| Project/Area Number |
06680768
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Jikei University School of Medicine |
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Principal Investigator |
OHKAWA Kiyoshi Jikei University School of Medicine, Professor, 医学部, 教授 (90112812)
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| Co-Investigator(Kenkyū-buntansha) |
HAYASHI Takashi Jikei University School of Medicine, Assistant, 医学部, 助手 (90221506)
ASAKURA Tadashi Jikei University School of Medicine, Instructor, 医学部, 講師 (30138705)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1995: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Vitamin B_6 / Pyridoxal kinase / Neurotransmitter / Pyridoxal phosphate |
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| Research Abstract |
Pyridoxal kinase (PLK) was purified to apparent homogeneity from bovine brain with high specific activity (2,105 nmol/min/mg). The native molecular weight estimated by gel filtration was approximeatly 80,000 and subunit determined by SDS-PAGE was 39,500. A polyclonal antibody against purified enzyme was generated by immunizing either mice or rats with a conventional method. Immunodistribution of PLK in rabbit brain was studied using a rat polyclonal antibody against bovine brain PLK.Relatively strong immunoreactivity was noticeable in neuronal cells and fibers of substantia nigra and area tegmentalis ventralis. Enzyme immunoassay of PLK was also established using the antibody. Regulatory mechanism (s) of PLK,with changing in the activity and/or alternating expression of the specific protein defined by anti-PLK antibody, on the fluctuating level of pyridoxal phosphate (PLP) was re-evaluated using hamster brains treated with reserpine or deoxypyridoxine. APLP catalyzes phosphoryl-group t
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ransfer from ATP to the hydroxymethyl group of pyridoxal (PL), forming PLP.However, extreme decrease or increase of the PLP did not induce any considerable change of the PLK-activity as well as -specific protein expression. By addition of dopamine or serotonin to the PLK-assay system, the enzymatic activity measured as phosphorylation rate of PLP from PL,was apparently inhibited because of irreversible, dose-dependent conjugation of an aldehyde group of PLP to an amino group of dopamine. These conjugated products did not express any active coenzymatic properties. A concentration of PLP,which was required for biosynthesis of some important neurotransmitters, may not be regulated by the "induced" PLK but regulated by the conjugation of PLP to these amines, so called endoproduct inhibition. The goal of this study is molecular cloning of the PLK gene and to determine the regulatory mechanism (s) with gene-expression of PLK during the fluctuating changes of neurotransmitters in the brain under influence by some biological environments. Less
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