Regulatory mechanism (s) of pyridoxal kinase on production of neurotransmitters in the brain.

吡哆醛激酶对大脑中神经递质产生的调节机制。

• 批准号: 06680768
• 负责人: OHKAWA Kiyoshi
• 金额: $ 1.09万
• 依托单位: Jikei University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06680768/
• 关键词: Vitamin B_6; Pyridoxal kinase; Neurotransmitter; Pyridoxal phosphate

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 … More 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

从牛脑中提纯了比活力高(2,105nmol/min/mg)的吡哆醛激酶(PLK)。凝胶过滤测得的天然相对分子质量约为80,000，SDS-PAGE测得亚基为39,500。用常规方法免疫小鼠或大鼠，制备抗纯化酶的多克隆抗体。用大鼠抗牛脑PLK多克隆抗体研究了PLK在兔脑内的免疫分布。在黑质和腹侧被盖的神经细胞和纤维中有较强的免疫反应。用该抗体建立了PLK的酶免疫分析方法。在利血平或脱氧吡哆醇处理的金黄地鼠脑中，通过改变抗PLK抗体所定义的特定蛋白的活性和/或交替表达，重新评估了PLK对磷酸吡哆醛(PLP)水平波动的调节机制(S)。APLP催化磷酰基t-…更多的ATP转移到吡哆醛的羟甲基(PL)，形成PLP，但PLP的极端降低或增加并未引起PLK活性和特异性蛋白表达的显著变化。在PLK测定体系中加入多巴胺或5-羟色胺，由于PLP的一个醛基团与一个多巴胺的氨基发生不可逆的、剂量依赖的偶联反应，以PLP的磷酸化率衡量的酶活性明显受到抑制。这些结合产物不表现出任何活性辅酶性质。一些重要神经递质的生物合成所需的PLP浓度可能不受诱导的PLK调节，而是由PLP与这些胺的结合调节，即所谓的内生抑制。本研究的目的是克隆plk基因，并探讨在某些生物环境影响下，脑内神经递质波动变化过程中plk基因表达的调控机制(S)。较少

项目成果

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