaAbstract:Murine polyclonal antibody against purified bovine brain Pyridoxal Kinase (EC 2.7.1.35) was generated and showed cross-reactivity with rabbit brain Pyridoxal Kinase. This antibody was used to immunohistochemically examine the distribution of Pyridoxal Kinase in the rabbit brain. The cytoplasm of neuronal cells and neuroglial cells in the cerebral cortex, hippocampal region, brain nuclei and cerebellar cortex showed positive staining with various degrees of intensity. The neuronal cells and surrounding fibers in some brain nuclei, such as the area tegmentalis ventralis or the substantia nigra, showed intense staining. The neuronal cells of the hippocampal region showed somewhat weak reactivity, but some with intense reactivity were found sparsely distributed and positive staining fiber networks of a very low density were also observed.
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Purification and Properties of Pyridoxal Kinase from Bovine Brain
Molecular and Cellular Biochemistry, 1993Co-Authors: Tae Hirakawa-sakurai, Kiyoshi Ohkawa, Makoto MatsudaAbstract:A 27,000-fold purification of Pyridoxal Kinase from bovine brain tissue has been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-150 gel filtration, Blue Sepharose CL-6B chromatography, and Phenyl-Superose chromatography. The final chromatography step yields a homogeneous preparation of high specific activity (2105 nmol/min/mg protein). The molecular mass of the native enzyme was estimated to be approximately 80,000 on gel filtration. The subunit molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be approximately 39,500. This indicates that Pyridoxal Kinase is a dimeric enzyme.
Soo Young Choi - One of the best experts on this subject based on the ideXlab platform.
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vigabatrin inhibits pyridoxine 5 phosphate oxidase not Pyridoxal Kinase in the hippocampus of seizure prone gerbils
Neurochemistry International, 2004Co-Authors: Seung Kook Park, Oh-shin Kwon, Soo Young Choi, In Koo Hwang, Mooho Won, Tae-cheon KangAbstract:To identify the effects of vigabatrin (VGB) on the metabolism of Pyridoxal 5'-phosphate (PLP) in the seizure prone gerbil hippocampus, we conducted a chronological and comparative analysis of Pyridoxal Kinase (PLK) and pyridoxine-5'-phosphate oxidase (PNP oxidase) expression. In the VGB treated animals, PNP oxidase immunoreactivity was reduced, although the distribution and immunodensity of PLK were unaltered, as compared with control animals. In a Western blot study, the densities of PNP oxidase immunoreactivities in VGB treated animals were found to have decreased significantly. However, no differences in PLK immunoreactive bands were observed in controls or in VGB treated animals. By enzyme activity assay, and in contrast to PLK, the specific activity of PNP oxidase in the VGB treated gerbils was significantly reduced. In conclusion, the present data presents a piece of in vivo evidence that supports the anti-epileptic effects mediated by pyridoxamine-5'-phosphate (PMP) metabolism, and which may be helpful in the development of an anti-epileptic drug.
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Changes in Pyridoxal Kinase immunoreactivity in the gerbil hippocampus following spontaneous seizure.
Brain research, 2002Co-Authors: Tae-cheon Kang, Oh-shin Kwon, Soo Young Choi, Jae Hoon Bahn, Seung Kook Park, In Koo Hwang, Dae Won Kim, Nam-in Baek, Hyeon Yong LeeAbstract:To identify the roles of Pyridoxal Kinase (PLK) in epileptogenesis and the recovery mechanisms in spontaneous seizure, a chronological and comparative analysis of PLK expression in the gerbil hippocampus was conducted. PLK immunoreactivity in a pre-seizure group of seizure sensitive (SS) gerbils was more strongly detected than that in a seizure resistant (SR) group. The density of PLK immunoreactivity in a 30-min postictal group was significantly lower than that of a pre-seizure group. In a 12 h postictal group, PLK immunodensity recovered to pre-seizure level. The over-expression of PLK in the hippocampus of pre-seizure SS gerbils suggests that PLP play an important role in the modulation of GAD activity and GABA reuptake as mediated by membrane transporter via neurons.
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Molecular Cloning and Catalytic Properties of Human Brain Pyridoxal Kinase
Biochemistry and Molecular Biology of Vitamin B6 and PQQ-dependent Proteins, 2000Co-Authors: Hyun-shik Lee, Soo Young Choi, Byung Jo Moon, Oh-shin KwonAbstract:cDNA fragments of ovine liver Pyridoxal Kinase were amplified by PCR using degenerate oligonucleotide primers based on partial amino acid sequence data. Using the PCR product as probes, we have isolated a full-length cDNA encoding the human brain Pyridoxal Kinase. The recombinant enzyme was overexpressed inE. coliand the recombinant enzyme has maximum catalytic activity in the narrow pH range of 5.5-6.0. The Km values for two substrates Pyridoxal and ATP are 97 µM and 12 µM, respectively. Zn2+ is the most effective divalent cation in the phosphorylation of Pyridoxal. Results from intrinsic fluorescence quenching measurements showed that the tryptophanyl residues in the recombinant protein are more accessible to the neutral acrylamide than to the negatively charged iodide.
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Production and characterization of monoclonal antibodies to porcine brain Pyridoxal Kinase.
BioFactors (Oxford England), 1999Co-Authors: Soo Young Choi, Francis Kwok, Jae Hoon Bahn, Seong Gyu Jeon, Yoon Kyung Ahn, Byung Hak Yoon, Byung Ryong Lee, Kyung Soon Choi, G. Z. GaoAbstract:Six monoclonal antibodies that recognize porcine brain Pyridoxal Kinase have been selected and designated as PK67, PK86, PK91, PK144, PK252 and PK275. A total of six monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which four inhibited the enzyme activity. When total proteins of porcine brain homogenate separated by SDS-PAGE were subjected to monoclonal antibodies, a single reactive protein band of molecular weight 39 kDa which comigrated with purified porcine Pyridoxal Kinase was detected. Using the anti-Pyridoxal Kinase antibodies as probes, the cross reactivities of brain Pyridoxal Kinase from human and other mammalian tissues and from avian sources were also investigated. Among human and all animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 39 kDa. These results indicate that mammalian brains contain only one major type of immunologically similar Pyridoxal Kinase, although some properties of the enzymes reported previously differed from one another.
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isolation and characterization of cdna encoding Pyridoxal Kinase from ovine liver
Journal of Biochemistry and Molecular Biology, 1999Co-Authors: Hyun-shik Lee, Soo Young Choi, Oh-shin KwonAbstract:cDNA fragments of ovine liver Pyridoxal Kinase were amplified by PCR using degenerate oligonucleotide primers derived from partial amino acids sequences of the enzyme. Using PCR products as probes, several overlapping cDNA clones were isolated independently from an ovine liver and a human brain cDNA library. The largest cDNA clone for each was selected for sequence analysis. The ovine liver cDNA encodes a polypeptide of 297 amino acid residues with Mr of 32,925, whereas the human clone is comprised of an open reading frame encoding 312 amino acid residues with Mr of 35,102. The deduced sequence of the human brain enzyme is completely identical to that of human testes cDNA recently reported (Hanna et al., 1997). The ovine enzymes have approximately 77% sequence identity with the human enzyme although the two sequences are completely different in the N-terminus comprising 32 residues. This result suggests that Pyridoxal Kinase is highly homologous in mammalian species.
