To study the mechanisms of the non-coenzyme action of thiamine and its diphosphate (ThDP) on brain proteins, proteins of acetone extract of bovine brain synaptosomes or the homogenate of rat brain cortex were subjected to affinity chromatography on thiamine-modified Sepharose. In the step-wise eluates by thiamine (at pH 7.4 or 5.6), NaCl, and urea, the occurrence of glutamate dehydrogenase (GDH) and isoenzymes of malate dehydrogenase (MDH) along with the influence of thiamine and/or ThDP on the enzymatic activities were characterized using mass spectrometry and kinetic experiments. Maximal activation of the malate dehydrogenase reaction by thiamine is observed after the protein elution with the acidic thiamine solution, which does not elute the MDH1 isoenzyme. Effects of exogenous thiamine or ThDP on the GDH activity may depend on endogenous enzyme regulators. For example, thiamine and/or ThDP activate the brain GDH in eluates from thiamine-Sepharose but inhibit the enzyme in the crude preparations applied to the sorbent. Inhibition of GDH by ThDP is observed using the ADP-activated enzyme. Compared to the affinity chromatography employing the elu-tion by thiamine at pH 7.4, the procedure at pH 5.6 decreases the activation of GDH by thiamine (but not ThDP) in the eluates with NaCl and urea. Simultaneously, the MDH2 content and total GDH activity are higher after the affinity elution at pH 5.6 than at pH 7.4, suggesting the role of the known interaction of GDH with MDH2 in stabilizing the activity of GDH and in the regulation of GDH by thiamine. The biological potential of thiamine-dependent regulation of the brain GDH is confirmed in vivo by demonstration of changes in regulatory properties of GDH after administration of a high dose of thiamine to rats. Bioinformatics analysis of the thiamine-eluted brain proteins shows a specific enrichment of their annotation terms with “phosphoprotein”, “acetylation”, and “methylation”. The relationship between thiamine and the post-translational modifications in brain may contribute to the neuroprotective effects of high doses of thiamine, including the regulation of oxidation of the major excitatory neurotransmitter in brain - glutamate.

中文翻译：

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硫胺素在体内外对哺乳动物脑苹果酸脱氢酶和谷氨酸脱氢酶的调控

为了研究硫胺素及其二磷酸酯 (ThDP) 对脑蛋白的非辅酶作用机制，在硫胺素修饰的琼脂糖凝胶上对牛脑突触体丙酮提取物或大鼠脑皮层匀浆的蛋白质进行亲和层析。在硫胺素（pH 7.4 或 5.6）、NaCl 和尿素的逐步洗脱液中，谷氨酸脱氢酶 (GDH) 和苹果酸脱氢酶 (MDH) 同工酶的出现以及硫胺素和/或 ThDP 对酶促反应的影响使用质谱和动力学实验表征活性。在用酸性硫胺素溶液洗脱蛋白质后，观察到硫胺素对苹果酸脱氢酶反应的最大激活，这不会洗脱 MDH1 同工酶。外源性硫胺素或 ThDP 对 GDH 活性的影响可能取决于内源性酶调节剂。例如，硫胺素和/或 ThDP 会激活来自硫胺素-Sepharose 的洗脱液中的脑 GDH，但会抑制应用于吸附剂的粗制品中的酶。使用 ADP 激活的酶观察到 ThDP 对 GDH 的抑制。与使用 pH 7.4 硫胺素洗脱的亲和色谱法相比，pH 5.6 的程序降低了硫胺素（但不是 ThDP）在 NaCl 和尿素洗脱液中对 GDH 的活化。同时，在 pH 5.6 亲和洗脱后，MDH2 含量和总 GDH 活性高于 pH 7.4，表明 GDH 与 MDH2 的已知相互作用在稳定 GDH 活性和硫胺素调节 GDH 中的作用。通过向大鼠施用高剂量硫胺素后 GDH 调节特性的变化证明在体内证实了硫胺素依赖性调节脑 GDH 的生物学潜力。硫胺素洗脱的脑蛋白的生物信息学分析显示，它们的注释术语特别丰富，包括“磷蛋白”、“乙酰化”和“甲基化”。硫胺素与大脑中翻译后修饰之间的关系可能有助于高剂量硫胺素的神经保护作用，包括调节大脑中主要兴奋性神经递质谷氨酸的氧化。硫胺素洗脱的脑蛋白的生物信息学分析显示，它们的注释术语特别丰富，包括“磷蛋白”、“乙酰化”和“甲基化”。硫胺素与大脑中翻译后修饰之间的关系可能有助于高剂量硫胺素的神经保护作用，包括调节大脑中主要兴奋性神经递质谷氨酸的氧化。硫胺素洗脱的脑蛋白的生物信息学分析显示，它们的注释术语特别丰富，包括“磷蛋白”、“乙酰化”和“甲基化”。硫胺素与大脑中翻译后修饰之间的关系可能有助于高剂量硫胺素的神经保护作用，包括调节大脑中主要兴奋性神经递质谷氨酸的氧化。