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

O. A. Mezhenska1, V. A. Aleshin2,3, T. Kaehne4, A. V. Artiukhov2,3, and V. I. Bunik2,3,a*

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

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

3Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

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

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

* To whom correspondence should be addressed.

Received August 5, 2019; Revised September 24, 2019; Accepted September 24, 2019
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.
KEY WORDS: thiamine, malate dehydrogenase, glutamate dehydrogenase, thiamine-Sepharose, phosphoprotein, acetylation, methylation

DOI: 10.1134/S0006297920010034