Pierre Wins

Studies on red-cell ghost ATPase systems: properties of a (Mg2+ + Ca2+)-dependent ATPase.

1. 1. In the presence of Mg2+, Ca2+, Sr2+ and Ba2+ have an activating effect on the ATPase activity of human red-cell ghosts, the greatest activity being obtained with Sr2+ as the activator. 2. 2. The presence of Ca2+ and Sr2+ tends to prevent the activation by Na+ and K+. 3. 3. The activating effects of Mg2+ and Ca2+ appear to differ in their point of attack; Sr2+ and Ba2+ seem to be bound by the same sites as Ca2+. 4. 4. There is an optimal ratio between the concentrations of Mg2+ and Ca2+ (or Sr2); excess Ca2+ (or Sr2+) has an inhibiting effect, presumably by interfering with Mg2+ activation. Excess Mg2+ may also interfere with Ca2+ (or Sr2+) activation. 5. 5. The optimal pH is somewhat higher for the (Na+ + K+)-dependen activity than for the (Mg2+ + Ca2+)-dependent activity and the curves of the activity as a function of pH have different shapes. 6. 6. The (Mg2+ + Ca2+)-dependent activity is inhibited by Salygran, Atebrin and more specifically by sodium Amytal and 2,4-dinitrophenol. In the presence of Mg2+ alone, the ATPase activity is inhibited by Atebrin, Salygran and, more specifically, by guanidin. The (Mg2+ + Na+ + K+)-dependent component is inhibited by Atebrin and Salyrgan but is insenstivie to guanidin and 2,4-dinitrophenol. 7. 7. The properties of the (Mg2 + Ca2+)-dependent enzymic system are analogous to those of myofibrillar ATPase; thus, this system may be identified with the actomyosin-like proteins described in red-cell ghosts by Ohnishi. 8. 8. In the presence of 2,4-dinitrophenol in amounts sufficient to inhibit the (Mg2+ + Ca2+)-dependent ATPase, one observes in intact cells a marked increase in the passive permeability to Na+ but no effect on active transport. This suggests that contractile proteins of red cells may be involved in the regulation of passive permeability to Na+.

Thiamine status in humans and content of phosphorylated thiamine derivatives in biopsies and cultured cells.

Background Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines. Methodology and Principal Findings Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines. Conclusions and Significance The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.

Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors

Prokaryotes, yeasts and plants synthesize thiamin (vitamin B1) via complex pathways. Animal cells capture the vitamin through specific high‐affinity transporters essential for internal thiamin homeostasis. Inside the cells, thiamin is phosphorylated to higher phosphate derivatives. Thiamin diphosphate (ThDP) is the best‐known thiamin compound because of its role as an enzymatic cofactor. However, in addition to ThDP, at least three other thiamin phosphates occur naturally in most cells: thiamin monophosphate, thiamin triphosphate (ThTP) and the recently discovered adenosine thiamin triphosphate. It has been suggested that ThTP has a specific neurophysiological role, but recent data favor a much more basic metabolic function. During amino acid starvation, Escherichia coli accumulate ThTP, possibly acting as a signal involved in the adaptation of the bacteria to changing nutritional conditions. In animal cells, ThTP can phosphorylate some proteins, but the physiological significance of this mechanism remains unknown. Adenosine thiamin triphosphate, recently discovered in E. coli, accumulates during carbon starvation and might act as an alarmone. Among the proteins involved in thiamin metabolism, thiamin transporters, thiamin pyrophosphokinase and a soluble 25‐kDa thiamin triphosphatase have been characterized at the molecular level, in contrast to thiamin mono‐ and diphosphatases whose specificities remain to be proven. A soluble enzyme catalyzing the synthesis of adenosine thiamin triphosphate from ThDP and ADP or ATP has been partially characterized in E. coli, but the mechanism of ThTP synthesis remains elusive. The data reviewed here illustrate the complexity of thiamin biochemistry, which is not restricted to the cofactor role of ThDP.

Thiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals

AbstractIn most organisms, the main form of thiamine is the coenzyme thiamine diphosphate. Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain proteins. Here we show that ThTP exists not only in vertebrates but is present in bacteria, fungi, plants and invertebrates. Unexpectedly, we found that in Escherichia coli as well as in Arabidopsis thaliana, ThTP was synthesized only under particular circumstances such as hypoxia (E. coli) or withering (A. thaliana). In mammalian tissues, ThTP concentrations are regulated by a specific thiamine triphosphatase that we have recently characterized. This enzyme was found only in mammals. In other organisms, ThTP can be hydrolyzed by unspecific phosphohydrolases. The occurrence of ThTP from prokaryotes to mammals suggests that it may have a basic role in cell metabolism or cell signaling. A decreased content may contribute to the symptoms observed during thiamine deficiency.

Determination of Thiamin and Its Phosphate Esters in Cultured Neurons and Astrocytes Using an Ion-Pair Reversed-Phase High-Performance Liquid Chromatographic Method

A sensitive method, based on fluorescence detection, for the determination of thiamin derivatives after precolumn derivatization is described. The separation is achieved on a PRP-1 column using ion-pair reversed-phase HPLC. This method is especially well adapted to the detection of thiamin triphosphate in complex mixtures such as tissue extracts. The detection limit for TTP is 50 fmol. The contents of thiamin derivatives were determined in primary cultures of rat cerebellar granule neurons and cerebral astrocytes. The amount of TTP is about five times higher in neurons than in astrocytes. Thus in rat brain TTP seems to be essentially associated with neurons and the intracellular concentration is estimated to be about 0.2 microM. Our results suggest the existence, in nerve cells, of specific regulatory mechanisms not related to the blood-brain barrier and responsible for the maintenance of thiamin homeostasis in brain.

Metabolism of thiamine triphosphate in rat brain : correlation with chloride permeability

Abstract: Our results show that a net synthesis of thiamine triphosphate (TTP) can be demonstrated in vitro using rat brain extracts. The total homogenate was preincubated with thiamine or its diphosphate derivative (TDP), centrifuged, and washed twice. With TDP (1 mM) as substrate, a 10‐fold increase in TTP content was observed in this fraction (nuclear fraction, membrane vesicles). A smaller, but significant, increase was observed in the P2 fraction (mitochondrial/synaptosomal fraction). In view of the low TTP content of our fractions, it was carefully assessed that authentic TTP was being formed. Incorporation of radioactivity from [β‐32P]TDP and [γ‐32P]ATP in TTP suggests that these two compounds are its precursors. Furthermore, TTP synthesis was inhibited by ADP and relatively low concentrations of Zn2+. These results suggest that TTP synthesis is catalyzed by an ATP:TDP transphosphorylase rather than by the cytoplasmic adenylate kinase that may be present in the vesicles. After osmotic lysis of the vesicles at alkaline pH, TTP was recovered in protein‐bound form. Concomitantly, a soluble thiamine triphosphatase, with alkaline pH optimum, was also released from the vesicles. No net synthesis could be obtained in the cytosolic fraction or in detergent‐solubilized systems. Like TTP synthesis, chloride permeability of the vesicles was increased when the homogenate had been incubated with thiamine and particularly with TDP. Our results suggest a regulatory role of TTP on chloride permeability, but the target remains to be characterized.

Thiamine Deficiency in Cultured Neuroblastoma Cells: Effect on Mitochondrial Function and Peripheral Benzodiazepine Receptors

Abstract: When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16‐fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 ± 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high‐affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low‐affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine‐deficient neuroblastoma cells.

Thiamine Deficiency‐Induced Partial Necrosis and Mitochondrial Uncoupling in Neuroblastoma Cells Are Rapidly Reversed by Addition of Thiamine

Abstract: Culture of neuroblastoma cells in a medium of low‐thiamine concentration (6 nM) and in the presence of the transport inhibitor amprolium leads to the appearance of overt signs of necrosis; i.e., the chromatin condenses in dark patches, the oxygen consumption decreases, mitochondria are uncoupled, and their cristae are disorganized. Glutamate formed from glutamine is no longer oxidized and accumulates, suggesting that the thiamine diphosphate‐dependent α‐ketoglutarate dehydrogenase activity is impaired. When thiamine (10 µM) is added to the cells, the O2 consumption increases, respiratory control is restored, and normal cell and mitochondrial morphology is recovered within 1 h. Succinate, which is oxidized via the thiamine diphosphate‐independent succinate dehydrogenase, is also able to restore a normal O2 consumption (with respiratory control) in digitonin‐permeabilized thiamine‐deficient cells. Our results therefore suggest that the slowing of the citric acid cycle is the main cause of the biochemical lesion induced by thiamine deficiency as observed in Wernickes encephalopathy.

Subcellular localization and compartmentation of thiamine derivatives in rat brain

The subcellular distribution of thiamine derivatives in rat brain was studied. Thiamine diphosphate content was highest in the mitochondrial and synaptosomal fractions, and lowest in microsomal, myelin and cytosolic fractions. Only 3-5% of total thiamine diphosphate was bound to transketolase, a cytosolic enzyme. Thiamine triphosphate was barely detectable in the microsomal and cytosolic fraction, but synaptosomes were slightly enriched in this compound compared to the crude homogenate. Both myelin and mitochondrial fractions contained significant amounts of thiamine triphosphate. In order to estimate the relative turnover rates of these compounds, the animals received an intraperitoneal injection of either [14C]thiamine or [14C]sulbutiamine (isobutyrylthiamine disulfide) 1 h before decapitation. The specific radioactivities of thiamine compounds found in the brain decreased in the order: thiamine > thiamine triphosphate > thiamine monophosphate > thiamine diphosphate. Incorporation of radioactivity into thiamine triphosphate was more marked with [14C]sulbutiamine than with [14C]thiamine. The highest specific radioactivity of thiamine diphosphate was found in the cytosolic fraction of the brain, though this pool represents less than 10% of total thiamine diphosphate. Cytosolic thiamine diphosphate had a twice higher specific radioactivity when [14C]sulbutiamine was used as precursor compared with thiamine though no significant differences were found in the other cellular compartments. Our results suggest the existence of two thiamine diphosphate pools: the bound cofactor pool is essentially mitochondrial and has a low turnover; a much smaller cytosolic pool (6-7% of total TDP) of high turnover is the likely precursor of thiamine triphosphate.

Astroglial Cells Express Large Amounts of GABAA Receptor Proteins in Mature Brain

Abstract: GABAA receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high‐affinity binding sites for [3H]flunitrazepam and other “central‐type” benzodiazepine ligands. The amount of specific [3H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [3H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABAA receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin‐like convulsants were characterized. Allosteric interactions between the different sites were typical of central‐type GABAA receptors. The presence of α‐subunit(s), as revealed by [3H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mass 51–53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABAA receptors are extrasynaptic and expressed in astroglia.