In vitro studies on the biosynthesis and reduction of ubiquinone
Author: Nordman, Tomas
Date: 2003-04-11
Location: Birkeaula 2, Forskningsgatan 1, plan 5, Huddinge Universitetssjukhus
Time: 9.15
Department: Institutionen för laboratoriemedicin / Department of Laboratory Medicine
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Thesis (620.9Kb)
Abstract
This thesis concerns the role of ubiquinone, the only endogenously synthesised lipid soluble antioxidant, in the cellular defence against peroxidation of proteins and lipids.
The aims of the present investigations were to study the biosynthesis of ubiquinone in two different organelle fractions, i.e. microsomes and peroxisomes and to characterise the enzyme reactions of the three flavoenzymes, hpoamide dehydrogenase, glutathione reductase and thioredoxin reductase in the reduction of ubiquinone.
A semipreparative HPLC method was established to rapidly isolate different polyprenols with high purity. The isolated compounds could be used for studies of different enzyme reactions in the mevalonate pathway, and as standards for quantitative HPLC-analysis. Compared to conventional chromatographic methods this new technique was much more rapid and polyprenols with higher purity was isolated.
It was demonstrated that both peroxisomes and microsomes were involved in the biosynthesis of ubiquinone. Two enzymes involved in the synthesis of ubiquinone, trans-prenyltransferase and nonaprenyl-4-hydroxybenzoate (NPHB)-transferase, were investigated. The results clearly showed differences in the regulation of the synthesis of ubiquinone in those organelles. The specific activity of trans-prenyltransferase in peroxisomes was 30% of the total activity found in both organelles.
The characteristics of the regeneration of ubiquinol by the flavoenzymes, hpoamide dehydrogenase, glutathione reductase and thioredoxin reductase were investigated. These enzymes belong to the same family of enzymes and are defined as homodimeric pyridine nucleotide-disulfide oxidoreductases. The reduction of ubiquinone by Hpoamide dehydrogenase and glutathione reductase was shown to be highly elevated by addition of zinc to the reaction mixture, whereas this reaction by thioredoxin reductase was inhibited by zinc. For Hpoamide dehydrogenase and glutathione reductase the pH optimum for the reaction was found at acidic pH, but at physiological pH for thioredoxin reductase.
The reduction of ubiquinone by thioredoxin reductase was confirmed to be selenium dependent by use of full-length bovine and rat, E. coli (lacking selenocysteine), recombinant human (selenocysteine replaced by alanine), and truncated rat thioredoxin reductases, as well as with stable cell lines overexpressing thioredoxin reductase.
Altogether, the novel biological findings in this thesis are that; ubiquinone is not only synthesised in microsomes but also to a high extent in peroxisomes; ubiquinone is efficiently reduced by glutathione reductase and thioredoxin reductase; the reduction of ubiquinone by thioredoxin reductase is entirely selenium dependent.
The aims of the present investigations were to study the biosynthesis of ubiquinone in two different organelle fractions, i.e. microsomes and peroxisomes and to characterise the enzyme reactions of the three flavoenzymes, hpoamide dehydrogenase, glutathione reductase and thioredoxin reductase in the reduction of ubiquinone.
A semipreparative HPLC method was established to rapidly isolate different polyprenols with high purity. The isolated compounds could be used for studies of different enzyme reactions in the mevalonate pathway, and as standards for quantitative HPLC-analysis. Compared to conventional chromatographic methods this new technique was much more rapid and polyprenols with higher purity was isolated.
It was demonstrated that both peroxisomes and microsomes were involved in the biosynthesis of ubiquinone. Two enzymes involved in the synthesis of ubiquinone, trans-prenyltransferase and nonaprenyl-4-hydroxybenzoate (NPHB)-transferase, were investigated. The results clearly showed differences in the regulation of the synthesis of ubiquinone in those organelles. The specific activity of trans-prenyltransferase in peroxisomes was 30% of the total activity found in both organelles.
The characteristics of the regeneration of ubiquinol by the flavoenzymes, hpoamide dehydrogenase, glutathione reductase and thioredoxin reductase were investigated. These enzymes belong to the same family of enzymes and are defined as homodimeric pyridine nucleotide-disulfide oxidoreductases. The reduction of ubiquinone by Hpoamide dehydrogenase and glutathione reductase was shown to be highly elevated by addition of zinc to the reaction mixture, whereas this reaction by thioredoxin reductase was inhibited by zinc. For Hpoamide dehydrogenase and glutathione reductase the pH optimum for the reaction was found at acidic pH, but at physiological pH for thioredoxin reductase.
The reduction of ubiquinone by thioredoxin reductase was confirmed to be selenium dependent by use of full-length bovine and rat, E. coli (lacking selenocysteine), recombinant human (selenocysteine replaced by alanine), and truncated rat thioredoxin reductases, as well as with stable cell lines overexpressing thioredoxin reductase.
Altogether, the novel biological findings in this thesis are that; ubiquinone is not only synthesised in microsomes but also to a high extent in peroxisomes; ubiquinone is efficiently reduced by glutathione reductase and thioredoxin reductase; the reduction of ubiquinone by thioredoxin reductase is entirely selenium dependent.
List of papers:
I. Carlson T, Skorupinska-Tudek K, Hertel J, Chojnacki T, Olsson JM, Swiezewska E (2000). Single polyprenol and dolichol isolation by semipreparative high-performance liquid chromatography technique. J Lipid Res. 41(7): 1177-80.
Pubmed
II. Tekle M, Bentinger M, Nordman T, Appelkvist EL, Chojnacki T, Olsson JM (2002). Ubiquinone biosynthesis in rat liver peroxisomes. Biochem Biophys Res Commun. 291(5): 1128-33.
Pubmed
III. Xia L, Bjornstedt M, Nordman T, Eriksson LC, Olsson JM (2001). Reduction of ubiquinone by lipoamide dehydrogenase. An antioxidant regenerating pathway. Eur J Biochem. 268(5): 1486-90.
Pubmed
IV. Xia L, Nordman T, Olsson JM, Damdimopoulos A, Bjorkhem-Bergman L, Nalvarte I, Eriksson LC, Arner ES, Spyrou G, Bjornstedt M (2003). The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress. J Biol Chem. 278(4): 2141-6.
Pubmed
V. Nordman T, Bjornstedt M, Olsson JM (2003). Reduction of ubiquinone by glutathione reductase. [Submitted]
I. Carlson T, Skorupinska-Tudek K, Hertel J, Chojnacki T, Olsson JM, Swiezewska E (2000). Single polyprenol and dolichol isolation by semipreparative high-performance liquid chromatography technique. J Lipid Res. 41(7): 1177-80.
Pubmed
II. Tekle M, Bentinger M, Nordman T, Appelkvist EL, Chojnacki T, Olsson JM (2002). Ubiquinone biosynthesis in rat liver peroxisomes. Biochem Biophys Res Commun. 291(5): 1128-33.
Pubmed
III. Xia L, Bjornstedt M, Nordman T, Eriksson LC, Olsson JM (2001). Reduction of ubiquinone by lipoamide dehydrogenase. An antioxidant regenerating pathway. Eur J Biochem. 268(5): 1486-90.
Pubmed
IV. Xia L, Nordman T, Olsson JM, Damdimopoulos A, Bjorkhem-Bergman L, Nalvarte I, Eriksson LC, Arner ES, Spyrou G, Bjornstedt M (2003). The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress. J Biol Chem. 278(4): 2141-6.
Pubmed
V. Nordman T, Bjornstedt M, Olsson JM (2003). Reduction of ubiquinone by glutathione reductase. [Submitted]
Issue date: 2003-03-21
Rights:
Publication year: 2003
ISBN: 91-7349-475-5
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