A hydroxide ion carrier in planar phospholipid bilayer membranes: (C6F5)2Hg (dipentafluorophenylmercury)

Although a number of molecules are known to function as current-carrying proton carriers across lipid bilayer membranes, no such hydroxide ion carriers have been found to date. We report that (C₆F₅)₂ Hg, which can function as a chloride ion carrier, can also carry a hydroxide ion. In 100 mM Na₂SO₄ solutions, membranes treated with (C₆F₅)₂Hg are almost ideally selective for H⁺/OH⁻ between pH 6.0 and 9.5. Membrane conductance varies linearly with [OH⁻] over this pH range and with the square of the (C₆F₅)₂Hg concentration. The presumed current-carrying species is the dimer [(C₆F₅)₂Hg]₂OH⁻, which, along with the neutral molecule (C₆F₅)₂Hg, shuttles back and forth within the bilayer. In 0.2 M NaCl at pH 9.5, the OH⁻ and Cl⁻ conductances are approximately equal. Thus, the carrier displays an approximately 10⁴-fold preference for OH⁻ over Cl⁻.     

Proton/hydroxide conductance through phospholipid bilayer membranes: effects of phytanic acid

Mechanisms of proton/hydroxide conductance (GH/OH) were investigated in planar (Mueller-Rudin) bilayer membranes made from decane solutions of phospholipids or phospholipids plus phytanic acid (a 20-carbon, branched chain fatty acid). At neutral pH, membranes made from diphytanoylphosphatidylcholine or bacterial phosphatidylethanolamine had GH/OH values in the range of (2-5) X 10(-9) S X cm-2, corresponding to H+/OH- 'net' permeabilities of about (0.4-1.0) X 10(-5) cm X s-1. GH/OH was inhibited by serum albumin, phloretin, glycerol and low pH, but was increased by chlorodecane and voltage greater than 80 mV. Water permeability and GH/OH were not correlated, suggesting that water and H+/OH- cross the membrane by separate pathways. Addition of phytanic acid to the phospholipids caused an increase in GH/OH which was proportional to the first power of the phytanic acid concentration. In membranes containing phytanic acid, GH/OH was inhibited by albumin, phloretin, glycerol and low pH, but was increased by chlorodecane and voltages greater than 80 mV. The results suggest that phytanic acid acts as a simple (A- type) proton carrier. The qualitative similarities between the behavior of GH/OH in unmodified and phytanic-acid containing membranes suggest that phospholipids may contain weakly acidic contaminants which cause most of GH/OH at pH greater than 4. However, there is also a significant background (pH independent) GH/OH which may be due to hydrogen-bonded water chains. The ability of phytanic acid to act as a proton carrier may help to explain the toxicity of phytanic acid in Refsum's disease, a metabolic disorder in which phytanic acid accumulates to high levels in plasma, cells and tissues.     

Lactic acid secretion by human neutrophils. Evidence for an H+ + lactate- cotransport system.

The pathway by which L-lactate (Lac) crosses the plasma membrane of isolated human neutrophils was investigated. The influx of [14C]Lac from a 2 mM Lac, 145 mM Cl-, 5.6 mM glucose medium was approximately 1.5 meq/liter of cell water.min and was sensitive to the organomercurial agent mersalyl (apparent Ki approximately 20 microM), to alpha-cyano-4-hydroxycinnamate (CHC), the classical inhibitor of monocarboxylate transport in mitochondria, and to UK-5099 (apparent Ki approximately 40 microM), a more potent analogue of CHC. Transport was also strongly blocked (greater than 80%) by 1 mM of either 3,5-diiodosalicylic acid, MK-473 (an indanyloxyacetate derivative), or diphenyl-amine-2-carboxylate, and by 0.4 mM pentachlorophenol, but not by 1 mM ethacrynic acid, furosemide, or the disulfonic stilbenes SITS or H2DIDS. One-way [14C]Lac efflux from steady-state cells amounted to approximately 6 meq/liter.min and was likewise affected by the agents listed above. Influx, which was membrane potential insensitive and Na+ independent, displayed a strong pH dependence: extracellular acidification enhanced uptake while alkalinization inhibited the process (pK' approximately 5.7 at 2 mM external Lac). The rate of [14C]Lac influx was a saturable function of external Lac, the Km being approximately 7 mM. Steady-state cells exhibited an intracellular Lac content of approximately 5 mM and secreted lactic acid into the bathing medium a a rate of approximately 4 meq/liter.min. Secretion was completely suppressed by 1 mM mersalyl which inactivates the carrier, leading to an internal accumulation of Lac. That the Lac carrier truly mediates an H+ + Lac- cotransport (or formally equivalent Lac-/OH- exchange) was documented by pH-stat techniques wherein an alkalinization of poorly buffered medium could be detected upon the addition of Lac; these pH changes were sensitive to mersalyl. Thus, the Lac carrier of neutrophils possesses several features in common with other monocarboxylate transport systems in erythrocytes and epithelia.     

Hg2+ and Cu+ are ionophores, mediating Cl-/OH- exchange in liposomes and rabbit renal brush border membranes.

Organometals, including organomercurials, are capable of mediating Cl-/OH- exchange across lipid membranes by forming neutral ion pairs. In this study, the ability of inorganic metals to catalyze Cl-/OH- exchange was examined. In the presence of an inwardly directed chloride gradient, HgCl2 at concentrations as low as 30 nM resulted in quenching of acridine orange fluorescence in liposomes, indicating liposomal acidification. In the presence of the reducing agent, ascorbate, CuSO4 at concentrations as low as 0.6 microM also mediated chloride-dependent liposomal acidification. Copper in the absence of ascorbate, iron (with or without ascorbate), cobalt, cadmium, zinc, nickel, and lead were without an effect. 36Cl efflux from rabbit renal brush border membrane vesicles was also markedly stimulated by micromolar concentrations of mercury or copper plus ascorbate. Vesicle integrity was not altered by the concentrations of mercury or copper employed in these studies. In the absence of ascorbate, CuCl stimulated chloride efflux only under anaerobic conditions, confirming that it is the reduced form of copper (Cu+) that mediates chloride transport across the membrane. In the presence of mercury or reduced copper, an inside alkaline pH gradient stimulated the uphill accumulation of 36Cl and 82Br, respectively, confirming Cl-/OH- exchange. Studies in liposomes and brush border membranes demonstrate that this is an electroneutral process. These results show that Hg2+ and Cu+ are capable of acting as ionophores, mediating electroneutral Cl-/OH- exchange in liposomes and brush border membrane vesicles. This effect could contribute to the toxicity of these two metals.     

Modulation of Cl-/OH- exchange activity in Caco-2 cells by nitric oxide

The present studies were undertaken to determine the direct effects of nitric oxide (NO) released from an exogenous donor, S-nitroso-N-acetyl pencillamine (SNAP) on Cl-/OH- exchange activity in human Caco-2 cells. Our results demonstrate that NO inhibits Cl-/OH- exchange activity in Caco-2 cells via cGMP-dependent protein kinases G (PKG) and C (PKC) signal-transduction pathways. Our data in support of this conclusion can be outlined as follows: 1) incubation of Caco-2 cells with SNAP (500 microM) for 30 min resulted in approximately 50% inhibition of DIDS-sensitive 36Cl uptake; 2) soluble guanylate cyclase inhibitors Ly-83583 and (1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one significantly blocked the inhibition of Cl-/OH- exchange activity by SNAP; 3) addition of 8-bromo-cGMP (8-BrcGMP) mimicked the effects of SNAP; 4) specific PKG inhibitor KT-5823 significantly inhibited the decrease in Cl-/OH- exchange activity in response to either SNAP or 8-BrcGMP; 5) Cl-/OH-exchange activity in Caco-2 cells in response to SNAP was not altered in the presence of protein kinase A (PKA) inhibitor (Rp-cAMPS), demonstrating that the PKA pathway was not involved; 6) the effect of NO on Cl-/OH- exchange activity was mediated by PKC, because each of the two PKC inhibitors chelerythrine chloride and calphostin C blocked the SNAP-mediated inhibition of Cl-/OH- exchange activity; 7) SO/OH- exchange in Caco-2 cells was unaffected by SNAP. Our results suggest that NO-induced inhibition of Cl-/OH- exchange may play an important role in the pathophysiology of diarrhea associated with inflammatory bowel diseases.     

The physiological basis for altered Na+ and Cl- movements across the gills of rainbow trout (Oncorhynchus mykiss) in alkaline (pH = 9.5) water

To test the hypothesis that internal ion imbalances at high pH are caused by altered branchial ion transporting capacity and permeability, radiotracers (24Na+ and 36Cl-) were used to measure ion movements across the gills of intact rainbow trout (Oncorhynchus mykiss) during 3 d exposure to pH 9.5. At control pH (pH 8.0), the trout were in net ion balance, but by 8 h at high pH, 60%-70% reductions in Cl- influx (JClin) and Na+ influx (JNain) led to net Cl- and Na+ losses of -200 micromol kg-1 h-1. Outflux (diffusive efflux plus renal ion losses) was not initially altered. By 72 h, net Cl- balance was reestablished because of a restoration of JClin. Although JNain remained 50% lower at this time, counterbalancing reductions in Na+ outflux restored net Na+ balance. One-substrate ion-uptake kinetics analyses indicated that reduced ion influx after 8 h at pH 9.5 was caused by 50% decreases in Cl- and Na+ maximal transport rates (JClmax, JNamax), likely reflecting decreased numbers of functional transport sites. Two-substrate kinetic analyses indicated that reduced internal HCO-3 and H+ supply for respective branchial Cl-/base and Na+/acid transport systems also contributed to lower JClin and, to a lesser extent, lower JNain at pH 9.5. Recovery of JClmax after 3 d accounted for restoration of Cl- balance and likely reflected increased numbers of transport sites. In contrast, JNamax remained 33% lower after 3 d, but a lower affinity of the gills for Na+ (fourfold greater KNam) accounted for the chronic reduction in Na+ influx at pH 9.5. Thus, reestablishment of Cl- uptake capacity and counterbalancing reductions in Na+ outflux allows rainbow trout to reestablish net ion balance in alkaline waters.     

Triorganotins inhibit the mitochondrial inner membrane anion channel.

The inner membrane of liver and heart mitochondria possesses an anion uniport pathway, known as the inner membrane anion channel (IMAC). IMAC is inhibited by matrix Mg2+, matrix H+, N,N'-dicyclohexycarbodiimide, mercurials and amphiphilic amines such as propranolol. Most of these agents react with a number of different mitochondrial proteins and, therefore, more selective inhibitors have been sought. In this paper, we report the discovery of a new class of inhibitors, triorganotin compounds, which block IMAC completely. One of the most potent, tributyltin (TBT) inhibits malonate uniport via IMAC 95% at 0.9 nmol/mg. The only other mitochondrial protein reported to react with triorganotins, the F1F0ATPase, is inhibited by about 0.75 nmol/mg. The potency of inhibition of IMAC increases with hydrophobicity in the sequence trimethyltin much less than triethyltin much less than tripropyltin less than triphenyltin less than tributyltin; which suggests that the binding site is accessible from the lipid bilayer. It has long been established that triorganotins are anionophores able to catalyze Cl-/OH- exchange; however, TBT is able to inhibit Cl- and NO3- transport via IMAC at doses below those required to catalyze rapid rates of Cl-/OH- exchange. Consistent with previous reports, the data indicate that about 0.8 nmol of TBT per mg of mitochondrial protein is tightly bound and not available to mediate Cl-/OH- exchange. We have also shown that the mercurials, p-chloromercuribenzene sulfonate and mersalyl, which only partially inhibit Cl- and NO3- transport can increase the IC50 for TBT 10-fold. This effect appears to result from a reaction at a previously unidentified mercurial reactive site. The inhibitory dose is also increased by raising the pH and inhibition by TBT can be reversed by S2- and dithiols but not by monothiols.     

Basolateral membrane Na(+)-independent Cl-/HCO3- exchange in the inner stripe of the rabbit outer medullary collecting tubule

The inner stripe of the outer medullary collecting tubule is a major distal nephron segment in urinary acidification. To examine the mechanism of basolateral membrane H+/OH-/HCO3- transport in this segment, cell pH was measured microfluorometrically in the inner stripe of the rabbit outer medullary collecting tubule perfused in vitro using the pH-sensitive fluorescent dye, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. Decreasing peritubular pH from 7.4 to 6.8 (changing [HCO3-] from 25 to 5 mM) caused a cell acidification of 0.25 +/- 0.02 pH units, while a similar luminal change resulted in a smaller cell acidification of only 0.04 +/- 0.01 pH units. Total replacement of peritubular Cl- with gluconate caused cell pH to increase by 0.18 +/- 0.04 pH units, an effect inhibited by 100 microM peritubular DIDS and independent of Na+. Direct coupling between Cl- and base was suggested by the continued presence of peritubular Cl- removal-induced cell alkalinization under the condition of a cell voltage clamp (K(+)-valinomycin). In addition, 90% of basolateral membrane H+/OH-/HCO3- permeability was inhibited by complete removal of luminal and peritubular Cl-. Peritubular Cl(-)-induced cell pH changes were inhibited two-thirds by removal of exogenous CO2/HCO3- from the system. The apparent Km for peritubular Cl- determined in the presence of 25 mM luminal and peritubular [HCO3-] was 113.5 +/- 14.8 mM. These results demonstrate that the basolateral membrane of the inner stripe of the outer medullary collecting tubule possesses a stilbene-sensitive Cl-/HCO3- exchanger which mediates 90% of basolateral membrane H+/OH-/HCO3- permeability and may be regulated by physiologic Cl- concentrations.     

Transport of protons and hydrochloric acid through lipid bilayer membranes

Transport of protons and hydrochloric acid through lipid bilayer membranes was studied by a combination of electrical conductance and pH electrode techniques. In the presence of large pH gradients, proton transport occurs primarily by diffusion of molecular HCl. The permeability of egg phosphatidylcholine/decane bilayers to HCl is about 3 cm . s-1, seven to nine order of magnitude higher than the permeability to H+, OH- or Cl-. The HCl permeability of phosphatidylserine or egg phosphatidylcholine/cholesterol (1 : 1) bilayers is about 50% lower than the permeability of egg phosphatidylcholine bilayers. Diffusion of molecular HCl may be an important process in tissues exposed to high HCl concentrations, e.g., gastric mucosa. However, at neutral pH the diffusion of molecular HCl is too slow to contribute significantly to net movements of H+ or Cl-.     

Effects of pH-induced variations of the charge of the transmembrane alpha-helical peptide Ac-K2(LA)12K2-amide on the organization and dynamics of the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the transmembrane alpha-helical peptide Ac-K(2)(LA)(12)K(2)-amide ((LA)(12)) on the phase transition and dynamics of saturated dimyristoylphosphatidylcholine (DMPC) membranes were investigated at different pH using conventional and saturation-recovery EPR observations of phosphatidylcholine spin labels. At a peptide-to-DMPC ratio of 1/10, the main phase-transition temperature of the DMPC bilayer is decreased by 4.0 degrees C when measured at pH 7.0, by 1.6 degrees C when measured at pH 9.5, and not affected when measured at pH 11.5. This reversible pH effect is due to the subsequent neutralization of the positive charges of lysine side chains at both ends of (LA)(12). Apparent pK(a)s of the lysine side chain amino groups of (LA)(12) in DMPC bilayer are 8.6 and approximately 10.9, as compared with the pK(a) value of 10.5 for these groups when lysine is dissolved in water. Saturation-recovery curves as a function of oxygen concentration using phosphatidylcholine spin labels in DMPC bilayer containing (LA)(12) are always mono-exponential when measured at pH 7.0 and 9.5. This observation is consistent with the hypothesis that the lipid exchange rates among the bulk, boundary, and (LA)(12)-rich regions are faster than 0.5 micros, the electron spin-lattice relaxation time in the presence of molecular oxygen, suggesting that stable oligomers of (LA)(12) do not form. Neutralization of one lysine side chain positive charge on each end of the peptide significantly decreases the ordering effect of (LA)(12) on the lipid hydrocarbon chains, while its effect on the reorientational motion of terminal groups of lipid hydrocarbon chains is rather moderate. It does not affect the local diffusion-solubility product of oxygen measured in the DMPC-(LA)(12) membrane interior.     

Anomalous driving force for renal brush border H+/OH-transport characterized by using 6-carboxyfluorescein

The pH, delta pH, and membrane potential dependences of H+/OH-permeability in renal brush border membrane vesicles (BBMV) were studied by using the entrapped pH indicator 6-carboxyfluorescein (6CF). Quantitative H+/OH-fluxes (JH) were obtained from a calibration of the fluorescence response of 6CF to intravesicular pH using vesicles prepared with varying intravesicular and solution pHs. Intravesicular buffer capacity, determined by titration of lysed vesicles, increased monotonically from 140 to 260 mequiv/L in the pH range 5-8. JH was measured by subjecting voltage-clamped BBMV (K+/valinomycin) to preformed pH gradients over the pH range 5-8 and measuring the rate of change of intravesicular pH. For small preformed pH gradients (0.4 pH unit) JH [6 nequiv s-1 (mg of protein)-1] was nearly independent of pH (5-8), predicting a highly pH dependent H+ permeability coefficient. JH increased in a curvilinear manner from 6 to 104 nequiv s-1 (mg of protein)-1 as delta pH increased from 0.4 to 2.5. JH increased linearly [1.6-7.3 nequiv s-1 (mg of protein)-1] with induced K+ diffusion potentials (21-83 mV) in the absence of a pH gradient. These findings cannot be explained by simple diffusion of H+ or OH- or by mobile carrier models. Two mechanisms are proposed, including a lipid diffusion mechanism, facilitated by binding of H+/OH- to fixed sites in the membrane, and a linear H2O strand model, where dissociation of H2O in the membrane fixes H+ and OH- concentrations in strands, which can result in net H+/OH- transport.     

Effect of ionizing radiation on artificial (planar) lipid membranes. II. The ion carriers valinomycin and nonactin as probes for radiation induced structural changes of the membrane

Planar lipid membranes in the presence of the ion carriers valinomycin or nonactin were irradiated with 14 MeV electrons from a linear accelerator. A large increase of the membrane conductance by up to more than two orders of magnitude was found. The effect is virtually abolished either at high pH, or in the absence of oxygen, or in the presence of the radical scavenger ethanol. A further prerequisite for the effect is the presence of unsaturated fatty acid residues. A kinetic analysis of the carrier transport model based on current-voltage curves and on voltage-jump relaxation experiments was performed as a function of radiation dose. Only the translocation rate constant, kMS, of the charged carrier-ion complex was found to be influenced by irradiation. The effect is interpreted as an increase of the polarity (dielectric constant) of the membrane interior induced by the presence of polar products of lipid peroxidation. A combined action of OH- and HO2-radicals seems to be responsible for the phenomena. At large radiation doses (greater than or equal to 10(3) Gy) a reduction of the membrane conductance was observed. This is interpreted as an increased microviscosity, possibly caused by cross-linking of fatty acid residues. Ion carriers represent sensitive probes of radiation induced membrane damage.     

Electroneutral, HCO3(-)-independent, pH gradient-dependent uphill transport of Cl- by ileal brush-border membrane vesicles. Possible role in the pathogenesis of chloridorrhea.

By applying a rapid filtration technique to isolated brush border membrane vesicles from guinea pig ileum, 36Cl uptake was quantified in the presence and absence of electrical, pH and alkali-metal ion gradients. A mixture of 20 mM-Hepes and 40 mM-citric acid, adjusted to the desired pH with Tris base, was found to be the most suitable buffer. Malate and Mes could be used to replace the citrate, but succinate, acetate and maleate proved to be unsuitable. In the absence of a pH gradient (pHout:pHin = 7.5:7.5), Cl- uptake increased slightly when an inside-positive membrane potential was applied, but uphill transport was never observed. A pH gradient (pHout:pHin = 5.0:7.5) induced both a 400% increase in the initial Cl- influx rate and a long-lasting (20 to 300 s) overshoot, indicating that a proton gradient can furnish the driving force for uphill Cl- transport. Under pH gradient conditions, initial Cl- entry rates had the following characteristics. (1) They were unaffected by cis-Na+ and/or -K+, indicating the absence of Cl-/K+, Cl-/Na+ or Cl-/K+/Na+ symport activity. (2) Inhibition by 20-100 mM-trans-Na+ and/or -K+ occurred, independent of the existence of an ion gradient. (3) Cl- entry was practically unaffected by short-circuiting the membrane potential with equilibrated potassium and valinomycin. (4) Carbonyl cyanide m-chlorophenylhydrazone was strongly inhibitory and so, to a lesser extent, was 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid [(SITS)], independent of the sign and size of the membrane potential. (5) Cl- entry was negligibly increased (less than 30%) by either trans-Cl- or -HCO3-, indicating the absence of an obligatory Cl-/anion antiport activity. In contrast, the height of the overshoot at 60 s was increased by trans-Cl-, indicating time-dependent inhibition of 36Cl efflux. That competitive inhibition of 36Cl fluxes by anions is involved here is supported by initial influx rate experiments demonstrating: (1) the saturability of Cl- influx, which was found to exhibit Michaelis-Menten kinetics; and (2) competitive inhibition of influx by cis-Cl- and -Br-. Quantitatively, the conclusion is warranted that over 85% of the total initial Cl- uptake energized by a pH gradient involves an electroneutral Cl-/H+ symporter or its physicochemical equivalent, a Cl-/OH- antiporter, exhibiting little Cl- uniport and either Cl-/Cl- or Cl-/HCO3- antiport activities.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of apical Cl-/OH- exchange activity in Caco-2 cells by phorbol esters is mediated by PKCepsilon

The present studies were undertaken to examine the possible regulation of apical membrane Cl-/OH- exchanger in Caco-2 cells by protein kinase C (PKC). The effect of the phorbol ester phorbol 12-myristate 13-acetate (PMA), an in vitro PKC agonist, on OH- gradient-driven 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive 36Cl uptake in Caco-2 cells was assessed. The results demonstrated that PMA decreased apical Cl-/OH- exchanger activity via phosphatidylinositol 3-kinase (PI3-kinase)-mediated activation of PKCepsilon. The data consistent with these conclusions are as follows: 1) short-term treatment of cells for 1-2 h with PMA (100 nM) significantly decreased Cl-/OH- exchange activity compared with control (4alpha-PMA); 2) pretreatment of cells with specific PKC inhibitors chelerythrine chloride, calphostin C, and GF-109203X completely blocked the inhibition of Cl-/OH- exchange activity by PMA; 3) specific inhibitors for PKCepsilon (Ro-318220) but not PKCalpha (Go-6976) significantly blocked the PMA-mediated inhibition; 4) specific PI3-kinase inhibitors wortmannin and LY-294002 significantly attenuated the inhibitory effect of PMA; and 5) PI3-kinase activators IRS-1 peptide and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] mimicked the effects of PMA. These findings provide the first evidence for PKCepsilon-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells and indicate the involvement of the PI3-kinase-mediated pathways in the regulation of Cl- absorption in intestinal epithelial cells.     

Interpretation of effects of pH on rate constants for the oxidation of three ferrocytochromes c-551 with [Fe(CN)6]3- and [Co(phen)3]3+, and assignment of pKa values

Effects of pH on second-order rate constants, k (25 degrees C), have been determined for the [Fe(CN)6]3- and [Co(phen)3]3+ oxidations of ferrocytochrome c-551 from Pseudomonas aeruginosa, Pseudomonas stutzeri, and Azotobacter vinelandii. For each oxidant similar directional trends are observed. With [Fe(CN)6]3-, rate constants over the pH 4-9.5 range first decrease, and then increase to plateau pH approximately equal to 9 k values of 0.96.10(5), 4.4.10(5) and 1.05.10(5) M-1.s-1, respectively. With [Co(phen)3]3+, rate constants increase in two separate well-defined stages from pH 2.5-9.5 to plateau pH approximately equal to 9 k values of 1.35.10(5), 3.6.10(5) and 1.37.10(5) M-1.s-1, respectively. From these trends, and consistent with previous NMR studies, protein pKa values of 7.16, 8.00 and 6.67, respectively, for the three reduced cytochromes c-551 are assigned to the buried propionic acid at position 7 on the haem ring. Since at pH greater than 6 the trends with pH for both [Fe(CN)6]3- and [Co(phen)3]3+ are in the same direction, it is concluded that this deprotonation results in a decrease in protein reduction potential. At pH less than 6, the trends with [Co(phen)3]3+ and [Fe(CN)6]3- are in opposite directions. Well defined pKa values of 3.6, 3.80 and 3.80 for P. aeruginosa, P. stutzeri and A. vinelandii, respectively, are observed with [Co(phen)3]3+ as oxidant. Upper limits only of pKa values less than 5.0, less than 4.1 and less than 4.5, respectively, are observed with [Fe(CN)6]3- as oxidant, which may or may not be the same as those observed for [Co(phen)3]3+. These latter pKa values are assigned to carboxylate residues at or near to the binding site(s). It is noted that charged residues are invariant on the front face (incorporating the exposed haem edge) of all three cytochromes c-551, and that there are only two carboxylates. One possibility is that the locality including both carboxylates defined by residues Asp-19, Lys-21, Lys-28 and Asp/Glu-29, serves as a binding site for both 3+ and 3- oxidants.     

Pseudo cross-link of human hemoglobin with mono-(3,5-dibromosalicyl)fumarate

The reaction of human oxyhemoglobin with mono(3,5-dibromosalicyl)fumarate, produces a derivative specifically acylated at the two lysines beta 82, which can be purified with a 70% yield. The oxygen affinity of this derivative at 37 degrees C at pH 7.4, 0.1 M Cl- is of 12 mm Hg, and is not affected by organic phosphate. In the presence of 5% CO2, the oxygen affinity decreases to 25 mm Hg. In all cases the cooperativity is lowered, with a value of n in the Hill plots near 2. Sedimentation velocity measurements indicate that, contrary to normal hemoglobin, this derivative fails to dissociate into dimers upon exposure to pH 5.5. The stability of the tetrameric structure is probably due to a modification of the beta-beta interface, resulting from electrostatic and hydrophobic interactions introduced in the beta cleft by the fumaryl residues. These new interactions are probably the origin of a new reverse Bohr effect group at alkaline pH. Consistent with the stabilization of the tetrameric structure, the half-time of retention of this compound in the rat is increased 4-fold with respect to that of normal hemoglobin. These characteristics cast a favorable light on the usage of this compound as an oxygen carrier in transfusional and perfusional fluids.     

C1/HCO3 exchange in human placental brush border membrane vesicles

Membrane transport pathways for transplacental transfer of CO2/HCO3 were investigated by assessing the possible presence of a Cl/HCO3 exchange mechanism in the maternal-facing membrane of human placental epithelial cells. Cl/HCO3 exchange was tested for in preparations of purified brush border membrane vesicles by 36Cl tracer flux measurements and determinations of acridine orange fluorescence changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake to levels approximately 2-fold greater than observed at equilibrium. Maneuvers designed to offset the development of ion gradient-induced diffusion potentials (valinomycin, Ko = Ki) significantly reduced HCO3- gradient-driven Cl- uptake but concentrative accumulation of Cl- persisted. Early time point determinations performed in the presumed absence of membrane potential suggests the reduced level of HCO3- gradient-driven Cl- uptake resulted from a more rapid dissipation of the HCO3- concentration gradient. Concentrative accumulation of Cl- was not observed in the presence of a pH gradient alone under 100% N2, suggesting a preference of HCO3- over OH- as a substrate for transport. As monitored by acridine orange fluorescence the Cl- gradient-dependent collapse of an imposed pH gradient (pHo 8.5/pHi 6) was accelerated in the presence of CO2/HCO3 when compared with its absence, indicating coupling of HCO3- influx to Cl- efflux. Increasing concentrations of the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid were observed to cause a stepwise reduction in HCO3- gradient-driven Cl- uptake (I50 approximately 25 microM) further suggesting the presence of a Cl/HCO3 exchange mechanism. The results of this study provide evidence for a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive Cl/HCO3 exchange mechanism in the maternal-facing membrane of human placental epithelial cells. The identification of an ion-coupled HCO3- transport pathway in placental epithelia may suggest functional roles in mediating transplacental transfer of CO2 as well as maintenance of fetal acid/base balance.     

Determinants of [Cl-] in recycling and late endosomes and Golgi complex measured using fluorescent ligands

Chloride concentration ([Cl-]) was measured in defined organellar compartments using fluorescently labeled transferrin, alpha2-macroglobulin, and cholera toxin B-subunit conjugated with Cl--sensitive and -insensitive dyes. In pulse-chase experiments, [Cl-] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over approximately 10 min in parallel to a drop in pH from 6.91 to 6.05. The low [Cl-] just after internalization (compared with 137 mM solution [Cl-]) was prevented by reducing the interior-negative Donnan potential. [Cl-] in alpha2-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20. Cl- accumulation was prevented by bafilomycin but restored by valinomycin. A Cl- channel inhibitor slowed endosomal acidification and Cl- accumulation by approximately 2.5-fold. [Cl-] was 49 mM and pH was 6.42 in cholera toxin B subunit-labeled Golgi complex in Vero cells; Golgi compartment Cl- accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that Cl- is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [Cl-] early after internalization. We propose that reduced [Cl-] and volume in early endosomes permits endosomal acidification and [Cl-] accumulation without lysis.     

The effect of Cl- depletion and X- reconstitution on the oxygen-evolution rate, the yield of the multiline manganese EPR signal and EPR signal II in the isolated Photosystem-II complex

The role of Cl- in photosynthetic O2 evolution has been investigated by measurement of the steady-state O2 rate and EPR of the electron donors responsible for the S2 multiline signal and Signal IIs upon Cl- depletion and substitution in Photosystem II membranes. Cl- removal has three effects upon the donor side of Photosystem II. (1) It abolishes O2 evolution reversibly, while decreasing the yield of the S2 multiline signal indicative of the manganese site of the O2-evolving complex in the S2 oxidation state. This decrease is brought about by (2) the reversible disconnection of the manganese complex from the reaction center; and by (3) deactivation of S1 centers having reduced primary acceptor QA to form SO centers having a reduced Signal IIs species. Reactivation of O2 evolution by anions confirms earlier work showing a requirement for a univalent anion of optimum charge density. The observed order of reactivation is Cl- greater than Br- approximately NO3- much greater than OH- approximately F-. Reactivation of the S2 multiline signal follows Cl- approximately Br- greater than NO3- approximately OH- greater than F-, in near correspondence with reactivation of O2-evolution rates. Cl- titrations of F- -inhibited samples reveal two binding sites for Cl- which differ in binding affinity by 11-fold. The higher-affinity site reactivates the S1----S2 light reaction, while the lower-affinity site reactivates the S3----S0 light reaction. The high affinity site is located within the O2-evolving complex at an undetermined site, while the lower-affinity site functions in coupling the reaction center photochemistry to the O2-evolving complex. The results are compared with Cl-/F- exchange equilibria for Mn3+ in solution. A model for the lower S-state transitions is presented in which specific oxidation state assignments are made for some of the donors and acceptors of Photosystem II.