Effect of acute pH change on serum anion gap

The serum anion gap is decreased in hyperchloremic (HCl) acidosis and increased in diuretic-induced alkalosis. These anion gap changes have been largely attributed to titration-induced variations in the net negative charge of the serum proteins, which are the predominant non-HCO3 buffers of serum. It has recently been shown, however, that albumin has all of the net protein charge, and titration-induced changes in charge are smaller than have been widely believed. Because the non-HCO3 buffers are also titrated in acute hypocapnia and hypercapnia, these disorders were induced in 16 anesthetized dogs for 10 min in order to assess the effect of acute changes in pH on the anion gap. Although the mean arterial pH varied from 7.04 to 7.65, the calculated mean albumin charge only varied from 6.8 to 9.0 mEq/L. When the anion gap was computed with HCO3 (AGHCO3 = Na + K - Cl - HCO3), the change in AGHCO3 per 0.1 change in pH (delta AGHCO3/ delta pH) was only 0.15 mEq/L per 0.1 pH. When the anion gap was computed with total CO2 content (AGTCO2 = Na + K - Cl - TCO2), delta AGTCO2/delta pH was larger (0.51 mEq/L per 0.1 pH) because of the effect of variable PCO2 levels on TCO2. In a review of 22 previous studies in humans and dogs, similar estimates of delta AG/delta pH were obtained (after adjusting for the lower albumin level in dogs). These results show that simple titration processes that occur within 10 min of a change in pH cause minimal changes in the anion gap. Titration of the known non-HCO3 buffers of serum does not explain the much larger anion gap changes of HCl acidosis and diuretic alkalosis.     

Intramolecular charge transfer in the bacteriorhodopsin mutants Asp85-->Asn and Asp212-->Asn: effects of pH and anions

The photovoltage kinetics of the bacteriorhodopsin mutants Asp212-->Asn and Asp85-->Asn after excitation at 580 nm have been investigated in the pH range from 0 to 11. With the mutant Asp85-->Asn (D85N) at pH 7 no net charge translocation is observed and the signal is the same, both in the presence of Cl- (150 mM) and in its absence (75 mM SO4(2-)). Under both conditions the color of the pigment is blue (lambda max = 615 nm). The time course of the photovoltage kinetics is similar to that of the acid-blue form of wild-type, except that an additional transient charge motion occurs with time constants of 60 microseconds and 1.3 ms, indicating the transient deprotonation and reprotonation of an unknown group to and from the extracellular side of the membrane. It is suggested that this is the group XH, which is responsible for proton release in wild-type. At pH 1, the photovoltage signal of D85N changes upon the addition of Cl- from that characteristic for the acid-blue state of wild-type to that characteristic for the acid-purple state. Therefore, the protonation of the group at position at 85 is necessary, but not sufficient for the chloride-binding. At pH 11, well above the pKa of the Schiff base, there is a mixture of "M-like" and "N-like" states. Net proton transport in the same direction as in wild-type is restored in D85N from this N-like state. With the mutant Asp212-->Asn (D212N), time-resolved photovoltage measurements show that in the absence of halide ions the signal is similar to that of the acid-blue form of wild-type and that no net charge translocation occurs in the entire pH range from 0 to 11. Upon addition of Cl- in the pH range from 3.8 to 7.2 the color of the pigment returns to purple and the photovoltage experiments indicate that net proton pumping is restored. However, this Cl(-)-induced activation of net charge-transport in D212N is only partial. Outside this pH range, no net charge transport is observed even in the presence of chloride, and the photovoltage shows the same chloride-dependent features as those accompanying the acid-blue to acid-purple transition of the wild-type.     

Electrochemical modeling of electron and proton transfer to ubiquinone-10 in a self-assembled phospholipid monolayer

Ubiquinone-10 (UQ) was incorporated at concentrations ranging from 0.5 to 2 mol% in a self-assembled monolayer of dioleoylphosphatidylcholine (DOPC) deposited on a mercury drop electrode, and its electroreduction to ubiquinol (UQH2) was investigated in phosphate and borate buffers over the pH range from 7 to 9.5 by a computerized chronocoulometric technique. The dependence of the applied potential for a constant value of the faradaic charge due to UQ reduction upon the electrolysis time t at constant pH and upon pH at constant t was examined on the basis of a general kinetion approach. This permitted us to conclude that the reduction of UQ to UQH2 in DOPC monolayers takes place via the reversible uptake of one electron with the formation of the semiubiquinone radical anion UQ.-, followed by the rate-determining protonation of this anion with UQH. formation; this neutral radical is more easily reduced than UQ, yielding the ubiquinol UQH2. In spite of the very low concentration of hydrogen ions as compared with that of the acidic component of the buffer, the only effective proton donor is the proton itself; this strongly suggests that the protonation step takes place inside the polar head region of the DOPC monolayer, which is only accessible to protons.     

The pH-induced release of iron from transferrin investigated with a continuum electrostatic model

A reduction in pH induces the release of iron from transferrin in a process that involves a conformational change in the protein from a closed to an open form. Experimental evidence suggests that there must be changes in the protonation states of certain, as yet not clearly identified, residues in the protein accompanying this conformational change. Such changes in protonation states of residues and the consequent changes in electrostatic interactions are assumed to play a large part in the mechanism of release of iron from transferrin. Using the x-ray crystal structures of human ferri- and apo-lactoferrin, we calculated the pKa values of the titratable residues in both the closed (iron-loaded) and open (iron-free) conformations with a continuum electrostatic model. With the knowledge of a residue's pKa value, its most probable protonation state at any specified pH may be determined. The preliminary results presented here are in good agreement with the experimental observation that the binding of ferric iron and the synergistic anion bicarbonate/carbonate results in the release of approximately three H+ ions. It is suggested that the release of these three H+ ions may be accounted for, in most part, by the deprotonation of the bicarbonate and residues Tyr-92, Lys-243, Lys-282, and Lys-285 together with the protonation of residues Asp-217 and Lys-277.     

Microprobe analysis of element distribution in rabbit and dog erythrocytes as examples of "high" and "low" potassium cells

The concentrations of Na, Mg, P, S, Cl, K and Fe were determined by microprobe in near 100% hematocrit suspensions of rabbit and dog erythrocytes prepared by freezing and drying. These cells are representative, respectively, of "high" potassium, "low" sodium, and "high" sodium, "low" potassium cells. Water contents of the cells were the same, as were, approximately, the levels of Cl, S and Fe. Rabbit P was nearly double that of the dog. For the rabbit, the cell Na/K ratio was 0.21 and for the dog 15.4, illustrating the major diffusible electrolyte difference between these two types of cell. The rabbit erythrocytes showed an apparent negative immobile charge density of 95 meq/kg of cell water and the dog 56 meq/kg cell water, a distinct difference. Serum electrolytes in the two species are exactly comparable (Standard Tables). Ionic distribution in these cell types was treated by the Gibbs-Duhem equation representing two heterogeneous systems in thermodynamic equilibrium with the blood serum. Factors to be considered are: (1) the composition of the erythrocyte and its net immobile charge; (2) the physicochemical properties of the individual ions (charge, ionic radius, hydration energy, standard chemical potential); (3) the dielectric constant of the dispersion medium (in this case, water); and (4) the binding constants of the ions. The hypothesis of "active transport" (the sodium-potassium pump) is specifically rejected as an explanation of ionic differences.     

Self-consistent framework for standardising mobilities in free solution capillary electrophoresis: applications to oligoglycines and oligoalanines

A theoretical analysis of deviations from ideality in ionic transport is presented to correct mobilities, mu, measured in free solution capillary electrophoresis (CE) to mobility at infinite dilution, mu degree (limiting mobility). Non-ideality is treated at the same level of approximation as in equilibrium, using a correction factor for the sum of the analyte and counter-ion radius originally suggested by Robinson and Stokes (Electrolyte Solutions, 1961). Unlike previous corrections using Debye-Hückel-Onsager theory, which are strictly applicable only at very low ionic strengths, this treatment is expected to be valid for univalent ions migrating in a uni-univalent background electrolyte for ionic strengths up to 0.075 mol kg-1, a range typical of CE experiments. The analysis is applied to the determination of mu degree in acidic and basic buffers for oligoalanines and oligoglycines with degree of polymerisation 2 to 6. Limiting mobilities for the fully protonated and deprotonated peptides are found to be numerically equal but opposite in sign, consistent with a change in charge from +1 to -1. In all uni-univalent buffers studied (borate, citrate, low pH lithium phosphate and sodium phosphate) mu degree values established using data over a range of pH and ionic strength are found to be identical and in excellent agreement with previous values from isotachophoresis. Values of mu degree in high pH sodium phosphate buffer are systematically 0.2.10(-8) m2 V-1 s-1 higher than those in other buffers; this may be attributed to limitations of the model for a buffer with 1+:2- and 1+:3- ions. This self-consistent framework for standardising mobilities in free solution CE is expected to be widely applicable to univalent analytes migrating in a 1:1 background electrolyte.     

催化动力学光度法测定合金中镍

在pH 9.00的NH3·H2O-NH4Cl介质中,镍(Ⅱ)对H2O2氧化茜素红S(ARS)的褪色反应存在明显的催化作用,由此建立了一个光度法测定镍(Ⅱ)的新体系。通过试验酸度、试剂用量、表面活性剂、温度和时间的影响,确定了最佳测定条件。考察了大量常见离子对反应的影响,发现大部分常见离子对测定无干扰,Fe3+、Cu2+的干扰可通过加入NH3F溶液和KI溶液消除。体系的最大吸收峰位于520 nm,线性范围为0.02~2.8 μg/mL,检出限为0.008 μg/mL 。反应的表观活化能为71.68 kJ/mol。方法用于合金标准样品中镍的测定,结果与认定值一致。     

Direct high affinity modulation of connexin channel activity by cyclic nucleotides

Protonated aminosulfonate compounds directly inhibit connexin channel activity. This was demonstrated by pH-dependent connexin channel activity in Good's pH buffers (MES (4-morpholineethanesulfonic acid)), HEPES, and TAPS (3-({[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]amino]-1-propanesulfonic acid)) that have an aminosulfonate moiety in common and by the absence of pH-dependent channel activity in pH buffers without an aminosulfonate moiety (maleate, Tris, and bicarbonate). The pH-activity relation was shifted according to the pKa of each aminosulfonate pH buffer. At constant pH, increased aminosulfonate concentration inhibited channel activity. Taurine, a ubiquitous cytoplasmic aminosulfonic acid, had the same effect at physiological concentrations. These data raise the possibility that effects on connexin channel activity previously attributed to protonation of connexin may be mediated instead by protonation of cytoplasmic regulators, such as taurine. Modulation by aminosulfonates is specific for heteromeric connexin channels containing connexin-26; it does not occur significantly for homomeric connexin-32 channels. The identification of taurine as a cytoplasmic compound that directly interacts with and modulates connexin channel activity is likely to facilitate understanding of cellular modulation of connexin channels and lead to the development of reagents for use in structure-function studies of connexin protein.     

Complete structural elucidation of triacylglycerols by tandem sector mass spectrometry

We developed a method to elucidate the complete structure of triacylglycerols by means of high-energy collisional activation tandem mass spectrometry (MS/MS). Both ESI- and FAB-produced [M + NH4]+ and [M + met.]+ ions (where met. = Li, Na, and Cs) of triacylglycerols undergo charge-remote and charge-driven fragmentations. We emphasize the study of fragment ions from ESI-produced [M + NH4]+ and [M + Na]+ ions and FAB-produced [M + Na]+ ions. ESI-produced [M + NH4]+ ions fragment to produce four types of ions, [M + NH4 - RnCOONH4]+, [RnCO + 128]+, [RnCO + 74]+, and RnCO+ ions, from which the carbon number and the degree of unsaturation of each acyl group are obtained. In addition, three series of ions are produced by charge-remote fragmentations (CRFs), and analysis of their patterns gives the position and the number of double bonds on the acyl groups. Information about the position of each acyl group on the glycerol backbone, however, is not provided by collisionally activated dissociation of [M + NH4]+ ions. On the other hand, ESI- and FAB-produced [M + Na]+ ions fragment to form eight types of ions (named A-J ions) that, like those produced by CRF, are highly structurally informative. The absence of certain series members also carries useful structural information. Interpretation of these patterns enables one to obtain the number of carbons, degrees of unsaturation, and location of double bonds, as well as the positions of acyl groups on the glycerol backbone.     

Chloride-dependent transport of NH4+ into bee retinal glial cells

Mammalian astrocytes convert glutamate to glutamine and bee retinal glial cells convert pyruvate to alanine. To maintain such amination reactions these glial cells may take up NH4+/NH3. We have studied the entry of NH4+/NH3 into bundles of glial cells isolated from bee retina by using the fluorescent dye BCECF to measure pH. Ammonium caused intracellular pH to decrease by a saturable process: the rate of change of pH was maximal for an ammonium concentration of about 5 mM. This acidifying response to ammonium was abolished by the loop diuretic bumetanide (100 microM) and by removal of extracellular Cl-. These results strongly suggest that ammonium enters the cell by contransport of NH4+ with Cl-. Removal of extracellular Na+ did not abolish the NH(4+)-induced acidification. The NH(4+)-induced pH change was unaffected when nearly all K+ conductance was blocked with 5 mM Ba2+ showing that NH4+ did not enter through Ba(2+)-sensitive ion channels. Application of 2 mM NH4+ led to a large increase in total intracellular proton concentration estimated to exceed 13.5 mEq/L. As the cell membrane appeared to be permeable to NH3, we suggest that when NH4+ entered the cells, NH3 left, so that protons were shuttled into the cell. This shuttle, which was strongly dependent on internal and external pH, was quantitatively modelled. In retinal slices, 2 mM NH4+ alkalinized the extracellular space: this alkalinization was reduced in the absence of bath Cl-. We conclude that NH4+ enters the glial cells in bee retina on a cotransporter with functional similarities to the NH4+(K+)-Cl- cotransporter described in kidney cells.     

Ribosomal protein L7/L12 is required for optimal translation

We have tested the performance in vitro of Escherichia coli ribosomes containing or lacking the protein L7/L12. When the experiments are performed in an optimized mixture of ions (polymix), L7/L12 is required for maximal rate of synthesis as well as for minimal missense error frequency. The results in conventional Tris/Mg2+/NH4Cl buffers are different; in these buffers, only the rate of synthesis is strongly dependent on the presence of L7/L12. In addition, we show that there is a large difference between the optimal Mg2+ concentration required for speed of translation and that for accuracy of translation in conventional buffer. These optima are very close in polymix. Finally, we show that the contribution of L7/L12 to the speed of translation is obscured in translation systems that are limited by substrates. We conclude that it is not possible to analyze details of the mechanism of translation in conventional buffers.     

Factors influencing the choice of buffer in background electrolytes for indirect detection of fast anions by capillary electrophoresis

The suitability of relatively slow (low absolute value of mobility) coanionic buffers in background electrolytes (BGEs) for indirect photometric detection of anions by capillary electrophoresis was investigated. As a model system, 2-(cyclohexylamino)ethanesulfonic acid (CHES) was used to buffer the indirect detection electrolyte of sodium chromate. CHES (PKa 9.55) is a zwitterionic molecule carrying a net negative charge depending on the pH (effective charge -0.5 at pH = pKa). Within its useful pH buffering range CHES acted as a competing probe coanion. System peaks were induced which had deleterious effects on the detection sensitivity of slow to medium mobility anions. The mobility of the system peak was determined by the effective mobility of CHES, both of which increased with increasing pH. The peaks of analytes that migrated near or on the system peak were distorted and lost all quantitative properties. Analytes that migrated after the system peak either were not detected or reversed their responses. Analytes that migrated well before the system peak were unaffected. Consequently, the suitability of slow coanionic buffers is limited either to (i) fast anions or, (ii) a pH range much below the PKa, where the buffering capacity is not optimal.     

Multicenter study of noninvasive monitoring systems as alternatives to invasive monitoring of acutely ill emergency patients

Responses in dry matter intake (DMI) and acidbase balance to three sources of anionic salts (dietary cation-anion difference = -63 to -40 meq/kg of dry matter), an acidified fermentation by-product, MgSO4.7H2O + NH4Cl, and MgSO4.7H2O + CaCl2.2H2O + CaSO4, were evaluated relative to the responses of cows fed a control diet (dietary cationanion difference = 203 meq/kg of dry matter) that did not contain anionic salts. Diets were fed for 1-wk periods to eight nonlactating Holsteins assigned to two replicated 4 x 4 Latin squares. Daily DMI increased as time of access to the diet increased up to d 5; mean DMI over d 5 to 7 was reduced by dietary anionic salts. Diets containing anionic salts induced a mild metabolic acidosis that was completely compensated by nonrespiratory mechanisms (decreased blood bicarbonate and base excess; pCO2 and pH values were unaffected). Urinary pH values and bicarbonate excretion were reduced, and urinary NH4+ and titratable acidity excretion were increased, for cows fed diets containing anionic salts. Strong ion difference in urine was decreased by dietary anionic salts because of the relatively greater excretions of Cl- and S2- versus Na+ and K+ by cows fed these diets. Dietary anionic salts decreased mean ruminal pH by 0.12 units, possibly because of the reduced strong ion difference of ruminal fluid. Dietary anionic salts increased mean ruminal NH3 concentration by 2.2 mM, probably because of the higher nonprotein N content of these diets. The strong negative relationship (r2 = 0.95) between urinary pH and net acid excretion by cows fed the diets containing anionic salts suggested that urinary pH measurement might be a useful tool to assess the degree of metabolic acidosis that was imposed by dietary anionic salts.     

The multi-ion nature of the pore in Shaker K+ channels

We have investigated some of the permeation properties of the pore in Shaker K channels. We determined the apparent permeability ratio of K+, Rb+, and NH4+ ions and block of the pore by external Cs+ ions. Shaker channels were expressed with the baculovirus/Sf9 expression system and the channel currents measured with the whole-cell variant of the patch clamp technique. The apparent permeability ratio, PRb/PK, determined in biionic conditions with internal K+, was a function of external Rb+ concentration. A large change in PRb/PK occurred with reversed ionic conditions (internal Rb+ and external K+). These changes in apparent permeability were not due to differences in membrane potential. With internal K+, PNH4/PK was not a function of external NH4+ concentration (at least over the range 50-120 mM). We also investigated block of the pore by external Cs+ ions. At a concentration of 20 mM, Cs+ block had a voltage dependence equivalent to that of an ion with a valence of 0.91; this increased to 1.3 at 40 mM Cs+. We show that a 4-barrier, 3-site permeation model can simulate these and many of the other known properties of ion permeation in Shaker channels.     

Cloning and characterization of the NAD-linked glycerol-3-phosphate dehydrogenases of Trypanosoma brucei brucei and Leishmania mexicana mexicana and expression of the trypanosome enzyme in Escherichia coli

A polyclonal antiserum raised against the purified glycosomal glycerol-3-phosphate dehydrogenase of Trypanosoma brucei brucei has been used to identify the corresponding cDNA clone in a T.b. brucei expression library. This cDNA was subsequently used to obtain genomic clones containing glycerol-3-phosphate dehydrogenase genes. Two tandemly arranged genes were detected in these clones. Characterization of one of the genes showed that it codes for a polypeptide of 353 amino acids, with a molecular mass of 37,651 Da and a calculated net charge of +8. Using the T.b. brucei gene as a probe, a corresponding glycerol-3-phosphate dehydrogenase gene was also identified in a genomic library of Leishmania mexicana mexicana. The L.m. mexicana gene codes for a polypeptide of 365 amino acids, with a molecular mass of 39,140 Da and a calculated net charge of +8. The amino-acid sequences of both polypeptides are 63% identical and carry a type-1 peroxisomal targeting signal (PTS1) SKM and -SKL at their respective C-termini. Moreover, the L.m. mexicana polypeptide also carries a short N-terminal extension reminiscent of a mitochondrial transit sequence. Subcellular localisation analysis showed that in L.m. mexicana the glycerol-3-phosphate dehydrogenase activity co-fractionated both with mitochondria and with glycosomes. This is not the case in T. brucei, where the enzyme is predominantly glycosomal. The two trypanosomatid sequences resemble their prokaryotic homologues (32-36%) more than their eukaryotic counterparts (25-31%) and carry typical prokaryotic signatures. The possible reason for this prokaryotic nature of a trypanosomatid glycerol-3-phosphate dehydrogenase is discussed.     

A novel buffer system for separation of metal cations by capillary electrophoresis with indirect UV detection

Generally, the buffers used for metal ion separations in capillary electrophoresis (CE) consist of a UV-active substance, pH-adjuster, and weak complexing reagent. This paper describes the successful separation of metal ions with a new buffer that contains no complexing reagent. Of several weakly basic compounds tested, 2-aminopyridine was selected as the most useful UV-active substance. It was used at a concentration of 15 mM with pH adjusted to 5.0 +/- 0.1 by acetic acid. The degree of protonation of the UV-active substance played an important role in detection. The stacking phenomenon was a significant contributor to efficiency in this buffer system, and water-diluted samples gave especially high efficiencies. When a 75-micron-i.d. fused-silica capillary was used, a separation efficiency of 1.8 x 10(5) was observed. Quantitative determinations of Ca2+, Mn2+, Zn2+, and Cd2+ were achieved with good linear calibration curves over the range of concentration from a few milligrams per liter to 100 mg/L. The detection limits were 0.2 mg/L for Ca2+, 0.4 mg/L for Mn2+ and Zn2+, and 0.6 mg/L for Cd2+, based on three times the baseline noise.     

Channeling of the intermediates and catalytic facilitation to Rubisco in a multienzyme complex of Calvin cycle enzymes

Calvin cycle multienzyme complex, consisting of phosphoriboisomerase, phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase (Rubisco), shows ribose-5-phosphate + ATP dependent CO2 fixation activity with a small but discernible lag. Transient time analysis showed that the lag at pH 7 was independent of multienzyme concentration and was significantly lower than the expected transient time calculated from Km and Vmax of the individual enzymes, indicative of channeling of the intermediates in the enzyme complex. Channeling of ribulose-1,5-bisphosphate was found to offer a catalytic advantage to Rubisco. Rubisco shows a decrease in activity during catalysis in ribulose-1,5-bisphosphate dependent CO2 fixation reaction, due to the formation of the catalytic inhibitor. Such a decrease of Rubisco activity was not observed in ribose-5-phosphate + ATP dependent CO2 fixation reaction and the catalytic inhibitor was also not detected. These results suggested that the intermediates are channeled in the complex and channeling offers a catalytic facilitation to Rubisco.     

Ammonia acquisition in enteric bacteria: physiological role of the ammonium/methylammonium transport B (AmtB) protein

Homologues of the amtB gene of enteric bacteria exist in all three domains of life. Although their products are required for transport of the ammonium analogue methylammonium in washed cells, only in Saccharomyces cerevisiae have they been shown to be necessary for growth at low NH4+ concentrations. We now demonstrate that an amtB strain of Escherichia coli also grows slowly at low NH4+ concentrations in batch culture, but only at pH values below 7. In addition, we find that the growth defect of an S. cerevisiae triple-mutant strain lacking the function of three homologues of the ammonium/methylammonium transport B (AmtB) protein [called methylammonium/ammonium permeases (MEP)] that was observed at pH 6.1 is relieved at pH 7.1. These results provide direct evidence that AmtB participates in acquisition of NH4+/NH3 in bacteria as well as eucarya. Because NH3 is the species limiting at low pH for a given total concentration of NH4+ + NH3, results with both organisms indicate that AmtB/MEP proteins function in acquisition of the uncharged form. We confirmed that accumulation of [14C]methylammonium depends on its conversion to gamma-N-methylglutamine, an energy-requiring reaction catalyzed by glutamine synthetase, and found that at pH 7, constitutive expression of AmtB did not relieve the growth defects of a mutant strain of Salmonella typhimurium that appears to require a high internal concentration of NH4+/NH3. Hence, contrary to previous views, we propose that AmtB/MEP proteins increase the rate of equilibration of the uncharged species, NH3, across the cytoplasmic membrane rather than actively transporting-that is, concentrating-the charged species, NH4+.     

K+/NH4+ antiporter: a unique ammonium carrying transporter in the kidney inner medulla

The mechanism of NH4+ transport in inner medulla is not known. The purpose of these experiments was to study the process that is involved in ammonium (NH4+) transport in cultured inner medullary collecting duct (mIMCD-3) cells. Cells grown on coverslips were exposed to NH4+ and monitored for pHi changes by the use of the pH-sensitive dye BCECF. The rate of cell acidification following the initial cell alkalinization was measured as an index of NH4+ transport. The rate of NH4+ transport was the same in the presence or absence of sodium in the media (0.052 +/- 0.003 vs 0.048 +/- 0.004 pH/min. P > 0.05), indicating that NH4+ entry into the cells was independent of sodium. The presence of ouabain, bumetanide, amiloride, barium, or 4,4'-di-isothiocyanostilbene-2-2'-disulfonic acid (DIDS) did not block the NH4(+)-induced cell acidification, indicating lack of involvement of Na+:K(+)-ATPase, Na+:K+:2Cl- transport, Na+:H+ exchange, K+ channel, or Cl-/base exchange, respectively, in NH4+ transport. The NH4(+)-induced cell acidification was significantly inhibited in the presence of high external [K+] as compared to low external [K+] (0.018 +/- 0.001 vs. 0.049 +/- 0.003 pH/min for 140 mM K+ vs. 1.8 mM K+ in the media, respectively, P < 0.001). Inducing K+ efflux by imposing an outward K+ gradient caused intracellular acidification by approximately 0.3 pH unit in the presence but not the absence of NH4+. This K+ efflux-induced NH4+ entry increased by extracellular NH4+ in a saturable manner with a Km of approximately 5 mM, blocked by increasing extracellular K+ and was not inhibited by barium. The K+ efflux-coupled NH4+ entry was electroneutral as monitored by the use of cell membrane potential probe 3,3'-dipropylthiadicarbocyanine. These results are consistent with the exchange of internal K+ with external NH4+ in a 1:1 ratio. The K(+)-NH4+ antiporter was inhibited by verapamil and Schering 28080 in a dose-dependent manner, was able to work in reverse mode, and did not show any affinity for H+ as a substrate, indicating that it is distinct from other NH4(+)-carrying transporters. We conclude that a unique transporter, a potassium-ammonium (K+/NH4+) antiport, is responsible for NH4+ transport in renal inner medullary collecting duct cells. This antiporter is sensitive to verapamil and Schering 28080, is electroneutral, and is selective for NH4+ and K+ as substrates. The K+/NH4+ antiporter may play a significant role in acid-base regulation by excretion of ammonium and elimination of acid.     

Calorimetric studies of carbon monoxide and inositol hexaphosphate binding to hemoglobin A

Heats of CO and IHP binding to hemoglobin A have been determined under a variety of buffer and pH conditions. From these data heats of ion binding linked to hemoglobin oxygenation have been estimated. For IHP binding to deoxyhemoglobin the buffer-corrected enthalpies are surprisingly large, reaching -25 kcal/mol of IHP at pH 7.4. These values correspond to approximately -11 kcal/mol of proton absorbed upon IHP binding and may rise largely from the protonation of hitidine and NH2-terminal groups in the binding site (Arnone, A., and Perutz, M.F. (1974) Nature 249, 34-36). The decreased magnitude of delta HIHP observed at low pH parallels the decreased proton uptake at low pH. In 0.1 M chloride (pH 7.4) the reaction Hb(aq) + IHP leads to Hb x IHP(aq) has a standard free energy change (Edalji, R., Benesch, R.E., and Benesch, R. (1976) J. Biol. Chem. 251, 7720-7721) of -10 kcal and an enthalpy change of -25 kcal. Therefore, enthalpic forces provide the dominant driving force of this process. The origin of these large negative enthalpy changes is attributed to the exothermic protonation of protein basic groups induced by the proximity of phosphate negative charges. The importance of protonation in the binding of organic phosphates to hemoglobin may well extend to the specific binding of other phosphate substrates to enzyme reaction sites.