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Thiamine Mono- and Diphosphate Phosphatases in Bovine Brain Synaptosomes

V. A. Aleshin1,2,a, O. A. Mezhenska3, Y. M. Parkhomenko3, T. Kaehne4, and V. I. Bunik1,2,5,b*

1Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics, 119991 Moscow, Russia

2Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

3Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, 01601 Kyiv, Ukraine

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

5Sechenov First Moscow State Medical University, Ministry of Health of the Russian Federation, 119992 Moscow, Russia

* To whom correspondence should be addressed.

Received December 31, 2019; Revised January 8, 2020; Accepted January 9, 2020
Neurodegenerative diseases are accompanied by changes in the activity of thiamine mono- and diphosphate phosphatases, but molecular identification of these mammalian enzymes is incomplete. In this work, the protein fraction of bovine brain synaptosomes displaying phosphatase activity toward thiamine derivatives was subjected to affinity chromatography on thiamine-Sepharose. Protein fractions eluted with thiamine (pH 7.4 or 5.6), NaCl, and urea were assayed for the phosphatase activity against thiamine monophosphate (ThMP), thiamine diphosphate (ThDP), and structurally similar purine nucleotides. Proteins in each fraction were identified by mass spectrometry using the SwissProt database for all organisms because of insufficient annotation of the bovine genome. Peptides of two annotated bacterial phosphatases, alkaline phosphatase L from the DING protein family and exopolyphosphatase, were identified in the acidic thiamine eluate. The abundance of peptides of alkaline phosphatase L and exopolyphosphatase in the eluted fractions correlated with ThMPase and ThDPase activities, respectively. The elution profiles of the ThMPase and ThDPase activities differed from the elution profiles of nucleotide phosphatases, thus indicating the specificity of these enzymes toward thiamine derivatives. The search for mammalian DING phosphatases in the eluates from thiamine-Sepharose revealed X-DING-CD4, mostly eluted by the acidic thiamine solution (pH 5.6). The identified exopolyphosphatase demonstrated structural similarity with apyrases possessing the ThDPase activity. The obtained results demonstrate that mammalian DING proteins and apyrases exhibit ThMPase and ThDPase activity, respectively.
KEY WORDS: apyrase, DING proteins, HPBP, thiamine, thiamine monophosphatase, thiamine diphosphatase, X-DING-CD4

DOI: 10.1134/S000629792003013X