Redox interaction of Mn-bicarbonate complexes with reaction centres of purple bacteria

It is found that dark reduction of photooxidized primary electron donor P870+ in reaction centres from purple anoxygenic bacteria (two non-sulphur Fe-oxidizing Rhodovulum iodosum and Rhodovulum robiginosum, Rhodobacter sphaeroides R-26 and sulphur alkaliphilic Thiorhodospira sibirica) is accelerated upon the addition of Mn2+ jointly with bicarbonate (30-75 mM). The effect is not observed if Mn2+ and HCO3(-) have been replaced by Mg2+ and HCO2(-), respectively. The dependence of the effect on bicarbonate concentration suggests that formation of Mn2+-bicarbonate complexes, Mn(HCO3)+ and/or Mn(HCO3)2, is required for re-reduction of P870+ with Mn2+. The results are considered as experimental evidence for a hypothesis on possible participation of Mn-bicarbonate complexes in the evolutionary origin of oxygenic photosynthesis in the Archean era.     

Bicarbonate requirement for the water-oxidizing complex of photosystem II

It is well established that bicarbonate stimulates electron transfer between the primary and secondary electron acceptors, Q(A) and Q(B), in formate-inhibited photosystem II; the non-heme Fe between Q(A) and Q(B) plays an essential role in the bicarbonate binding. Strong evidence of a bicarbonate requirement for the water-oxidizing complex (WOC), both O2 evolving and assembling from apo-WOC and Mn2+, of photosystem II (PSII) preparations has been presented in a number of publications during the last 5 years. The following explanations for the involvement of bicarbonate in the events on the donor side of PSII are considered: (1) bicarbonate serves as an electron donor (alternative to water or as a way of involvement of water molecules in the oxidative reactions) to the Mn-containing O2 center; (2) bicarbonate facilitates reassembly of the WOC from apo-WOC and Mn2+ due to formation of the complexes MnHCO3+ and Mn(HCO3)2 leading to an easier oxidation of Mn2+ with PSII; (3) bicarbonate is an integral component of the WOC essential for its function and stability; it may be considered a direct ligand to the Mn cluster; (4) the WOC is stabilized by bicarbonate through its binding to other components of PSII.     

On-line preparation of peroxymonocarbonate and its application for the study of energy transfer chemiluminescence to lanthanide inorganic coordinate complexes.

It has been shown that peroxymonocarbonate ion (HCO(4) (-)) is a potent oxidant. In this study, a flow-injection system was developed in order to prepare on-line HCO(4) (-) ion and the optimum conditions for the on-line preparation of HCO(4) (-) were studied in detail. We used 99% (13)C-enriched NaHCO(3) to examine peroxymonocarbonate by (13)C-NMR at 25 degrees C. An ultra-weak chemiluminescence (CL) was observed after mixing H(2)O(2) and sodium bicarbonate in an organic co-solvent that can accelerate the formation of HCO(4) (-) ion. When lanthanide inorganic coordinate complex, Eu(II)-EDTA, was added into this HCO(4) (-) system, the CL intensity was significantly enhanced. The CL mechanism was investigated by various methods. The experimental results indicate that peroxymonocarbonate oxidizes Eu(II) to Eu(III) and produces singlet oxygen; meanwhile, the energy originating from dimers of singlet oxygen is accepted by the Eu(III)-EDTA(-) complex. The excited Eu(III) ions undergo radiative deactivation and emit CL.     

Thermodynamics of reactions catalyzed by anthranilate synthase

Microcalorimetry and high performance liquid chromatography have been used to conduct a thermodynamic investigation of reactions catalyzed by anthranilate synthase, the enzyme located at the first step in the biosynthetic pathway leading from chorismate to tryptophan. One of the overall biochemical reactions catalyzed by anthranilate synthase is: chorismate(aq) + ammonia(aq) = anthranilate(aq) + pyruvate(aq) + H2O(l). This reaction can be divided into two partial reactions involving the intermediate 2-amino-4-deoxyisochorismate (ADIC): chorismate(aq) + ammonia(aq) = ADIC(aq) + H2O(l) and ADIC(aq) = anthranilate(aq) + pyruvate(aq). The native anthranilate synthase and a mutant form of it that is deficient in ADIC lyase activity but has ADIC synthase activity were used to study the overall ammonia-dependent reaction and the first of the above two partial reactions, respectively. Microcalorimetric measurements were performed on the overall reaction at a temperature of 298.15 K and pH 7.79. Equilibrium measurements were performed on the first partial (ADIC synthase) reaction at temperatures ranging from 288.15 to 302.65 K, and at pH values from 7.76 to 8.08. The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms. For the reaction: chorismate2-(aq) + NH4+(aq) = anthranilate-(aq) + pyruvate-(aq) + H+(aq) + H2O(l), delta rHmo = -(116.3 +/- 5.4) kJ mol-1. For the reaction: chorismate2-(aq) + NH4+(aq) = ADIC-(aq) + H2O(l), K = (20.3 +/- 4.5) and delta rHmo = (7.5 +/- 0.6) kJ mol-1. Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that are pertinent to this branch point of the chorismate pathway. The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength. Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes delta rG'mo under approximately physiological conditions are given.     

Bicarbonate is a native cofactor for assembly of the manganese cluster of the photosynthetic water oxidizing complex. Kinetics of reconstitution of O2 evolution by photoactivation

Assembly of the inorganic core (Mn(4)O(x)Ca(1)Cl(y)) of the water oxidizing enzyme of oxygenic photosynthesis generates O(2) evolution capacity via the photodriven binding and photooxidation of the free inorganic cofactors within the cofactor-depleted enzyme (apo-WOC-PSII) by a process called photoactivation. Using in vitro photoactivation of spinach PSII membranes, we identify a new lower affinity site for bicarbonate interaction in the WOC. Bicarbonate addition causes a 300% stimulation of the rate and a 50% increase in yield of photoassembled PSII centers when using Mn(2+) and Ca(2+) concentrations that are 10-50-fold larger range than previously examined. Maintenance of a fixed Mn(2+)/Ca(2+) ratio (1:500) produces the fastest rates and highest yields of photoactivation, which has implications for intracellular cofactor homeostasis. A two-step (biexponential) model is shown to accurately fit the assembly kinetics over a 200-fold range of Mn(2+) concentrations. The first step, the binding and photooxidation of Mn(2+) to Mn(3+), is specifically stimulated via formation of a ternary complex between Mn(2+), bicarbonate, and apo-WOC-PSII, having a proposed stoichiometry of [Mn(2+)(HCO(3)(-))]. This low-affinity bicarbonate complex is thermodynamically easier to oxidize than the aqua precursor, [Mn(2+)(OH(2))]. The photooxidized intermediate, [Mn(3+)(HCO(3)(-))], is longer lived and increases the photoactivation yield by suppressing irreversible photodamage to the cofactor-free apo-WOC-PSII (photoinhibition).Bicarbonate does not affect the second (rate-limiting) dark step of photoactivation, attributed to a protein conformational change. Together with the previously characterized high-affinity site, these results reveal that bicarbonate is a multifunctional "native" cofactor important for photoactivation and photoprotection of the WOC-PSII complex.     

Transepithelial potential in the Magadi tilapia, a fish living in extreme alkalinity

We investigated the transepithelial potential (TEP) and its responses to changes in the external medium in Alcolapia grahami, a small cichlid fish living in Lake Magadi, Kenya. Magadi water is extremely alkaline (pH = 9.92) and otherwise unusual: titratable alkalinity (290 mequiv L(-1), i.e. HCO(3) (-) and CO(3) (2-)) rather than Cl(-) (112 mmol L(-1)) represents the major anion matching Na(+) = 356 mmol L(-1), with very low concentrations of Ca(2+) and Mg(2+) (<1 mmol L(-1)). Immediately after fish capture, TEP was +4 mV (inside positive), but stabilized at +7 mV at 10-30 h post-capture when experiments were performed in Magadi water. Transfer to 250% Magadi water increased the TEP to +9.5 mV, and transfer to fresh water and deionized water decreased the TEP to -13 and -28 mV, respectively, effects which were not due to changes in pH or osmolality. The very negative TEP in deionized water was attenuated in a linear fashion by log elevations in [Ca(2+)]. Extreme cold (1 vs. 28°C) reduced the positive TEP in Magadi water by 60%, suggesting blockade of an electrogenic component, but did not alter the negative TEP in dilute solution. When fish were transferred to 350 mmol L(-1) solutions of NaHCO(3), NaCl, NaNO(3), or choline Cl, only the 350 mmol L(-1) NaHCO(3) solution sustained the TEP unchanged at +7 mV; in all others, the TEP fell. Furthermore, after transfer to 50, 10, and 2% dilutions of 350 mmol L(-1) NaHCO(3), the TEPs remained identical to those in comparable dilutions of Magadi water, whereas this did not occur with comparable dilutions of 350 mmol L(-1) NaCl-i.e. the fish behaves electrically as if living in an NaHCO(3) solution equimolar to Magadi water. We conclude that the TEP is largely a Na(+) diffusion potential attenuated by some permeability to anions. In Magadi water, the net electrochemical forces driving Na(+) inwards (+9.9 mV) and Cl(-) outwards (+3.4 mV) are small relative to the strong gradient driving HCO(3) (-) inwards (-82.7 mV). Estimated permeability ratios are P (Cl)/P (Na) = 0.51-0.68 and [Formula: see text] = 0.10-0.33. The low permeability to HCO(3) (-) is unusual, and reflects a unique adaptation to life in extreme alkalinity. Cl(-) is distributed close to Nernst equilibrium in Magadi water, so there is no need for lower P (Cl). The higher P (Na) likely facilitates Na(+) efflux through the paracellular pathway. The positive electrogenic component is probably due to active HCO(3) (-) excretion.     

Bicarbonate transport mechanisms in the Ambystoma kidney proximal tubule: transepithelial potential measurements

Modes of bicarbonate entry from tubule lumen to cell were examined in isolated Ambystoma proximal tubules, using determinations of transepithelial potential differences (V3). (1) Upon removal of luminal substrate, tubules first equilibrated in bilateral (lumen and bath) 94.72 mM Cl- and 10 mM HCO3- yielded a change in V3 between the experimental and control circumstances of +1.8 mV (delta V3). (2) The identical experiment conducted under the condition of symmetrical 4.72 mM Cl- produced a delta V3 of +7.6 mV. This reduction of luminal and bath Cl- generates an amplification of delta V3 by a factor of 4.4 and reflects a substantial increase in the paracellular Cl- shunt resistance. Ensuing experiments were conducted in bilateral nominally Cl(-)-free solutions and in the absence of luminal substrate. The experimental protocols are divided into several situations where HCO3- is removed from the lumen, bath, or lumen and bath; the HCO3- removal sequences are repeated in the presence of luminal SITS and then after SITS washout. 0.5 mM SITS (4-acetoamido-4-isothiocyanostilbene-2,2'-disulfonate) was applied exclusively to the luminal perfusate. (1) Removal of luminal HCO3- in the absence of SITS produces a delta V3 of -1.9 mV, whereas, in the presence of SITS, the delta V3 measures -1.3 mV. Subsequent removal of luminal HCO3- in the presence of bath HCO3- (in the presence of luminal SITS) yields a delta V3 of -1.0 mV. All of these measurements reflect a decrease in HCO3- current across the basolateral membrane Na+ (HCO3-)n co-transporter; the role of a possible Cl-/Anion- antiport cannot be assessed. (2) Removal of bath HCO3- in the absence of SITS yields a delta V3 of +1.5 mV, whereas, in the presence of SITS, the delta V3 value measures +1.2 mV. Subsequent removal of bath HCO3- in the absence of luminal HCO3- (in the presence of SITS) yields a delta V3 of +0.8 mV. These experiments are consistent with an increase in HCO3- current across the basolateral Na+(HCO3-)n co-transporter, do not rule out the possibility of an apical HCO3- conductance pathway, and diminish the likelihood of an apical Cl-/HCO3- antiport system.     

A novel missense mutation in the sodium bicarbonate cotransporter (NBCe1/SLC4A4) causes proximal tubular acidosis and glaucoma through ion transport defects

In humans and terrestrial vertebrates, the kidney controls systemic pH in part by absorbing filtered bicarbonate in the proximal tubule via an electrogenic Na+/HCO3- cotransporter (NBCe1/SLC4A4). Recently, human genetics revealed that NBCe1 is the major renal contributor to this process. Homozygous point mutations in NBCe1 cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts (Igarashi, T., Inatomi, J., Sekine, T., Cha, S. H., Kanai, Y., Kunimi, M., Tsukamoto, K., Satoh, H., Shimadzu, M., Tozawa, F., Mori, T., Shiobara, M., Seki, G., and Endou, H. (1999) Nat. Genet. 23, 264-266). We have identified and functionally characterized a novel, homozygous, missense mutation (S427L) in NBCe1, also resulting in pRTA and similar eye defects without mental retardation. To understand the pathophysiology of the syndrome, we expressed wild-type (WT) NBCe1 and S427L-NBCe1 in Xenopus oocytes. Function was evaluated by measuring intracellular pH (HCO3- transport) and membrane currents using microelectrodes. HCO3- -elicited currents for S427L were approximately 10% of WT NBCe1, and CO2-induced acidification was approximately 4-fold faster. Na+ -dependent HCO3- transport (currents and acidification) was also approximately 10% of WT. Current-voltage (I-V) analysis reveals that S427L has no reversal potential in HCO3-, indicating that under physiological ion gradient conditions, NaHCO3 could not move out of cells as is needed for renal HCO3- absorption and ocular pressure homeostasis. I-V analysis without Na+ further shows that the S427L-mediated NaHCO3 efflux mode is depressed or absent. These experiments reveal that voltage- and Na+ -dependent transport by S427L-hkNBCe1 is unfavorably altered, thereby causing both insufficient HCO3- absorption by the kidney (proximal RTA) and inappropriate anterior chamber fluid transport (glaucoma).     

Thermodynamics of hydrolysis of oligosaccharides

Microcalorimetry has been used to determine enthalpy changes for the hydrolysis of a series of oligosaccharides. High-pressure liquid chromatography was used to determine the extents of reaction and to check for any possible side reactions. The enzyme glucan 1,4-alpha-glucosidase was used to bring about the following hydrolysis reactions: (A) maltose(aq) + H2O(liq) = 2D-glucose(aq); (B) maltotriose(aq) + 2H2O(liq) = 3D-glucose(aq); (C) maltotetraose(aq) + 3H2O(liq) = 4D-glucose(aq); (D) maltopentaose(aq) + 4H2O(liq) = 5D-glucose(aq); (E) maltohexaose(aq) + 5H2O(liq) = 6D-glucose(aq); (F) maltoheptaose(aq) + 6H2O(liq) = 7D-glucose(aq); (G) amylose(aq) + nH2O(liq) = (n + 1) D-glucose(aq); and (H) panose(aq) + 2H2O(liq) = 3D-glucose(aq); (J) isomaltotriose(aq) + 2H2O(liq) = 3D-glucose(aq). The enzyme beta-fructofuranosidase was used for the reactions: (K) raffinose(aq) + H2O(liq) = alpha-D-melibiose(aq) + D-fructose(aq); and (L) stachyose(aq) + H2O(liq) = o-alpha-D-galactopyranosyl-(1----6)- alpha-o-D-galactopyranosyl-(1----6)-alpha-D-glucopyranose + D-fructose(aq). The results of the calorimetric measurements (298.15 K, 0.1 M sodium acetate buffer, pH 4.44-6.00) are: delta H0A = -4.55 +/- 0.10, delta H0B = -9.03 +/- 0.10, delta H0C = -13.79 +/- 0.15, delta H0D = -18.12 +/- 0.10, delta H0E = -22.40 +/- 0.15, delta H0F = -26.81 +/- 0.20, delta H0H = 1.46 +/- 0.40, delta H0J = 11.4 +/- 2.0, delta H0K = -15.25 +/- 0.20, and delta H0L = -14.93 +/- 0.20 kJ mol-1. The enthalpies of hydrolysis of two different samples of amylose were 1062 +/- 20 and 2719 +/- 100 kJ mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apical electrogenic NaHCO3 cotransport. A mechanism for HCO3 absorption across the retinal pigment epithelium

Intracellular microelectrode techniques and intracellular pH (pHi) measurements using the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were employed to characterize an electrogenic bicarbonate transport mechanism at the apical membrane of the frog retinal pigment epithelium (RPE). Reductions in apical concentrations of both [HCO3]o (at constant Pco2 or pHo) or [Na]o caused rapid depolarization of the apical membrane potential (Vap). Both of these voltage responses were inhibited when the concentration of the other ion was reduced or when 1 mM diisothiocyano-2-2 disulfonic acid stilbene (DIDS) was present in the apical bath. Reductions in apical [HCO3]o or [Na]o also produced a rapid acidification of the cell interior that was inhibited by apical DIDS. Elevating pHi at constant Pco2 (and consequently [HCO3]i) by the addition of apical NH4 (20 mM) produced an immediate depolarization of Vap. This response was much smaller when either apical [HCO3]o or [Na]o was reduced or when DIDS was added apically. These results strongly suggest the presence of an electrogenic NaHCO3 cotransporter at the apical membrane. Apical DIDS rapidly depolarized Vap by 2-3 mV and decreased pHi (and [HCO3]i), indicating that the transporter moves NaHCO3 and net negative charge into the cell. The voltage dependence of the transporter was assessed by altering Vap with transepithelial current and then measuring the DIDS-induced change in Vap. Depolarization of Vap increased the magnitude of the DIDS-induced depolarization, whereas hyperpolarization decreased it. Hyperpolarizing Vap beyond -114 mV caused the DIDS-induced voltage change to reverse direction. Based on this reversal potential, we calculate that the stoichiometry of the transporter is 1.6-2.4 (HCO3/Na).     

The photosystem II-associated Cah3 in Chlamydomonas enhances the O2 evolution rate by proton removal

Water oxidation in photosystem II (PSII) is still insufficiently understood and is assumed to involve HCO(3)(-). A Chlamydomonas mutant lacking a carbonic anhydrase associated with the PSII donor side shows impaired O(2) evolution in the absence of HCO(3)(-). The O(2) evolution for saturating, continuous illumination (R(O2)) was slower than in the wild type, but was elevated by HCO(3)(-) and increased further by Cah3. The R(O2) limitation in the absence of Cah3/HCO(3)(-) was amplified by H(2)O/D(2)O exchange, but relieved by an amphiphilic proton carrier, suggesting a role of Cah3/HCO(3)(-) in proton translocation. Chlorophyll fluorescence indicates a Cah3/HCO(3)(-) effect at the donor side of PSII. Time-resolved delayed fluorescence and O(2)-release measurements suggest specific effects on proton-release steps but not on electron transfer. We propose that Cah3 promotes proton removal from the Mn complex by locally providing HCO(3)(-), which may function as proton carrier. Without Cah3, proton removal could become rate limiting during O(2) formation and thus, limit water oxidation under high light. Our results underlie the general importance of proton release at the donor side of PSII during water oxidation.     

Thermodynamics of the hydrolysis of adenosine 5'-triphosphate to adenosine 5'-diphosphate

The enthalpy of hydrolysis of the enzyme-catalyzed (heavy meromyosin) conversion of adenosine 5'-triphosphate (ATP) to adenosine 5'-diphosphate (ADP) and inorganic phosphate has been investigated using heat-conduction microcalorimetry. Enthalpies of reaction were measured as a function of ionic strength (0.05-0.66 mol kg-1), pH (6.4-8.8), and temperature (25-37 degrees C) in Tris/HCl buffer. The measured enthalpies were adjusted for the effects of proton ionization and metal ion binding, protonation and interaction with the Tris buffer, and ionic strength effects to obtain a value of delta H0 = -20.5 +/- 0.4 kJ mol-1 at 25 degrees C for the process, ATP4-(aq) + H2O(l) = ADP3-(aq) + HPO2-4(aq) + H+(aq) where aq is aqueous and l is liquid. Heat measurements carried out at different temperatures lead to a value of delta C0p = -237 +/- 30 J mol-1 K-1 for the above process.     

Thermodynamics of hydrolysis of disaccharides. Lactulose, alpha-D-melibiose, palatinose, D-trehalose, D-turanose and 3-o-beta-D-galactopyranosyl-D-arabinose

High-pressure liquid chromatography and microcalorimetry have been used to study the thermodynamics of the hydrolysis reactions of a series of disaccharides. The enzymes used to bring about the hydrolyses were: beta-galactosidase for lactulose and 3-o-beta-D-galactopyranosyl-D-arabinose; beta-glucosidase for alpha-D-melibiose; beta-amylase for D-trehalose; isomaltase for palatinose; and alpha-glucosidase for D-turanose. The buffer used was sodium acetate (0.02-0.10 M and pH 4.44-5.65). For the following processes at 298.15 K: lactulose(aq) + H2O(liq) = D-galactose(aq) + D-fructose(aq), K0 = 128 +/- 10 and delta H0 = 2.21 +/- 0.10 kJ mol-1; alpha-D-melibiose(aq) + H2O(liq) = D-galactose(aq) + D-glucose(aq), K0 = 123 +/- 42 and delta H0 = -0.88 +/- 0.50 kJ mol-1; palatinose(aq) + H2O(liq) = D-glucose(aq) + D-fructose(aq), delta H0 = -4.44 +/- 1.1 kJ mol-1; D-trehalose(aq) + H2O(liq) = 2 D-glucose(aq), K0 = 119 +/- 10 and delta H0 = 4.73 +/- 0.41 kJ mol-1; D-turanose(aq) + H2O(liq) = D-glucose(aq) + D-fructose(aq), delta H0 = -2.68 +/- 0.75 kJ mol-1; and 3-o-beta-D-galactopyranosyl-D-arabinose(aq) + H2O(liq) = D-galactose(aq) + D- arabinose(aq),0H0 = 107 +/- 10 and delta H0 = 2.97 +/- 0.10 kJ mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

CO2-stimulated NaCl absorption in the mouse renal cortical thick ascending limb of Henle. Evidence for synchronous Na +/H+ and Cl-/HCO3- exchange in apical plasma membranes

These experiments evaluated salt transport processes in isolated cortical thick limbs of Henle (cTALH) obtained from mouse kidney. When the external solutions consisted of Krebs-Ringer bicarbonate (KRB), pH 7.4, and a 95% O2-5% CO2 gas phase, the spontaneous transepithelial voltage (Ve, mV, lumen-to-bath) was approximately mV; the net rate of Cl- absorption (JnetCl) was approximately 3,600 pmols s-1 cm-2; the net rate of osmotic solute absorption Jnetosm was twice JnetCl; and the net rate of total CO2 transport (JnetCO2) was indistinguishable from zero. Thus, net Cl- absorption was accompanied by the net absorption of a monovalent cation, presumably Na+, and net HCO3- absorption was negligible. This salt transport process was stimulated by (CO2 + HCO3- ): omission of CO2 from the gas phase and HCO3- from external solutions reduced JnetCl, Jnetosm, and Ve by 50%. Furthermore, 10(-4) M luminal furosemide abolished JnetCl and Ve entirely. The lipophilic carbonic anhydrase inhibitor ethoxzolamide (10(-4) M, either luminal or peritubular) inhibited (CO2 + HCO3-)-stimulated JnetCl, Jnetosm, and Ve by approximately 50%; however, when the combination (CO2 + HCO3-) was absent, ethoxzolamide had no detectable effect on salt transport. Ve was reduced or abolished entirely by omission of either Na+ or Cl- from external solutions, by peritubular K+ removal, by 10(-3) M peritubular ouabain, and by 10(-4) M luminal SITS. However, Ve was unaffected by 10(-3) M peritubular SITS, or by the hydrophilic carbonic anhydrase inhibitor acetazolamide (2.2 x 10(-4) M, lumen plus bath). We interpret these data to indicate that (CO2 + HCO3-)-stimulated NaCl absorption in the cTALH involved two synchronous apical membrane antiport processes: one exchanging luminal Na+ for cellular H+; and the other exchanging luminal Cl- for cellular HCO3- or OH-, operating in parallel with a (CO2+ HCO3-)-independent apical membrane NaCl cotransport mechanism.     

Bicarbonate is a recycling substrate for cyanase

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as substrate as opposed to activator); the following mechanism was proposed (Anderson, P. M. (1980) Biochemistry 19, 2282-2888; Anderson, P. M., and Little, R. M. (1986) Biochemistry 25, 1621-1626). (formula; see text) Direct evidence for this mechanism was obtained in this study by 1) determining whether CO2 or HCO3- serves as substrate and is formed as product, 2) identifying the products formed from [14C]HCO3- and [14C] OCN-, 3) identifying the products formed from [13C] HCO3- and [12C]OCN- in the presence of [18O]H2O, and 4) determining whether 18O from [18O]HCO3- is incorporated into CO2 derived from OCN-. Bicarbonate (not CO2) is the substrate. Carbon dioxide (not HCO3-) is produced in stoichiometric amounts from both HCO3- and OCN-. 18O from [18O]H2O is not incorporated into CO2 formed from either HCO3- or OCN-. Oxygen-18 from [18O]HCO3- is incorporated into CO2 derived from OCN-. These results support the above mechanism, indicating that decomposition of cyanate catalyzed by cyanase is not a hydrolysis reaction and that bicarbonate functions as a recycling substrate.     

Modeling the acid–base properties of glutathione in different ionic media, with particular reference to natural waters and biological fluids

The acid-base properties of γ-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH) were determined by potentiometry (ISE-H(+), glass electrode) in pure NaI((aq)) and in NaCl((aq))/MgCl(2(aq)), and NaCl((aq))/CaCl(2(aq)) mixtures, at T = 298.15 K and different ionic strengths (up to I(c) ~ 5.0 mol L(-1)). In addition, the activity coefficients of glutathione were also determined by the distribution method at the same temperature in various ionic media (LiCl((aq)), NaCl((aq)), KCl((aq)), CsCl((aq)), MgCl(2(aq)), CaCl(2(aq)), NaI((aq))). The results obtained were also used to calculate the Specific ion Interaction Theory (SIT) and Pitzer coefficients for the dependence on medium and ionic strength of glutathione species, as well as the formation constants of weak Mg(j)H( i )(GSH)((i+2j-3)) and Ca(j)H(i)(GSH)((i+2j-3)) complexes. Direct calorimetric titrations were also carried out in pure NaCl((aq)) and in NaCl((aq))/CaCl(2(aq)) mixtures at different ionic strengths (0.25 ≤ I (c )/mol L(-1) ≤ 5.0) in order to determine the enthalpy changes for the protonation and complex formation equilibria in these media at T = 298.15 K. Results obtained are useful for the definition of glutathione speciation in any aqueous media containing the main cations of natural waters and biological fluids, such as Na(+), K(+), Mg(2+), and Ca(2+). Finally, this kind of systematic studies, where a series of ionic media (e.g., all alkali metal chlorides) is taken into account in the determination of various thermodynamic parameters, is useful for the definition of some trends in the thermodynamic behavior of glutathione in aqueous solution.     

Bicarbonate accelerates assembly of the inorganic core of the water-oxidizing complex in manganese-depleted photosystem II: a proposed biogeochemical role for atmospheric carbon dioxide in oxygenic photosynthesis

The proposed role for bicarbonate (HCO(3)(-)) as an intrinsic cofactor within the water-oxidizing complex (WOC) of photosystem II (PSII) [Klimov et al. (1997) Biochemistry 36, 16277-16281] was tested by investigation of its influence on the kinetics and yield of photoactivation, the light-induced assembly of the functional inorganic core (Mn(4)O(y)Ca(1)Cl(x)) starting from the cofactor-depleted apo-WOC-PSII center and free Mn(2+), Ca(2+), and Cl(-). Two binding sites for bicarbonate were found that stimulate photoactivation by accelerating the formation and suppressing the decay, respectively, of the first light-induced assembly intermediate, IM(1) [apo-WOC-Mn(OH)(2)(+)]. A high-affinity bicarbonate site (K(D) 相似文献   

Facilitating an understanding of integrative physiology: emphasis on the composition of body fluid compartments

As a teaching exercise, we used deductive reasoning and a quantitative analysis to convert a number of facts into a series of concepts to facilitate an understanding of integrative physiology and shed light on the composition of the different body fluid compartments. The starting point was the central need to regenerate ATP to perform biologic work. Because a large quantity of O2 must be delivered to cells at a sufficiently high concentration to aid its diffusion into mitochondria, approximately one third of the O2 in inspired air was extracted; this led to a P(CO2) in arterial blood of 40 mmHg (1 mmHg = 133.322 Pa). Blood flow to individual organs must be adjusted precisely to avoid having too low or too high a P(O2) in mitochondria--the latter augments the formation of reactive O2 species. The extracellular fluid (ECF) bicarbonate concentration (E(HCO3)) must be high to minimize H+ buffering by proteins. This high E(HCO3) sets the ECF concentrations of ionized calcium (Ca2+) and inorganic phosphate (HPO4(2-)) because of solubility issues. Three features defined the intracellular fluid (ICF) volume and composition. First, expelling monovalent anions minimized its mass (volume). Second, controlling the tissue P(CO2) ensured a relatively constant net valence on intracellular proteins. Third, the range of ICF Ca2+ concentrations must both induce regulatory signals and avoid Ca3(PO4)2 formation. All the above were incorporated into the integrated response that optimized the capacity for vigorous exercise.     

Metabolic effects of induced alkalosis during progressive forearm exercise to fatigue.

Metabolic alkalosis induced by sodium bicarbonate (NaHCO(3)) ingestion has been shown to enhance performance during brief high-intensity exercise. The mechanisms associated with this increase in performance may include increased muscle phosphocreatine (PCr) breakdown, muscle glycogen utilization, and plasma lactate (Lac(-)(pl)) accumulation. Together, these changes would imply a shift toward a greater contribution of anaerobic energy production, but this statement has been subject to debate. In the present study, subjects (n = 6) performed a progressive wrist flexion exercise to volitional fatigue (0.5 Hz, 14-21 min) in a control condition (Con) and after an oral dose of NaHCO(3) (Alk: 0.3 g/kg; 1.5 h before testing) to evaluate muscle metabolism over a complete range of exercise intensities. Phosphorus-31 magnetic resonance spectroscopy was used to continuously monitor intracellular pH, [PCr], [P(i)], and [ATP] (brackets denote concentration). Blood samples drawn from a deep arm vein were analyzed with a blood gas-electrolyte analyzer to measure plasma pH, Pco(2), and [Lac(-)](pl), and plasma [HCO(3)(-)] was calculated from pH and Pco(2). NaHCO(3) ingestion resulted in an increased (P < 0.05) plasma pH and [HCO(3)(-)] throughout rest and exercise. Time to fatigue and peak power output were increased (P < 0.05) by approximately 12% in Alk. During exercise, a delayed (P < 0.05) onset of intracellular acidosis (1.17 +/- 0.26 vs. 1.28 +/- 0.22 W, Con vs. Alk) and a delayed (P < 0.05) onset of rapid increases in the [P(i)]-to-[PCr] ratio (1.21 +/- 0.30 vs. 1.30 +/- 0.30 W) were observed in Alk. No differences in total [H(+)], [P(i)], or [Lac(-)](pl) accumulation were detected. In conclusion, NaHCO(3) ingestion was shown to increase plasma pH at rest, which resulted in a delayed onset of intracellular acidification during incremental exercise. Conversely, NaHCO(3) was not associated with increased [Lac(-)](pl) accumulation or PCr breakdown.     

Energetics for oxidation of a bound manganese cofactor in modified bacterial reaction centers

The energetics of a Mn cofactor bound to modified reaction centers were determined, including the oxidation/reduction midpoint potential and free energy differences for electron transfer. To determine these properties, a series of mutants of Rhodobacter sphaeroides were designed that have a metal-ion binding site that binds Mn2+ with a dissociation constant of 1 μM at pH 9.0 (Thielges et al. (2005) Biochemistry 44, 7389-7394). In addition to the Mn binding site, each mutant had changes near the bacteriochlorophyll dimer, P, that resulted in altered P/P+ oxidation/reduction midpoint potentials, which ranged from 480 mV to above 800 mV compared to 505 mV for wild type. The bound Mn2+ is redox active and after light excitation can rapidly reduce the oxidized primary electron donor, P+. The extent of P+ reduction was found to systematically range from a full reduction in the mutants with high P/P+ midpoint potentials to no reduction in the mutant with a potential comparable to wild type. This dependence of the extent of Mn2+ oxidation on the P/P+ midpoint potential can be understood using an equilibrium model and the Nernst equation, yielding a Mn2+/Mn3+ oxidation/reduction midpoint potential of 625 mV at pH 9. In the presence of bicarbonate, the Mn2+/Mn3+ potential was found to be 90 mV lower with a value of 535 mV suggesting that the bicarbonate serves as a ligand to the bound Mn. Measurement of the electron transfer rates yielded rate constants for Mn2+ oxidation ranging from 30 to 120 s(-1) as the P/P+ midpoint potentials increased from 670 mV to approximately 805 mV in the absence of bicarbonate. In the presence of bicarbonate, the rates increased for each mutant with values ranging from 65 to 165 s(-1), reflecting an increase in the free energy difference due to the lower Mn2+/Mn3+ midpoint potential. This dependence of the rate constant on the P/P+ midpoint potential can be understood using a Marcus relationship that yielded limits of at least 150 s(-1) and 290 meV for the maximal rate constant and reorganization energy, respectively. The implications of these results are discussed in terms of the energetics of proteins with redox active Mn cofactors, in particular, the Mn4Ca cofactor of photosystem II.