Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, 01601, Ukraine.
Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics, Moscow, 119991, Russia.
Biochemistry (Mosc). 2020 Jan;85(1):27-39. doi: 10.1134/S0006297920010034.
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 elution 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 posttranslational 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.
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，将牛脑突触体丙酮提取物或大鼠大脑皮质匀浆的蛋白质进行硫胺素修饰的琼脂糖亲和层析。在分步洗脱液中，用硫胺素（在 pH7.4 或 5.6）、NaCl 和脲，通过质谱和动力学实验，研究谷氨酸脱氢酶（GDH）和苹果酸脱氢酶（MDH）同工酶的出现以及硫胺素和/或 ThDP 对酶活性的影响。在用酸性硫胺素溶液洗脱蛋白质后，观察到苹果酸脱氢酶反应的最大激活，而 MDH1 同工酶没有被洗脱出来。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 在硫胺素琼脂糖洗脱液中激活脑 GDH，但在应用于吸附剂的粗制剂中抑制酶。在 ADP 激活的酶中观察到 ThDP 对 GDH 的抑制作用。与采用 pH7.4 的硫胺素洗脱的亲和层析相比，在 pH5.6 的洗脱程序中，NaCl 和脲洗脱液中硫胺素对 GDH 的激活作用降低（但 ThDP 不降低）。同时，在 pH5.6 洗脱后，MDH2 含量和总 GDH 活性均高于 pH7.4，这表明 GDH 与 MDH2 的已知相互作用在稳定 GDH 活性和通过硫胺素调节 GDH 方面具有重要作用。通过向大鼠给予高剂量硫胺素来证明 GDH 调节性质的变化，体内证实了依赖硫胺素的脑 GDH 调节的生物学潜力。从洗脱的脑蛋白的生物信息学分析显示，其注释术语的“磷酸化蛋白”、“乙酰化”和“甲基化”有特定的富集。硫胺素与脑内的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，包括对脑内主要兴奋性神经递质谷氨酸的氧化的调节。
