Isolation and Characterization of Thiamin Pyrophosphotransferase from Glycine max Seedlings

Thiamin:ATP pyrophosphotransferase (EC2.7.6.2) activity from soybean (Merr.) seedlings grown for 48 hours was determined by measuring the rate of [2-¹⁴C]thiamin incorporation into thiamin pyrophosphate. With partially purified (11-fold) enzyme, optimal activity occurred between pH 7.1 and 7.3, depending on the buffer system that was used. Assays were routinely conducted at a final pH of 8.1 in order to minimize interference from competing reactions. Enzyme activity required the presence of a divalent cation, and a number of nucleoside triphosphates proved to be active as pyrophosphate donors. Apparent K_m values of 18.3 millimolar and 4.64 micromolar were obtained for Mg·ATP and thiamin, respectively. Among the compounds tested, pyrithiamin and thiamin pyrophosphate were most effective in inhibiting thiamin pyrophosphotransferase activity. Based on Sephadex G-100 gel filtration, soybean thiamin pyrophosphotransferase has a molecular weight of 49,000.     

Nuclear magnetic resonance studies of 5-aminolevulinate demonstrate multiple forms in aqueous solution

5-Aminolevulinic acid (ALA), the common precursor of heme and chlorophyll, can exist in a variety of forms at neutral pH. ¹³C NMR studies of [3-¹³C]ALA, [4-¹³C]ALA, and [5-¹³C]ALA have been used to demonstrate that the predominant species in solution under physiologic conditions is the ketone. The mole fraction of the hydrate is about 0.6%. To further substantiate the existence of the hydrate, ¹³C NMR was used to monitor ¹⁸O exchange at C₄ of [4-¹³C]ALA with H₂, ¹⁸O. Confirmation of the existence of the hydrate was achieved through direct observation by ¹H NMR. The mole fractions of the enol forms of ALA are each below 0.3%. Although direct observation of the enol forms of ALA has not been achieved, enol formation has been indirectly demonstrated by monitoring hydrogen exchange at the C₃ and C₅ methylene groups by ¹H NMR in D₂O. In neutral phosphate buffer, hydrogen exchange occurs readily at both C₃ and C₅ at a ratio of rates of 1:4. In N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid-KOH buffer the hydrogen exchange rates are more than an order of magnitude slower than in phosphate buffer, but the ratio of the exchange rates remains unchanged. The results suggest that phosphate catalyzes enolization at both C₃ and C₅. To evaluate the role of the C₅ substituent in the proton exchange reactions, levulinate and 5-chlorolevulinate (5-CLA) were also monitored for proton exchange at C₃ and C₅. For levulinate, the hydrogen exchange rates in phosphate buffer are two to three orders of magnitude slower than for ALA, and the rate of hydrogen exchange at C₅ is three times slower than hydrogen exchange at C₃. The enolization rate at C₅ of 5-CLA is identical to ALA while enolization at C₃ is about threefold slower for 5-CLA than ALA. These NMR and kinetic studies suggest that under physiologic conditions, ALA rapidly equilibrates between the ketone, the hydrate at C₄, and two or more different enols (C₃---C₄ and C₄---C₅). The alternative forms of ALA may be biologically significant as active site structures for ALA synthase, glutamate semialdehyde transaminase, or porphobilinogen synthase. These NMR studies have also elucidated the structures of condensation products of ALA which can be formed under physiologic conditions. The alternative forms of ALA, as well as the autocondensation products, may serve as the active toxin in porphyrias characterized by elevated ALA levels (e.g., lead poisoning).     

NMR Studies of the Tautomerism in Pseudoisocytidine

Abstract

The structure of pseudoisocytidine may have two isomers. We would like to designate them as K1 and K3 for N₁H and N₃H, respectively. The authenticity of these two isomers was judged by ¹H NMR. The chemical shift value of N₁H in K1 is found more upfield than N₃H in K3, whereas the chemical shifts of rest protons remain the same. Theoretical calculations show that K1 is less stable than K3 by ca. 9 Kcal/mol in gas phase while a methyl group replaces the furanose moiety. This energy reduces as low as 2 Kcal/mol in solution depending on the polarity of the solvent. Thus, the equilibrium of two tautomers occurs most likely in solution. The ¹H and ¹³C NMR studies have been carried out in the pH range of 1 to 12. The pKa's of deprotonation of N₁ and N₃ sites are found to be 9.36 and 9.42, for K1 and K3, respectively. On the other hand, the pKa's of protonation of the same sites corresponding to these two isomers are 3.79 and 3.69, respectively. A critical analysis of line broadening of C₂ in K1 and K3 in the pH range of 5 to 7 establishes the proton exchange phenomenon. The exchange rate, catalyzed by both [H⁺] and [OH^?], depends on the pH.     

On the mechanism of action of thiamin enzymes, Crystal structure of 2-(α-hydroxyethyl)thiamin pyrophosphate (HETPP). Complexes of HETPP with zinc(II) and cadmium(II)

The crystal structure of the 2-(α-hydroxethyl) thiamin pyrophosphate (LH₂) was solved by X-ray diffraction. Crystallographic data: space group F2dd, a=7.922(4) Å, b=33.11(2) Å, c=36.232(10) Å, V=9503(9) ų, z=16. Metal complexes of the general formula K₂{[M(LH)Cl₂]₂} (M=Zn²⁺, Cd²⁺) were isolated from methanolic solutions and characterized by elemental analysis, IR, Raman, and ¹³C CP MAS NMR spectra. They were also characterized by ¹³C NMR, ³¹P NMR, ¹¹³Cd NMR, ES-MS, and ¹H NMR ROESY spectra in D₂O solutions. The data provide evidence for the bonding of the metals to the N(1′) atom of the pyrimidine ring and to the pyrophosphate group. The free ligand and the metal-coordinated ligand adopt the S conformation. Since thiamin cofactor, substrate, and metal ions are present in our system, the extracted results directly refer to thiamin catalysis and possible functional implications are correlated and discussed.     

Hydrogen exchange of the tryptophan residues in bovine alpha-lactalbumin studied by UV spectroscopy

The effect of Ca²⁺ ion on structural fluctuation of a milk Ca²⁺-binding protein, α-lactalbumin, under native conditions was investigated by comparing hydrogen-exchange reactions of tryptophan residues in the apo-form without Ca²⁺ and in the holo-form at 1 mM CaCl₂ at pH 7.0 in the presence of 0.1M Na⁺. The reactions were followed by measuring time-dependent absorption changes at 298–300 nm due to the ²H-¹H exchange of the tryptophan imino protons and were found to be biphasic under all the conditions examined. Two of the four tryptophan protons are insensitive to Ca²⁺ concentration and show a relatively fast exchange rate. The other two protons are much more extensively protected (a protection degree of 10³–10⁵) and are markedly affected by the presence of Ca²⁺. Examinations of the temperature dependence and pH dependence of the individual exchange rates have been utilized for elucidating the exchange mechanism. The fast protons show a low activation energy reaction with so-called EX₂ kinetics. The exchange reaction of the slow protons is accompanied by a high activation energy, and the exchange mechanism of the protons depended on the presence or absence of stabilizing Ca²⁺ ions—the EX₁ kinetics for the apo-protein and the EX₂ kinetics for the holo-protein at 1 mM Ca²⁺. The exchange reaction in the thermally unfolded state was also found to be biphasic, but the fast phase, which has an exchange rate in the fully exposed state, becomes predominant with decreasing temperature. By taking this fact and using a structural unfolding model of hydrogen exchange, the present results are fully consistent with thermodynamic parameters of the thermal transition and kinetic parameters of refolding reactions induced by concentration jumps of guanidine hydrochloride obtained in previous studies. It is demonstrated that the reaction of the slow protons in the native state is mediated by a transient global unfolding equivalent to the “thermal” unfolding under a native condition and that switching of the exchange mechanism from the EX₁ to EX₂ kinetics results from acceleration of the refolding rate with an increase in Ca²⁺ concentration. The transient global unfolding takes place even under a strongly native condition, e.g., at a temperature 20° below the beginning of the thermal transition.     

A 31P and 23Na NMR and terbium(III) luminescence study of bistriphosphato-lanthanide(III) complexes including the cation shift reagent [Dy(PPP)2]7−

Measurement of the ³¹P NMR spectra of the cation shift reagent, dysprosium bistriphosphate, [Dy- (PPP)₂]⁷⁻, shows that bound and free ligand are in slow exchange at pH 6.75. As the pH is decreased the exchange rate increases but the central phosphate group is still coordinated at pH 2.1. The ³¹P spectra were measured for eleven other lanthanide bistriphosphate complexes and the contact and dipolar contributions to the shift were separated, enabling geometrical information to be obtained from the latter.The association constants for Na⁺ and limiting shift were obtained from the ²³Na spectra in the presence of six [Ln(PPP)₂]⁷⁻ complexes, the dipolar mechanism dominates, but there is a small contact shift. The observed shift decreases with pH, depending approximately on a group with pK_a 6.3, one of the phosphate residues.The hydration of [Tb(PPP)₂]⁷⁻, determined from measurements of the luminescence lifetimes in H₂O and D₂O, was found to increase from 2 to 4 as the pH decreased from 9 to 3, a rough parallel with the decrease in ²³Na shift is noted. These results, together with the geometrical information from the dipolar shifts, allow an 8 coordinate structure to be proposed for the dysprosium bistriphosphate complex which possesses a pseudo-axial binding site for Na⁺, accounting for the large shift and other observations.     

Thiamin transport in Escherichia coli: the mechanism of inhibition by the sulfhydryl-specific modifier N-ethylmaleimide

Active transport of thiamin (vitamin B₁) into Escherichia coli occurs through a member of the superfamily of transporters known as ATP-binding cassette (ABC) transporters. Although it was demonstrated that the sulfhydryl-specific modifier N-ethylmaleimide (NEM) inhibited thiamin transport, the exact mechanism of this inhibition is unknown. Therefore, we have carried out a kinetic analysis of thiamin transport to determine the mechanism of inhibition by NEM. Thiamin transport in vivo exhibits Michaelis-Menten kinetics with K_M=15 nM and V_max=46 U mg⁻¹. Treatment of intact E. coli KG33 with saturating NEM exhibited apparent noncompetitive inhibition, decreasing V_max by approximately 50% without effecting K_M or the apparent first-order rate constant (k_obsd). Apparent noncompetitive inhibition is consistent with an irreversible covalent modification of a cysteine(s) that is critical for the transport process. A primary amino acid analysis of the subunits of the thiamin permease combined with our kinetic analysis suggests that inhibition of thiamin transport by NEM is different from other ABC transporters and occurs at the level of protein-protein interactions between the membrane-bound carrier protein and the ATPase subunit.     

Kinetics of thiamin cleavage by sulphite

Results are presented on the rate of thiamin cleavage by sulphite in aqueous solutions as affected by temperature (20–70°), pH(2·5–7·0), and variation of the concentration of either thiamin (1–20μm) or sulphite (10–5000μm as sulphur dioxide). Plots of the logarithm of percentage of residual thiamin against time were found to be linear and cleavage thus was first-order with respect to thiamin. At pH5 the rate was also found to be proportional to the sulphite concentration. In the pH region 2·5–7·0 at 25° the rate constant was 50m⁻¹hr.⁻¹ at pH5·5–6·0, and decreased at higher or lower pH values. The rate of reaction increased between 20° and 70°, indicating a heat of activation of 13·6kcal./mole.     

Ionisations within a subtilisin–glyoxal inhibitor complex

Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of subtilisin with a K_i=2.3±0.2 μM at pH 7.0 and 25 °C. Using Z-Ala-Pro-[2-¹³C]Phe-glyoxal we have detected a signal at 107.3 ppm by ¹³C NMR, which we assign to the tetrahedral adduct formed between the hydroxy group of serine-195 and the ¹³C-enriched keto-carbon of the inhibitor. The chemical shift of this signal is pH independent from pH 4.2 to 7.0 and we conclude that the oxyanion pK_a<3. This is the first observation of oxyanion formation in a reversible subtilisin–inhibitor complex. The inhibitor is bound as a hemiketal which is in slow exchange with the free inhibitor. Inhibitor binding depends on a pK_a of ～6.5 in the free enzyme and on a pK_a<3.0 when the inhibitor is bound to subtilisin. Protonation of the oxyanion promotes the disassociation of the inhibitor. We show that oxyanion formation cannot be rate limiting during catalysis and that subtilisin stabilises the oxyanion by at least 45.1 kJ mol^?1. We conclude that if the energy required for oxyanion stabilisation is utilised as binding energy in drug design it should make a significant contribution to inhibitor potency.     

A study on the exchange rate of magnesium with ATP

The exchange process of Mg²⁺ with ATP was found to be, in many cases, dominated by Mg²⁺ exchange between ATP and ATP-Mg (a bimolecular reaction) rather than the Mg²⁺ off-process from ATP-Mg to solutino (a unimolecular reaction). The Mg²⁺ off-rate from ATP-Mg and the rate constant of the bimolecular reaction were determined at 10 and 25°C at pH. 7.3, using ³¹P-NMR at 145.7 MHz. At this resonance frequency intermediate to slow exchange phenomena with respect to the NMR time scale of 2.5·10³ s^?1 were observed in ATP resonances. Various implications of these results to studies of biological systems have been pointed out.     

Reduction of pentavalent antimony by trypanothione and formation of a binary and ternary complex of antimony(III) and trypanothione

Several pentavalent antimony compounds have been used for the treatment of leishmaniasis for decades. However, the mechanism of these antimony drugs still remains unclear. One of their targets is thought to be trypanothione, a major low molecular mass thiol inside the parasite. We show that pentavalent antimony (Sb^V) can be rapidly reduced to its trivalent state by trypanothione at mildly acidic conditions and 310 K (k=4.42 M^–1 min^–1 at pH 6.4), and that Sb^III can be bound to trypanothione to form an Sb^III-trypanothione complex. NMR data demonstrate that Sb^III binds to trypanothione at the two thiolates of the cysteine residues, and that the binding is pH dependent and is strongest at biological pH with a stability constant logK=23.6 at 298 K (0.1 M NaNO₃). The addition of low molecular monothiol ligands such as glutathione and cysteine to the Sb^III-trypanothione complex results in the formation of a ternary complex. Thiolates from both trypanothione and monothiol bind to the Sb^III center. The formation of the ternary complex is important, as the antileishmanial properties of the drugs are probably due to a complex between of Sb^III-trypanothione and enzymes. Although thermodynamically stable, the complex is kinetically labile and the free and bound forms of thiolates exchange on the ¹H NMR timescale. Such a facile exchange may be crucial for the transport of Sb^III within parasites.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations amastigote the parasites culture at pH 5.0 and 310 K to resume the intracellular form - BPR bromopyrogallol - ESI-MS electrospary ionization mass spectrometry - GSH glutathione - pH^* pH meter reading in D₂O without correction for isotope effects - promastigote the parasites culture at pH 7.4 and 298 K to resume the extracellular stage - T(SH)₂ reduced form of trypanothione - T(S-S) oxidized form of trypanothione (disulfide form) - TR trypanothione reductase - tart tartrate     

TmDOTA-tetraglycinate encapsulated liposomes as pH-sensitive LipoCEST agents

Lanthanide DOTA-tetraglycinate (LnDOTA-(gly)₄ ⁻) complexes contain four magnetically equivalent amide protons that exchange with protons of bulk water. The rate of this base catalyzed exchange process has been measured using chemical exchange saturation transfer (CEST) NMR techniques as a function of solution pH for various paramagnetic LnDOTA-(gly)₄ ⁻ complexes to evaluate the effects of lanthanide ion size on this process. Complexes with Tb(III), Dy(III), Tm(III) and Yb(III) were chosen because these ions induce large hyperfine shifts in all ligand protons, including the exchanging amide protons. The magnitude of the amide proton CEST exchange signal differed for the four paramagnetic complexes in order, Yb>Tm>Tb>Dy. Although the Dy(III) complex showed the largest hyperfine shift as expected, the combination of favorable chemical shift and amide proton CEST linewidth in the Tm(III) complex was deemed most favorable for future in vivo applications where tissue magnetization effects can interfere. TmDOTA-(gly)₄ ⁻ at various concentrations was encapsulated in the core interior of liposomes to yield lipoCEST particles for molecular imaging. The resulting nanoparticles showed less than 1% leakage of the agent from the interior over a range of temperatures and pH. The pH versus amide proton CEST curves differed for the free versus encapsulated agents over the acidic pH regions, consistent with a lower proton permeability across the liposomal bilayer for the encapsulated agent. Nevertheless, the resulting lipoCEST nanoparticles amplify the CEST sensitivity by a factor of ∼10⁴ compared to the free, un-encapsulated agent. Such pH sensitive nano-probes could prove useful for pH mapping of liposomes targeted to tumors.     

Concentration-dependent hydrogen exchange kinetics of 3H-labeled S-peptide in ribonuclease S.

The hydrogen exchange kinetics of the S-peptide in ribonuclease S can be measured by first tritiating the S-peptide in the absence of S-protein and then allowing it to recombine rapidly with S-protein. Afterwards the exchange reactions of this specific segment of ribonuclease S can be studied. The exchange kinetics of bound S-peptide are complex, indicating that different protons exchange at markedly different rates. The terminal exchange reaction, involving at least five highly protected protons, has been studied as a function of pH.At low concentrations of ribonuclease S the exchange kinetics become concentration-dependent, owing to the dissociation of the S-peptide. Although the fraction of free S-peptide is always very small, its rate of exchange is several orders of magnitude faster than that of bound S-peptide, and the concentration dependence of the exchange kinetics is readily measurable. It provides a highly sensitive method for determining small dissociation constants (K_D). Values of K_D ranging from 10^?6m at pH 2.7, 0 °C, to 2 × 10^?10m at pH 7.0, 0 °C, are reported here. Our value for K_D at pH 7.0, 0 °C, confirms the data and extrapolation to 0 °C of Hearn et al. (1971).At high concentrations of ribonuclease S the terminal exchange reaction is independent of concentration. It probably results from a local unfolding reaction of the bound S-peptide. Above pH 4 the strong pH dependence of K_D closely resembles that of the apparent equilibrium constant for this local unfolding reaction. The latter may be one step in the dissociation process and we present such a model for ribonuclease S dissociation.Measurement of concentration-dependent exchange kinetics should provide a useful method of determining small dissociation constants in other systems: for example, in studies of protein-nucleic acid interactions.     

An NMR method for studying the kinetics of metal exchange in biomolecular systems

The kinetics of lanthanide (III) exchange for calcium(II) in the C-terminal EF-hand of the protein calbindin D_9khave been studied by one-dimensional (1D) stopped-flow NMR. By choosing a paramagnetic lanthanide (Ce³⁺), kinetics in the sub-second range can be easily measured. This is made possible by the fact that (i) the kinetic behaviour of hyperfine shifted signals can be monitored in 1D NMR and (ii) fast repetition rates can be employed because these hyperfine shifted signals relax fast. It is found that the Ce³⁺-Ca²⁺exchange process indeed takes place on a sub-second timescale and can be easily monitored with this technique. As the rate of calcium-cerium substitution was found not to depend on the presence of excess calcium in solution, the kinetics of the process were interpreted in terms of a bimolecular associative mechanism, and the rate constants extracted. Interestingly, the dissociative mechanism involving the apoform of the protein, which is generally assumed for metal ion exchange at protein binding sites, was not in agreement with our data.     

Effect of thiamin on the content of fructose 2,6-bisphosphate in Euglena

• 1.1. Thiamin deprived Euglena cells contained twice as high a concentration of Fru-2,6-P₂ in sufficient cells and the rate of the uptake of glucose from the medium was twice as great as that found in sufficient cells, indicating that Fru-2,6-P₂ is responsible for the glycolysis.
• 2.2. Moreover, incubation of thiamin deprived cells with increasing thiamin concentrations caused a decrease of Fru-2,6-P₂. The activity of Fru-6-P, 2-kinase was affected by thiamin and the activity of PFK-2 in thiamin deprived cells was 2.3 times greater than that observed in sufficient cells.
• 3.3. Incubating thiamin-deficient cells in a thiamin-containing medium, the activity of Fru-6-P, 2-kinase decreased rapidly and became the same activity as that found in thiamin sufficient cells.
• 4.4. In contrast, the two thiamin dependent 2-OGDC and PNOR activities showed a reverse relationship, being higher in thiamin-sufficient cells and lower in thiamin deficient cells.
• 5.5. We concluded that the carbohydrate metabolism of Euglena is regulated by Fru-2,6-P₂ through an intracellular concentration of thiamin based on the present evidence.

     

Direct assessment of water exchange on a Gd(III) chelate bound to a protein

 The Gd(III) complex of 4-pentylbicyclo[2.2.2]octane-1-carboxyl-di-l-aspartyl-lysine-derived DTPA, [GdL(H₂O)]^2–, binds to serum albumin in vivo, through hydrophobic interaction. A variable temperature ¹⁷O NMR, EPR, and Nuclear Magnetic Relaxation Dispersion (NMRD) study resulted in a water exchange rate of k ²⁹⁸ _ex=4.2×10⁶ s^–1, and let us conclude that the GdL complex is identical to [Gd(DTPA)(H₂O)]^2– in respect to water exchange and electronic relaxation. The effect of albumin binding on the water exchange rate has been directly evaluated by ¹⁷O NMR. Contrary to expectations, the water exchange rate on GdL does not decrease considerably when bound to bovine serum albumin (BSA); the lowest limit can be given as k _{ex, GdL-BSA}=k _ex, GdL / 2. In the knowledge of the water exchange rate for the BSA-bound GdL complex, the analysis of its NMRD profile at 35  °C yielded a rotational correlation time of 1.0 ns, one order of magnitude shorter than that of the whole protein. This value is supported by the longitudinal ¹⁷O relaxation rates. This indicates a remarkable internal flexibility, probably due to the relatively large distance between the protein- and metal-binding moieties of the ligand. Received: 25 June 1998 / Accepted: 11 August 1998     

Effect of pH on H-2H exchange,H2 production and H2 uptake,catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans

(1) The kinetics of isotope exchange catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans have been studied by measuring H²H, H₂ or ²H₂ produced when the enzyme catalyses the exchange between ²H₂ and H₂O or H₂ and ²H₂O. (2) In the ²H₂-H₂O system the measured rate of H₂ production was always higher than that of H²H. The $\text{H}{}_2\text{H}{}^2\text{H}$ ratio remained constant (about 1.70) in the protein concentration range 0.08–1.32 mg. The very rapid formation of H₂ with respect to H²H is consistent with the hypothesis of a heterolytic cleavage of ²H₂ into a deuteron and an enzyme hydride that can exchange with the solvent. (3) In the H₂-²H₂O system, the exchange rate was much lower than in the ²H₂-H₂O system, indicating a marked isotopic effect of ²H₂O. (4) The H-²H exchange activity, determined from the initial velocity of H²H formation, is optimal at pH 4.5. A second maximum of activity is observed at pH 8.3. The pH value of 4.5 is also the pH optimum for H₂ production while at pH 8.3–8.5 there is a maximum of H₂ oxidation activity. (5) In ordinary H₂O the K_m for hydrogen uptake estimated either from H₂ consumption or from benzyl viologen reduction was 0.06–0.07 μM for both H₂ and ²H₂ indicating a strong affinity of the enzyme for hydrogen at pH 8.3–8.5. Shifting from H₂O to ²H₂O does not affect the K_m of the enzyme for H₂ but lowers the V_max value about 10-fold. The K_m for benzyl viologen and methyl viologen was 0.08 and 2 mM, respectively.     

Counterflow efflux of thiamin in Escherichia coli

A resting cell of Escherichia coli lacking thiamin kinase incorporated external thiamin with an energy-dependent counterflow efflux (C-efflux). This C-efflux could be separated from an energy-dependent exit by a selective inhibition of exit by $\text{2 · 10}{}^{\mathrm{?2}}\text{M}$ NaN₃. The extracellular thiamin could be replaced by thiamin diphosphate, resulting in the same rate of C-efflux, but the rate of C-efflux of intracellular thiamin diphosphate against the external thiamin was markedly low. This low rate of C-efflux of thiamin diphosphate could explain the higher accumulation of the compound than that of free thiamin in the thiamin-kinase-defective mutant as well as in its wild-type parent. Basic characteristics of free thiamin uptake and exit in E. coli W mutant were compared with those reported in K 12 mutant: a marked difference existed in the rate of exit. The low rate of exit in E. coli W 70-23-102 was inferred as the reason for the absence of an overshoot phenomenon of thiamin uptake in this strain.     

Control of Cell pH in the T84 Colon Cell Line

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011 pH units/min in the presence of Na⁺, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na⁺ independent H⁺ transport mechanisms in HEPES Ringer. In CO₂/HCO⁻ ₃ Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO₂, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl⁻ or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl⁻/HCO⁻ ₃ exchange. This exchange was not Na⁺ dependent. When Na⁺ was added to cells incubated in 0 Na⁺ Ringer while blocking Na⁺/H⁺ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na⁺/HCO⁻ ₃ cotransport carrying HCO⁻ ₃ into these cells, which was abolished by DIDS. These experiments, thus, show that Na⁺/H⁺ and Cl⁻/HCO⁻ ₃ exchange and Na⁺/HCO⁻ ₃ cotransport participate in cell pH regulation in T84 cells. Received: 3 April 2000/Revised: 22 June 2000     

A proton-deuterium exchange study of three types ofDesulfovibrio hydrogenases

Summary Hydrogenases are among the main enzymes involved in bacterial anaerobic corrosion of metals. The study of their mode of action is important for a full comprehension of this phenomenon. The three types ofDesulfovibrio hydrogenases [(Fe), (NiFe), (NiFeSe)] present different patterns in the pH dependence of their activity. The periplasmic enzyme fromDesulfovibrio salexigens and the cytoplasmic enzyme fromDesulfovibrio baculatus both have pH optima at 7.5 for H₂ uptake and 4.0 for H₂ evolution and H⁺–D₂ exchange reaction (measured by membrane-inlet mass-spectrometry). The H₂ to HD ratio at pH above 5.0 is higher than 1.0. The periplasmic hydrogenase fromD. gigas presents the same pH optimum (8.0) for the H⁺–D₂ exchange as for H₂ consumption. In contrast, the enzyme fromD. vulgaris has the highest activity in H₂ production and in the exchange at pH 5.0. Both hydrogenases have a H₂-to-HD ratio below 1.0.