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Regulation of Malate Dehydrogenases and Glutamate Dehydrogenase of Mammalian Brain by Thiamine in vitro and in vivo

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Abstract

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.

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Abbreviations

GDH:

glutamate dehydrogenase

MDH:

malate dehydrogenase

MS:

mass spectrometry

ThDP:

thiamine diphos-phate

TIC:

total ion current

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Acknowledgments

The authors acknowledge the work of Dr. A. V. Graf and Dr. M. V. Maslova (Moscow State University) with animals used in this study.

Funding

The work of a PhD student of the Palladin Institute of Biochemistry (Kyiv, Ukraine), O. A. Mezhenska (experimental results in Figs. 1, 2, 4 and Tables 1–3 excluding MS data) within this project performed at Lomonosov Moscow State University was supported by the Russian Foundation for Basic Research in 2015 (grant no. 15-34-50124) among projects done by young investigators under supervision of candidates and doctors of sciences in scientific institutes of the Russian Federation. Experiments shown in Figs. 3 and 5, the analysis (including the bioinformatics analysis) of the affinity chromatography and mass spectrometry data, and preparation of the data for publication were supported by the Russian Science Foundation (grant no. 18-14-00116).

Author information

Authors and Affiliations

  1. Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, 01601, Kyiv, Ukraine

    O. A. Mezhenska

  2. Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119991, Moscow, Russia

    V. A. Aleshin, T. Kaehne, A. V. Artiukhov & V. I. Bunik

  3. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 19991, Moscow, Russia

    V. A. Aleshin & A. V. Artiukhov

  4. Institute of Experimental Internal Medicine, Otto-von-Guericke University, 39120, Magdeburg, Germany

    V. I. Bunik

Authors
  1. O. A. Mezhenska
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  2. V. A. Aleshin
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  3. T. Kaehne
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  4. A. V. Artiukhov
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  5. V. I. Bunik
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Corresponding author

Correspondence to V. I. Bunik.

Additional information

This paper is dedicated to 80th anniversary of the Biochemistry Department of Lomonosov Moscow State University (see vol. 84, no. 11, 2019).

Conflict of interest.

The authors declare no conflict of interest.

Ethical statements

All the international, national, and institutional principles of the animal care and usage were satisfied. In particular, all the animal experiments were done according to the EU directive 2010/63/EU. The experiments were approved by the Bioethics Committee of Lomonosov Moscow State University.

Published in Russian in Biokhimiya, 2020, Vol. 85, No. 1, pp. 34–48.

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Mezhenska, O.A., Aleshin, V.A., Kaehne, T. et al. Regulation of Malate Dehydrogenases and Glutamate Dehydrogenase of Mammalian Brain by Thiamine in vitro and in vivo. Biochemistry Moscow 85, 27–39 (2020). https://doi.org/10.1134/S0006297920010034

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