NON-COENZYME PROPERTIES OF THIAMINE: EVALUATION OF BINDING AFFINITY TO MALATE DEHYDROGENASE ISOFORMS
The aim of this work was evaluation of binding affinity of thiamine to malate dehydrogenase isoforms. The methods of affinity chromatography, SDS PAGE electrophoresis, and MALDI-TOF mass spectroscopy, as well as molecular modeling in silico were used in the study. Affinity sorbent (T-AS) contained C...
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| Format: | Article |
| Language: | English |
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National Academy of Sciences of Ukraine, Palladin Institute of Biochemistry
2020-08-01
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| Series: | Biotechnologia Acta |
| Subjects: | |
| Online Access: | http://biotechnology.kiev.ua/images/storage/4_2020/mezhenska_4_2020.pdf |
| _version_ | 1827836638359715840 |
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| author | O. Mezhenska A. Rebriev O. Kobzar N. Zlatoust A. Vovk Yu. Parkhomenko |
| author_facet | O. Mezhenska A. Rebriev O. Kobzar N. Zlatoust A. Vovk Yu. Parkhomenko |
| author_sort | O. Mezhenska |
| collection | DOAJ |
| description | The aim of this work was evaluation of binding affinity of thiamine to malate dehydrogenase isoforms. The methods of affinity chromatography, SDS PAGE electrophoresis, and MALDI-TOF mass spectroscopy, as well as molecular modeling in silico were used in the study. Affinity sorbent (T-AS) contained C2-conjugated thiamine fragment as an anchor which was bound to activated Sepharose 4B via a spacer composed of N-4-azobenzoylcaproic acid hydrazide. A commercial preparation of MDH from the porcine heart was chosen for the experimental study. Analysis of the protein content in fractions performed by the Bradford method showed that three separate protein peaks with malate dehydrogenase activity were obtained after elution with a thiamine solution. The results of one-dimensional electrophoresis of the initial MDH preparation and pooled fraction of proteins which were eluted from the affinity sorbent with a thiamine solution demonstrated that almost all protein fractions detected in the commercial MDH preparation were also present in eluates obtained by T-AS affinity chromatography. Four isoforms of MDH, including cytoplasmic malate dehydrogenase (MDH1), mitochondrial malate dehydrogenase (MDH2) and its isoform, and malate dehydrogenase 1B (MDH1B) were specifically bound to the affinity sorbent. According to the molecular docking results, the most preferred for both monomeric and homodimeric MDH1 and MDH2 could be ligand position at the NAD (NADH) binding site. Additional binding site could be located between two subunits of the homodimeric form of enzyme. Our results confirm the previously obtained data and expand an idea of ability of MDH isoforms to interact with the thiamine molecule in vivo. These data can also be useful for identification of thiamine binding protein (ThBP) which was previously isolated from rat brain, taking into account the possible partial homology of this protein with proteins that show MDH activity. |
| first_indexed | 2024-03-12T06:30:47Z |
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| id | doaj.art-26a1017ccc7d41029a91f3c85d49a93f |
| institution | Directory Open Access Journal |
| issn | 2410-7751 2410-776X |
| language | English |
| last_indexed | 2024-03-12T06:30:47Z |
| publishDate | 2020-08-01 |
| publisher | National Academy of Sciences of Ukraine, Palladin Institute of Biochemistry |
| record_format | Article |
| series | Biotechnologia Acta |
| spelling | doaj.art-26a1017ccc7d41029a91f3c85d49a93f2023-09-03T01:38:54ZengNational Academy of Sciences of Ukraine, Palladin Institute of BiochemistryBiotechnologia Acta2410-77512410-776X2020-08-01134263810.15407/biotech13.04.026NON-COENZYME PROPERTIES OF THIAMINE: EVALUATION OF BINDING AFFINITY TO MALATE DEHYDROGENASE ISOFORMSO. Mezhenska0A. Rebriev1O. Kobzar2N. Zlatoust 3A. Vovk4Yu. Parkhomenko5 Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, KyivKukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine, Kyiv Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, KyivKukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine, Kyiv Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, KyivThe aim of this work was evaluation of binding affinity of thiamine to malate dehydrogenase isoforms. The methods of affinity chromatography, SDS PAGE electrophoresis, and MALDI-TOF mass spectroscopy, as well as molecular modeling in silico were used in the study. Affinity sorbent (T-AS) contained C2-conjugated thiamine fragment as an anchor which was bound to activated Sepharose 4B via a spacer composed of N-4-azobenzoylcaproic acid hydrazide. A commercial preparation of MDH from the porcine heart was chosen for the experimental study. Analysis of the protein content in fractions performed by the Bradford method showed that three separate protein peaks with malate dehydrogenase activity were obtained after elution with a thiamine solution. The results of one-dimensional electrophoresis of the initial MDH preparation and pooled fraction of proteins which were eluted from the affinity sorbent with a thiamine solution demonstrated that almost all protein fractions detected in the commercial MDH preparation were also present in eluates obtained by T-AS affinity chromatography. Four isoforms of MDH, including cytoplasmic malate dehydrogenase (MDH1), mitochondrial malate dehydrogenase (MDH2) and its isoform, and malate dehydrogenase 1B (MDH1B) were specifically bound to the affinity sorbent. According to the molecular docking results, the most preferred for both monomeric and homodimeric MDH1 and MDH2 could be ligand position at the NAD (NADH) binding site. Additional binding site could be located between two subunits of the homodimeric form of enzyme. Our results confirm the previously obtained data and expand an idea of ability of MDH isoforms to interact with the thiamine molecule in vivo. These data can also be useful for identification of thiamine binding protein (ThBP) which was previously isolated from rat brain, taking into account the possible partial homology of this protein with proteins that show MDH activity.http://biotechnology.kiev.ua/images/storage/4_2020/mezhenska_4_2020.pdfthiamineaffinity chromatographymalate dehydrogenaseprotein affinity for thiaminemolecular dockingthiamine binding protein. |
| spellingShingle | O. Mezhenska A. Rebriev O. Kobzar N. Zlatoust A. Vovk Yu. Parkhomenko NON-COENZYME PROPERTIES OF THIAMINE: EVALUATION OF BINDING AFFINITY TO MALATE DEHYDROGENASE ISOFORMS Biotechnologia Acta thiamine affinity chromatography malate dehydrogenase protein affinity for thiamine molecular docking thiamine binding protein. |
| title | NON-COENZYME PROPERTIES OF THIAMINE: EVALUATION OF BINDING AFFINITY TO MALATE DEHYDROGENASE ISOFORMS |
| title_full | NON-COENZYME PROPERTIES OF THIAMINE: EVALUATION OF BINDING AFFINITY TO MALATE DEHYDROGENASE ISOFORMS |
| title_fullStr | NON-COENZYME PROPERTIES OF THIAMINE: EVALUATION OF BINDING AFFINITY TO MALATE DEHYDROGENASE ISOFORMS |
| title_full_unstemmed | NON-COENZYME PROPERTIES OF THIAMINE: EVALUATION OF BINDING AFFINITY TO MALATE DEHYDROGENASE ISOFORMS |
| title_short | NON-COENZYME PROPERTIES OF THIAMINE: EVALUATION OF BINDING AFFINITY TO MALATE DEHYDROGENASE ISOFORMS |
| title_sort | non coenzyme properties of thiamine evaluation of binding affinity to malate dehydrogenase isoforms |
| topic | thiamine affinity chromatography malate dehydrogenase protein affinity for thiamine molecular docking thiamine binding protein. |
| url | http://biotechnology.kiev.ua/images/storage/4_2020/mezhenska_4_2020.pdf |
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