• BMB Reports
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• 제30권2호
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• pp.125-131
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• 1997

This work reports studies of the catalytic and structural properties of pyridoxal kinase (ATP: pyridoxal 5' -phosphotransferase, EC. 2.7.1.35), Pyridoxal kinase catalyzes the phosphorylation of vitamin $B_6$ (pyridoxal, pyridoxamine, pyridoxine) using ATP-Zn as a phosphoryl donor. The enzyme purified from brain tissues is made up of two identical subunits of 40 kDa each. Native enzyme was inhibited by a substrate analogue, pyridoxal-oxime. Limited chymotrypsin digestion of pyridoxal kinase yields two fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were isolated by DEAE ion exchange chromatography and used for binding studies with fluorescent ATP and pyridoxal analogues. The spectroscopic properties of both fluorescent pyridoxal analogue and Anthraniloyl ATP (Ant-ATP) bound to the 24 kDa fragment are indistinguishable from those of both pyridoxal analogue and Ant-ATP bound to the native pyridoxal kinase, respectively. The small 16 kDa fragment, generated by proteolytic cleavage of the kinase, does not bind any of the substrate analogues. Binding characteristics of Ant-ATP were extensively studied by measuring the changes in fluorescence spectra at various conditions. From the results presented herein, it is postulated that the structural domain associated with catalytic activity comprises approximately one-half of the molecular mass of pyridoxal kinase (24 kDa). whereas the remaining portion (16 kDa) of the enzyme contains a regulatory binding domain.

• BMB Reports
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• 제32권5호
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• pp.502-505
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• 1999

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.

• 한국어병학회지
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• 제6권2호
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• pp.133-142
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• 1993

비타민은 동물과 어류에 있어 성장, 발육, 및 대사기능에 비교적 적은 양으로 요구되는 필수 미량원소이다. 이들 미세 영양분이 결핍됨으로써 식욕 감퇴에서부터 심한 조직 변형등의 증상들을 일으킨다. 수용성 vitamin B6는 비교적 적은양이 요구되며, 주로 보조 효소 기능을 가지고 있다. 비타민 B6라는 명칭은 유사한 대사 기능들을 가진 화학적으로 관련된 pyridoxine, pyridoxamine, pyridoxal들을 의미한다. 비타민 B6는 비 반추 포유류, 조류와 어류의 음식물을 통해 섭취되는 성분이며, 비타민 B6 화합물들은 식물과 미생물 등에 의해 합성된다. 대사적으로 활성형인 비타민 B6 보조효소는 pyridoxal-5-phsphate(PLP)이며, decarboxylases, aminotransferases, sulfhydrases, tryptophanases를 포함한 아미노산 대사에 관여하는 여러 효소들(PLP-dependant)에 coenzyme으로 작용한다. 비타민 B6 요구량이 육식 동물보다 고단백질을 섭취하는 어류에서 더 높다. B6는 탄수화물과 지질의 대사에도 역시 관여하며 heme과 serotonin의 합성에 필수적이다. 어류에서 결핍증(현상)은 빨리 나타나는데, 이러한 증세로는 신경계 분열, 경련, 유영 부조화, 피부병변, 부종, 안구 돌출, 근 긴장성 경련 등이 포함된다. 비타민 B6는 pyridoxal kinase와 pyridoxine(pyridoxamine) oxidase의 촉매 반응에 의해 생체내 활성형인 PLP로 된다. PLP는 PLP-의존성 효소에서 보조 효소로 필수적인 역할을 하는 관계로 이 논문에서는 비타민 B6 생합성 및 대사와 비타민 B6 의존성 효소(aminotransferase)의 특성과 작용기작에 대한 효소학적 연구를 중점으로 이들 enzyme들의 구조와 기능에 대한 최근 연구 동향을 살펴보고자 한다.

• BMB Reports
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• 제39권1호
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• pp.76-83
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• 2006

Pyridoxine-5-P oxidase catalyses the terminal step in the biosynthesis of pyridoxal-S-P, the biologically active form of vitamin $B_6$ Which acts as an essential cofactor. Here, a human brain pyridoxine-5-P oxidase 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-pyridoxine-5-P oxidase fusion protein. Expressed and purified Tat-pyridoxine-5-P oxidase fusion protein transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-pyridoxine-5-P oxidase protein showed catalytic activity and was stable for 48 h. Moreover, the formation of pyridoxal-5-P was increased by adding exogenous Tat-pyridoxine-5-P oxidase to media pre-treated with the vitamin $B_6$ precursor pyridoxine. In addition, the intracellular concentration of pyridoxal-S-P was markedly increased when Tat-pyridoxal kinase was transduced together with Tat-pyridoxine-5-P oxidase into cells. These results suggest that the transduction of Tat-pyridoxine-5-P oxidase fusion protein presents a means of regulating the level of pyridoxal-5-P and of replenishing this enzyme in various neurological disorders related to vitamin $B_6$.

• BMB Reports
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• 제41권5호
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• pp.408-413
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• 2008

Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin $B_6$ precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin $B_6$.