Abstract
In animals, thiamine deficiency leads to specific brain lesions, generally attributed to decreased levels of thiamine diphosphate, an essential cofactor in brain energy metabolism. However, another far less abundant derivative, thiamine triphosphate (ThTP), may also have a neuronal function. Here, we show that in the rat brain, ThTP is essentially present and synthesized in mitochondria. In mitochondrial preparations from brain (but not liver), ThTP can be produced from thiamine diphosphate and P(i). This endergonic process is coupled to the oxidation of succinate or NADH through the respiratory chain but cannot be energized by ATP hydrolysis. ThTP synthesis is strongly inhibited by respiratory chain inhibitors, such as myxothiazol and inhibitors of the H(+) channel of F(0)F(1)-ATPase. It is also impaired by disruption of the mitochondria or by depolarization of the inner membrane (by protonophores or valinomycin), indicating that a proton-motive force (Deltap) is required. Collapsing Deltap after ThTP synthesis causes its rapid disappearance, suggesting that both synthesis and hydrolysis are catalyzed by a reversible H(+)-translocating ThTP synthase. The synthesized ThTP can be released from mitochondria in the presence of external P(i). However, ThTP probably does not accumulate in the cytoplasm in vivo, because it is not detected in the cytosolic fraction obtained from a brain homogenate. Our results show for the first time that a high energy triphosphate compound other than ATP can be produced by a chemiosmotic type of mechanism. This might shed a new light on our understanding of the mechanisms of thiamine deficiency-induced brain lesions.
Highlights
In the brain, as in most other mammalian tissues, thiamine diphosphate (ThDP) is the most abundant thiamine derivative, amounting to 80 –90% of total thiamine, but several other thiamine derivatives, such as thiamine triphosphate (ThTP), might play physiological roles [3, 4]
Except for adenylate kinase (AK), we have been unable to measure any ThTPsynthesizing activity involving a soluble enzyme, but we have previously shown that when rat brain homogenates were incubated with ThDP, a significant amount of ThTP was detected in the particulate fraction containing cell debris, synaptosomes, and mitochondria [29, 30]
The present study demonstrates that ThTP synthesis occurs in the mitochondrial matrix with ThDP as precursor, but we find, unexpectedly, that the phosphate donor is not ATP or ADP but Pi
Summary
ThTP hydrolysis has been rather well studied, the mechanism of its synthesis remains unclear. At the end of the 1980s, Kawasaki and co-workers (24 –26) demonstrated that adenylate kinase (AK; EC 2.7.4.3) 1 (AK1; a cytosolic form found mainly in skeletal muscle) was able to synthesize ThTP according to the reaction ThDP ϩ ADP % ThTP ϩ AMP. They suggested that this reaction, very slow (kcat ϳ 0.5 minϪ1 at physiological pH) [25] was of physiological significance. ThTP synthesis in brain mitochondria appears to be carried out through an utterly new type of mechanism reminiscent of ATP synthesis, which ceases to be operative when the organelle is disrupted
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