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E Schoffeniels

Publications and source records attributed to E Schoffeniels.

At least 19 recordsLinked to original sources
PubMed1991

Solubilization of thiamine triphosphatase from the electric organ of Electrophorus electricus.

The membrane-associated, anion-regulated thiamine triphosphatase from Electrophorus electricus electric organ can be solubilized by various neutral detergents. Polyoxyethylene ethers are the most effective. Anionic detergents readily inactivate the enzyme. A 6.4-fold increase in specific activity is obtained by successive treatment of crude membranes with octanoyl-N-methylglucamide, which solubilized other proteins, and Lubrol-PX with releases 60% of the thiamine triphosphatase (TTPase) activity. Solubilization by Lubrol-PX rapidly modifies kinetic parameters. The Km, Vmax and pH optimum are decreased. However, the solubilized TTPase may be kept at 0 degrees C for many hours without further change in specific activity. At 35 degrees C, the half-life is still 83 min at pH 5.0, but denaturation becomes rapid at pH greater than or equal to 7. Solubilization modifies anion effects on TTPase activity. The activating effect of nitrate is nearly lost, while inhibition by sulfate is no longer time-dependent.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

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.

Animals↗
PubMed1990

Regulation of ion uptake in membrane vesicles from rat brain by thiamine compounds.

We examined the effects of thiamine derivatives on ion uptake in rat brain membrane vesicles. Thiamine triphosphate (1 mM) and pyrithiamine (0.1 mM) increase chloride uptake. Preincubation of crude homogenate with thiamine or pyrithiamine increases chloride uptake while oxythiamine has the reverse effect. Thiamine and oxythiamine also affect 22Na+ and 86Rb+ uptake in the same way as for 36Cl- but to a lesser extent. Thiamine-dependent 36Cl- uptake is activated by sodium bicarbonate (10 mM) and partially inhibited by bumetanide (0.1 mM) and 2,4-dinitrophenol (0.1 mM). Preincubation with thiamine increases the thiamine triphosphate content of the vesicles. The hypothesis that TTP is the activator of a particular chloride uptake mechanism is discussed.

Animals↗
PubMed1990

Molecular aspects of human brain sodium channel.

The sodium channel content of human brain was measured by tritiated tetrodotoxin specific binding. After solubilization, the sodium channel was submitted to chromatography on diethylaminoethyl(cellulose) Sephadex, hydroxylapatite and wheat germ agglutinin sepharose. An increase of tritiated tetrodotoxin binding specific activity was subsequently observed. Eluted sodium channels from wheat germ agglutinin sepharose were overlaid on a sucrose gradient. Electrophoretical analysis of the material obtained after the sedimentation step revealed two co-purified peptides, alpha (Mr = 275,000 mol. wt) and beta (Mr = 30,000-36,000 mol. wt.). Alpha showed an exceptionally high free electrophoretic mobility, which is a common feature for all sodium channels previously described. However, the high denaturation rate of the solubilized tetrodotoxin receptor site 1 did not allow tetrodotoxin receptor quantification by the tritiated toxin binding in sucrose fractions. Sodium channel effective reconstitution in liposomes was demonstrated: (1) 22Na+ influx in proteoliposomes was sensitive to sodium channel-specific neurotoxins: (2) reconstituted proteins showed a cation selectivity similar to that previously described for animal sodium channels. The sodium channel preparation obtained after four chromatographic steps shows two peptides on the electrophoretic analysis. Reconstituted sodium channels displayed some physiological properties found in intact conducting membranes.

Brain↗
PubMed1989

Phosphorylated thiamine derivatives and cortical activity in the baboon Papio papio: effect of intermittent light stimulation.

The effect of intermittent light stimulation (ILS) on the distribution of thiamine derivatives in three brain areas (occipital, motor, and premotor) was compared in photosensitive and nonphotosensitive baboons. ILS induces paroxysmal discharges in the motor and premotor areas of photosensitive animals only. In baboons submitted to ILS, thiamine triphosphate (TTP) decreases in both photosensitive and nonphotosensitive animals; thiamine monophosphate (TMP) increases in photosensitive animals, which present ILS-induced paroxysmal discharges, whereas it is unaffected in nonphotosensitive animals. The variations are the most significant in the occipital (visual) cortex. A consumption of TTP may result from electrical activity induced by light stimulation in the occipital area. No correlation between ILS-induced paroxysmal activity and a decrease in TTP contents was found. However, photosensitive animals are affected differently from nonphotosensitive animals, as their content of TMP in the cerebral cortex increases on stimulation. However, as long as the exact role of thiamine compounds in relation to membrane excitability in the nervous system remains unknown, it is impossible to conclude whether the differences observed in the metabolism of thiamine compounds are the cause or the consequence of the photosensitivity in the baboon Papio papio.

Animals↗
PubMed1989

Solubilization of sodium channel from human brain.

[3H]Tetrodotoxin binds to a single class of receptor sites in homogenates of human brain with a KD of 9.1 nM at 0 degree C and a maximal binding capacity of 5.9 pmol/mg of protein. This tetrodotoxin receptor has been solubilized, and several parameters influencing the efficiency of this critical step have been studied. Treatment of brain membranes with 2% (wt/vol) Nonidet P-40 solubilizes up to 38% of the tetrodotoxin receptor sites. The duration of this solubilization step must not exceed 15 min at an optimal pH of 6.8. The binding activity is most stable when exogenous phosphatidylcholine is added to the soluble receptor with a phosphatidylcholine/detergent ratio of 1:5.

Adult↗
PubMed1989

Thiamine triphosphatase in the membranes of the main electric organ of Electrophorus electricus: substrate-enzyme interactions.

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate (TTP). Membrane fractions prepared from this tissue contain a thiamine triphosphatase that is strongly activated by anions and irreversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion transport inhibitor. Kinetic parameters of the enzyme are markedly affected by the conditions of enzyme preparation: In crude membranes, the apparent Km is 1.8 mM and the pH optimum is 6.8, but trypsin treatment of these membranes or their purification on a sucrose gradient decreases both the apparent Km (to 0.2 mM) and the pH optimum (to 5.0). Anions such as NO3- (250 mM) have the opposite effect, i.e., even in purified membranes, the pH optimum is now 7.8 and the Km is 1.1 mM; at pH 7.8, NO3- increases the Vmax 24-fold. TTP protects against inhibition by DIDS, and the KD for TTP could be estimated to be 0.25 mM, a value close to the apparent Km measured in the same purified membrane preparation. Thiamine pyrophosphate (0.1 mM) did not protect against DIDS inhibition. At lower (10(-5)-10(-6) M) substrate concentrations, Lineweaver-Burk plots of thiamine triphosphatase activity markedly deviate from linearity, with the curve being concave downward. This suggests either anticooperative binding or the existence of binding sites with different affinities for TTP. The latter possibility is supported by binding data obtained using [gamma-32P]TTP. Our data suggest the existence of a high-affinity binding site (KD of approximately 0.5 microM) for the Mg-TTP complex.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

[Molecular aspects of bioelectrogenesis].

The action potential is a dissipative process producing entropy and using free energy. This is well demonstrated by: 1) the evolution of the Na conductance under voltage clamping conditions, 2) the microcalorimetric measurements, 3) the analysis of heat evolution during the conductance changes. The most appropriate explanation must involve an exogenous energy source since the energy dissipated by the ionic flows or even the applied stimulus depolarization are far too small to account for the overall energy balance. Thiamine triphosphate is a likely candidate as specific operating substance. The more so, since it is specifically hydrolyzed by a triphosphatase the activity of which is modulated by various anions. It is therefore suggested that the control of the Cl-permeability, a process requiring the hydrolysis of thiamine triphosphate, is the key to our understanding of the energetics of the action potential.

Action Potentials↗
PubMed1988

Thiamine triphosphatase from Electrophorus electric organ is anion-dependent and irreversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'disulfonic acid.

Thiamine triphosphatase (TTPase) from membranes isolated from the main electric organ of E. electricus is activated about 8 fold by NO3-, I- and SCN- while SO42- is inhibitory. Activating anions shift the pH optimum of the enzyme from 5.0 to 8.0. The enzyme is irreversibly inactivated by low concentrations of 4,4'-diisothiocyano-2,2' disulfonic acid (DIDS), an inhibitor of anion transport. Anions protect from DIDS inactivation. These and other results suggest that the membrane-bound TTPase activity is tightly controlled, possibly through mechanisms involving anion transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Synthesis of [gamma-32P]thiamine triphosphate.

We developed a novel chemical synthesis of thiamine triphosphate which allows us to incorporate 32P in the gamma position. The reaction is based on the condensation of [32P]orthophosphoric acid and thiamine diphosphate in the presence of ethyl chloroformate. After purification by two ion-exchange purification steps, the thiamine derivative has a specific radioactivity of 10 Ci/mmol. The average final yield synthesis is about 10%.

Chromatography, Ion Exchange↗
PubMed1987

Thiamine triphosphate and membrane-associated thiamine phosphatases in the electric organ of Electrophorus electricus.

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate, which represents 87% of the total thiamine content in this tissue. The thiamine pyrophosphate concentration, however, is very low in the eel electric organ and skeletal muscle as compared with other eel or rat tissues. Furthermore, electroplax membranes contain a whole set of enzymes responsible for the dephosphorylation of thiamine tri-, pyro- and monophosphate. Thiamine triphosphatase has a pH optimum of 6.8 and is dependent on Mg2+. The real substrate of the enzyme is probably a 1:1 complex of Mg2+ and thiamine triphosphate. Thiamine pyrophosphatase is activated by Ca2+. The apparent Km for thiamine triphosphate and Vmax are found to be, respectively, 1.76 mM and 5.95 nmol/mg of protein/min. Thiamine triphosphatase activity is inhibited at physiological K+ concentrations (up to 90 mM) and increasing Na+ concentrations (50% inhibition at 300 mM). ZnCl2 (10 mM) inhibits 90% of the enzyme activity. ATP and ITP are also strongly inhibitory. No significant effect of neurotoxins is seen. Membrane-associated thiamine triphosphatase is affected differently by proteolytic enzymes and is partially inactivated by pretreatment with phospholipase C and neuraminidase. The physiological significance of thiamine triphosphatase is discussed in relation to a specific role of thiamine in the nervous system.

Animals↗