氏名

コンドウ ヨシタカ

近藤 嘉高

職名

講師(任期付)

所属

(人間科学部)

学歴・学位

学位

博士

学内研究制度

特定課題研究

動物におけるペントースリン酸副経路の同定

2019年度

研究成果概要:Pentose phosphate pathway (PPP) is a metabolic pathway parallel to glycolysis, which generates nicotinamide adenine...Pentose phosphate pathway (PPP) is a metabolic pathway parallel to glycolysis, which generates nicotinamide adenine dinucleotide phosphate (NADPH) for reductive biosynthesis reactions and pentoses as well as such as ribose 5-phosphate, a precursor for the synthesis of nucleotides. 6-phosphogluconate is an intermediate in PPP which metabolize glucose 6-phosphate to glyceraldehyde 3-phosphate. In bacteria, it is known that alternative pathway of PPP metabolize glucose to 6-phosphogluconate. However, it has not been reported in human and mouse. To reveal a novel alternative PPP, we identified gluconokinase (GNK) gene and characterized GNK activity for phosphorylating gluconte to 6-phosphogluconate.By NCBI-BLAST search, amino acid sequence of bacterial GNK in Pseudomonas putida KT2440 showed high homology with those of possible GNK gene (NM_001001551 and NM_198004) in human and mouse, respectively. We cloned cDNA of possible GNK gene from liver in human and mouse. Recombinant 6xHis-tagged proteins were purified from bacterial soluble fraction by using Ni-NTA agarose and DEAE-Sephacel column. GNK activity in reaction mixture containng gluconate, ATP, MgCl2, and recombinant GNK protein was measured by monitoring A340 of NADPH generated in coupled with resulting 6-phosphogluconate, NADP+, and 6-phosphogluconate dehydrogenase. Human and mouse recombinat proteins showed GNK activity in dose-dependent manner, respectively. Furthermore, GNK activity was detected in mouse liver extract.In this study, we identified GNK gene in human and mouse, suggesting that alternative PPP could exists in human and mouse.

動物におけるペストースリン酸副経路の同定

2019年度

研究成果概要:Pentose phosphate pathway (PPP) is a metabolic pathway parallel to glycolysis, which generates nicotinamide adenine...Pentose phosphate pathway (PPP) is a metabolic pathway parallel to glycolysis, which generates nicotinamide adenine dinucleotide phosphate (NADPH) for reductive biosynthesis reactions and pentoses as well as such as ribose 5-phosphate, a precursor for the synthesis of nucleotides. 6-phosphogluconate is an intermediate in PPP which metabolize glucose 6-phosphate to glyceraldehyde 3-phosphate. In bacteria, it is known that alternative pathway of PPP metabolize glucose to 6-phosphogluconate. However, it has not been reported in human and mouse. To reveal a novel alternative PPP, we identified gluconokinase (GNK) gene and characterized GNK activity for phosphorylating gluconte to 6-phosphogluconate.By NCBI-BLAST search, amino acid sequence of bacterial GNK in Pseudomonas putida KT2440 showed high homology with those of possible GNK gene (NM_001001551 and NM_198004) in human and mouse, respectively. We cloned cDNA of possible GNK gene from liver in human and mouse. Recombinant 6xHis-tagged proteins were purified from bacterial soluble fraction by using Ni-NTA agarose and DEAE-Sephacel column. GNK activity in reaction mixture containng gluconate, ATP, MgCl2, and recombinant GNK protein was measured by monitoring A340 of NADPH generated in coupled with resulting 6-phosphogluconate, NADP+, and 6-phosphogluconate dehydrogenase. Human and mouse recombinat proteins showed GNK activity in dose-dependent manner, respectively. Furthermore, GNK activity was detected in mouse liver extract.In this study, we identified GNK gene in human and mouse, suggesting that alternative PPP could exists in human and mouse.

新規ペントースリン酸経路で働くグルコン酸キナーゼのマウスにおける発現解析

2020年度

研究成果概要:Pentose phosphate pathway (PPP) produce NADPH which is utilized for fatty acid synthesis, cholesterol synthesis, an...Pentose phosphate pathway (PPP) produce NADPH which is utilized for fatty acid synthesis, cholesterol synthesis, and steroid biosynthesis, and redox maintenance, and ribose 5-phosphate which is necessary for synthesis of nucleic acid. It is known that alternative pathway of PPP from glucose to 6-phosphogluconate exists in bacteria, however, it remains unclear in animals. In our previous study, SMP30/gluconolactonase (GNL) hydrolyzed glucono 1,5-lactone to gluconate, and gluconokinase (GNK) catalyzed phosphorylation of gluconate to 6-phosphogluconate in human and mouse. To reveal tissue distribution of GNK in mouse, we measured gene expression levels of GNK, and compared with those of SMP30/GNL, glucose 6-phosphate dehydrogenase (G6PD), and 6-phosphogluconolactonase (6PGL).Six C57BL/6J male mice were dissected at 8 weeks of age, and 20 kinds of tissues were collected. Gene expression levels of GNK, SMP30/GNL, G6PD, and 6PGL were quantified by using real-time PCR. GNK, SMP30/GNL, G6PD and 6PGL genes were ubiquitously expressed in almost all tissues. GNK gene was highly expressed in liver, kidney, duodenum, and brown fat. Gene expression of SMP30/GNL was most abundant in liver, subsequently in kidney, duodenum, and adrenal gland. G6PD gene was highly expressed in epididymal fat, brown fat, adrenal gland, and testis. 6PGL gene showed higher expression in brown fat and testis.Overall, tissue distribution of GNK gene expression showed similarity to that of SMP30/GNL, which is distinct from those of G6PD and 6PGL. These results suggest that GNK and SMP30/GNL could cooperatively functions in alternative PPP.

動物におけるペントースリン酸副経路の同定

2020年度

研究成果概要:Pentose phosphate pathway (PPP) produce NADPH which is utilized for fatty acid synthesis, cholesterol synthesis, an...Pentose phosphate pathway (PPP) produce NADPH which is utilized for fatty acid synthesis, cholesterol synthesis, and steroid biosynthesis, and redox maintenance, and ribose 5-phosphate which is necessary for synthesis of nucleic acid. It is known that alternative pathway of PPP from glucose to 6-phosphogluconate exists in bacteria, however, it remains unclear in animals. In our previous study, SMP30/gluconolactonase (GNL) hydrolyzed glucono 1,5-lactone to gluconate, and gluconokinase (GNK) catalyzed phosphorylation of gluconate to 6-phosphogluconate in human and mouse. To reveal tissue distribution of GNK in mouse, we measured gene expression levels of GNK, and compared with those of SMP30/GNL, glucose 6-phosphate dehydrogenase (G6PD), and 6-phosphogluconolactonase (6PGL).Six C57BL/6J male mice were dissected at 8 weeks of age, and 20 kinds of tissues were collected. Gene expression levels of GNK, SMP30/GNL, G6PD, and 6PGL were quantified by using real-time PCR. GNK, SMP30/GNL, G6PD and 6PGL genes were ubiquitously expressed in almost all tissues. GNK gene was highly expressed in liver, kidney, duodenum, and brown fat. Gene expression of SMP30/GNL was most abundant in liver, subsequently in kidney, duodenum, and adrenal gland. G6PD gene was highly expressed in epididymal fat, brown fat, adrenal gland, and testis. 6PGL gene showed higher expression in brown fat and testis.Overall, tissue distribution of GNK gene expression showed similarity to that of SMP30/GNL, which is distinct from those of G6PD and 6PGL. These results suggest that GNK and SMP30/GNL could cooperatively functions in alternative PPP.

動物におけるペントースリン酸副経路の同定

2020年度

研究成果概要:Pentose phosphate pathway (PPP) produce NADPH which is utilized for fatty acid synthesis, cholesterol synthesis, an...Pentose phosphate pathway (PPP) produce NADPH which is utilized for fatty acid synthesis, cholesterol synthesis, and steroid biosynthesis, and redox maintenance, and ribose 5-phosphate which is necessary for synthesis of nucleic acid. It is known that alternative pathway of PPP from glucose to 6-phosphogluconate exists in bacteria, however, it remains unclear in animals. In our previous study, SMP30/gluconolactonase (GNL) hydrolyzed glucono 1,5-lactone to gluconate. To reveal a novel alternative PPP, we identified gluconokinase (GNK) gene and characterized GNK activity for phosphorylating gluconate to 6-phosphogluconate. To reveal tissue distribution of GNK in mouse, we measured gene expression levels of GNK, and compared with those of SMP30/GNL, glucose 6-phosphate dehydrogenase (G6PD), and 6-phosphogluconolactonase (6PGL). Six C57BL/6J male mice were dissected at 8 weeks of age, and 20 kinds of tissues were collected. Gene expression levels of GNK, SMP30/GNL, G6PD, and 6PGL were quantified by using real-time PCR. GNK, SMP30/GNL, G6PD and 6PGL genes were ubiquitously expressed in almost all tissues. GNK gene was highly expressed in liver, kidney, duodenum, and brown fat. Gene expression of SMP30/GNL was most abundant in liver, subsequently in kidney, duodenum, and adrenal gland. G6PD gene was highly expressed in epididymal fat, brown fat, adrenal gland, and testis. 6PGL gene showed higher expression in brown fat and testis. Overall, tissue distribution of GNK gene expression showed similarity to that of SMP30/GNL, which is distinct from those of G6PD and 6PGL. These results suggest that GNK and SMP30/GNL could cooperatively functions in alternative PPP.

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