Acid-base Transfer Research Articles

Carbinolamine Formation and Dehydration in a DNA Repair Enzyme Active Site

In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics–molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water–independent enzyme–catalyzed reaction had a bias–corrected Jarzynski–average barrier height of approximately for the carbinolamine formation reaction and ) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately in the forward (formation) reaction and for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water–independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N–terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site carboxylate and the N–terminal amine.

Molecular detection and immunological localization of gill Na+/H+exchanger in the dogfish (Squalus acanthias)

The dogfish (Squalus acanthias) can make rapid adjustments to gill acid-base transfers to compensate for internal acidosis/alkalosis. Branchial Na+/H+ exchange (NHE) has been postulated as one mechanism driving the excretion of H+ following acidosis. We have cloned gill cDNA that includes an open reading frame coding for a 770-residue protein most homologous (approximately 71%) to mammalian NHE2. RT-PCR revealed NHE2 transcripts predominantly in gill, stomach, rectal gland, intestine, and kidney. In situ hybridization with an antisense probe against NHE2 in gill sections revealed a strong mRNA signal from a subset of interlamellar and lamellae cells. We developed dogfish-specific polyclonal antibodies against NHE2 that detected a approximately 70-kDa protein in Western blots and immunologically recognized branchial cells having two patterns of protein expression. Cytoplasmic and apical NHE2 immunoreactivity were observed in cells coexpressing basolateral Na+-K+-ATPase. Other large ovoid cells more generally staining for NHE2 also were strongly positive for basolateral H+-ATPase. Gill mRNA levels for NHE2 and H+-ATPase did not change following systemic acidosis (as measured by quantitative PCR 2 h after a 1- or 2-meq/kg acid infusion). These data indicate that posttranslational adjustments of NHE2 and other transport systems (e.g., NHE3) following acidosis may be of importance in the short-term pH adjustment and net branchial H+ efflux observed in vivo. NHE2 may play multiple roles in the gills, involved with H+ efflux from acid-secreting cells, basolateral H+ reabsorption for pHi regulation, and in parallel with H+-ATPase for the generation of HCO3(-) in base-secreting cells.

Tribute to R. G. Boutilier: Acid–base transfer across fish gills

The gills are the major site of acid-base regulation in most fish. Acid-base transfer across fish gills is dominated by carbon dioxide and ammonia excretion, especially the former. Bicarbonate buffering in the blood is less than that found in mammals; regulation of ventilation has little effect on CO(2) levels in the blood and control of ventilation is not used to regulate body pH in fish. Proton ATPase (freshwater fish), Na(+)/H(+) exchangers (marine fish) and anion exchangers (marine and freshwater fish) are located in the gills. These transporters contribute to the regulation of internal pH, but little is known about how this is done in fish. Fish kept in confined water volumes acidify their environment, largely due to CO(2). This acidification augments ammonia excretion and reduces ammonia toxicity. The possible involvement of ammonia recycling in acid excretion is also discussed.

Acid-base regulation in fishes: cellular and molecular mechanisms.

The mechanisms underlying acid-base transfers across the branchial epithelium of fishes have been studied for more than 70 years. These animals are able to compensate for changes to internal pH following a wide range of acid-base challenges, and the gill epithelium is the primary site of acid-base transfers to the water. This paper reviews recent molecular, immunohistochemical, and functional studies that have begun to define the protein transporters involved in the acid-base relevant ion transfers. Both Na(+)/H(+) exchange (NHE) and vacuolar-type H(+)-ATPase transport H(+) from the fish to the environment. While NHEs have been thought to carry out this function mainly in seawater-adapted animals, these proteins have now been localized to mitochondrial-rich cells in the gill epithelium of both fresh and saltwater-adapted fishes. NHEs have been found in the gill epithelium of elasmobranchs, teleosts, and an agnathan. In several species, apical isoforms (NHE2 and NHE3) appear to be up-regulated following acidosis. In freshwater teleosts, H(+)-ATPase drives H(+) excretion and is indirectly coupled to Na(+) uptake (via Na(+) channels). It has been localized to respiratory pavement cells and chloride cells of the gill epithelium. In the marine elasmobranch, both branchial NHE and H(+)-ATPase have been identified, suggesting that a combination of these mechanisms may be utilized by marine elasmobranchs for acid-base regulation. An apically located Cl(-)/HCO(3)(-) anion exchanger in chloride cells may be responsible for base excretion in fresh and seawater-adapted fishes. While only a few species have been examined to date, new molecular approaches applied to a wider range of fishes will continue to improve our understanding of the roles of the various gill membrane transport processes in acid-base balance.

Branchial acid-base transfers in the euryhaline oyster toadfish during exposure to dilute sea water

Branchial acid-base transfers in the euryhaline oyster toadfish during exposure to dilute sea water

Mechanisms of acid-base excretion across the gills of a marine fish

Na+/H+ and Cl–/HCO3–exchanges in the branchial epithelium are thought to be primarily responsible for acid-base transfers in fish. Several different cellular mechanisms have been proposed to drive these exchanges in fresh water and marine species. We measured the acid-base balance and net H+ transfers (ΔH+) in the marine long-horned sculpin (Myoxocephalus octodecimspinosus) following acidosis. ΔH+ was determined in different groups of acid loaded (2–3 meq kg–1) animals which were: 1) adapted to seawater (SW); 2) adapted to 20% SW; 3) exposed to water with artificially low [Na+] or [Cl–]; 4) exposed to water containing 1 × 10–4 M amiloride, 5-(N,N-hexamethylene)-amiloride (HMA), or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). Both seawater and 20% SW adapted fish were able to completely compensate for the infused load and over 24 hours typically over-excreted more than 2× the amount infused. A 30% decrease in plasma PCO2 following the metabolic acidosis in sculpin adpated to 20% SW (presumably secondary to respiratory alterations) contributed to the rapid recovery of blood pH. Low ambient [Na+] reversed normal acid excretion to an uptake (HCO3– loss; even after acid infusion). 20–30 mM Na+ in the water was necessary to induce a positive ΔH+. A reversible inhibition of ΔH+ was also observed in sculpin exposed to either amiloride or HMA during the acidosis. In contrast, low [Cl–] or DIDS enhanced ΔH+ excretion. We conclude that net H+ excretion measured following acidosis in these seawater or brackish water adapted animals is the sum of parallel (and counter acting) apical gill Na+/H+ and Cl–/HCO3– exchanges. The Na+/H+ transfers are most likely via an antiporter of the NHE family and occur on the background of continued “band-3” Cl–/HCO3– exchange. J. Exp. Zool. 279:509–520, 1997.© 1997 Wiley-Liss, Inc.

ACID-BASE REGULATION, BRANCHIAL TRANSFERS AND RENAL OUTPUT IN A MARINE TELEOST FISH (THE LONG-HORNED SCULPIN MYOXOCEPHALUS OCTODECIMSPINOSUS) DURING EXPOSURE TO LOW SALINITIES

A number of studies have implied a linkage between acid­base and ion exchanges in both freshwater and seawater fish, although little is known about the branchial and renal acid­base transfers involved as the animals move between different salinities. To investigate the role of these transfers in a marine teleost fish as it is exposed to a dilute environment, we measured plasma acid­base values and net movements from fish to water of NH4+, HCO3- and H+ in long-horned sculpin (Myoxocephalus octodecimspinosus) placed in 100 %, 20 %, 8 % or 4 % sea water for 24­48 h. Renal excretion of H+ was also monitored in fish exposed to 4 % sea water. Sculpin proved to be somewhat euryhaline for they were able to maintain plasma ion and acid­base transfers in hypo-osmotic (20 %) sea water, but could not tolerate greater dilutions for more than several days. Plasma pH and carbon dioxide concentration (CCO2) increased in the 20 % and 8 % dilution groups, with CCO2 nearly doubling (control, 4.56 mmol l-1; 8 % group, 8.56 mmol l-1) as a result of a combined increase in the partial pressure of plasma CO2 (PCO2) and [HCO3-]. During a 44­46 h exposure, HCO3- transfers increased progressively in the most dilute water, with animals in the 8 % and 4 % groups exhibiting a net H+ loss that was smaller than that of seawater fish (control, 5.1 mmol kg-1; 8 %, 0.9 mmol kg-1; 4 %, -2.9 mmol kg-1). Animals exposed to 4 % sea water for 24 h and then returned to normal sea water had a variable plasma pH, an elevated CCO2 and a net efflux of H+ that effectively stopped (control, 0.10 mmol kg-1 h-1; 4 %, 0.02 mmol kg-1 h-1; seawater recovery, 0.20 mmol kg-1 h-1) during the low-salinity period. Renal acid excretion remained relatively constant throughout the experiment but only made up a significant portion (approximately 40 %) of the total acid transfers during the 4 % dilution period (control rate approximately 3 µmol kg-1 h-1: 3 % of branchial rate). We postulate that the increase in plasma CCO2 during exposure to low salinity may be due to mobilization of base from the intracellular bone compartment. The decrease in external salinity could induce base loss by alteration of gill ion exchanges (Na+/H+, Cl-/HCO3-) and/or changes in branchial HCO3- permeability. For the first time, we have shown that the effects of a dilute environment on acid­base transfers may be an important limitation to the survival of a euryhaline species in brackish or fresh water.

Branchial Ion and Acid-Base Transfer in Freshwater Teleost Fish: Environmental Hyperoxia as a Probe

In freshwater fish, linked ion and acid-base fluxes across the gills are dynamically manipulated to regulate internal acid-base status in the face of environmental challenges. Relevant theory, methodology, and a synthesis of current ideas are presented, based on experiments with hypercapnia, low pH, acid and base infusion, temperature change, enforced exercise, toxicants, and other challenges. Hyperoxia may be the most useful of these treatments, for it is devoid of confounding factors caused by other probes-high water [H⁺], catecholamine mobilization, and metabolic rate changes. In rainbow trout, hyperoxia (inspired \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage{wasysym} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document}${\mathrm{P_{O_{2}}}[{\mathrm{P_{IO_{2}}}}] > 500 {\mathrm{Tor...

Diffusion of inorganic materials in soil

Long-established theory concerning the diffusion of solutes in soil is described, with particular reference to the means by which pH changes are propagated through soil by acid-base transfer. The application of the theory is then demonstrated by way of models of the reaction with soil or two important fertilizer materials (urea and rock phosphate) and a model of the diffusion and oxidation of ferrous iron in the rhizosphere of lowland rice. Experimental verifications of the urea and rock phosphate models are given, in which reactant concentration profiles in the soil are compared with model predictions based on independently measured parameter values. No parameter-fitting is used. A sensitivity analysis of the urea model shows how loss of urea-derived nitrogen by ammonia volatilization depends on fertilizer, soil and atmospheric parameters. A sensitivity analysis of the rock phosphate model shows how the rate of rockphosphate dissolution and the amount of dissolved phosphorus taken up by plant roots depend on fertilizer, soil and plant parameters including acid-base release by roots. A sensitivity analysis of the iron diffusion and oxidation model shows how rice roots may greatly alter chemical, conditions in the soil near them, with major implications for nutrient and toxin transformations.

Respiratory control of arterial pH as temperature changes in rainbow trout Salmo gairdneri.

Respiratory control of arterial pH as temperature changes in rainbow trout Salmo gairdneri.