Ouabain-sensitive 86Rb(K) influx is linked to transepithelial Na transport in pig kidney cell line

The pig kidney cell line, LLC-PK₁, exhibits rheogenic d-glucose coupled transepithelial Na⁺ transport that is inhibited by phlorizin. By measuring the difference in initial rates of influx of ⁸⁶Rb⁺ with and without coupled Na⁺ transport, we can demonstrate an ⁸⁶Rb⁺ uptake linked to Na⁺ transport. The simultaneous determination of phlorizin-inhibited Na coupled d-[³H]glucose uptake and ⁸⁶Rb⁺ influx allows calculation of an Na⁺/Rb⁺ stoichiometry that is consistent with an electrogenic Na⁺ for Rb⁺ exchange.     

Potassium and Phosphate Uptake in Corn Roots: Further Evidence for an Electrogenic H/K Exchanger and an OH/Pi Antiporter

Evidence is presented that K⁺ uptake in corn root segments is coupled to an electrogenic H⁺/K⁺ -exchanging plasmalemma ATPase while phosphate uptake is coupled to an OH⁻/Pi antiporter. The plasmalemma ATPase inhibitor, diethylstilbestrol, or the stimulator, fusicoccin, altered K⁺ uptake directly and phosphate uptake indirectly. On the other hand, mersalyl, an OH⁻/Pi antiporter inhibitor, inhibited phosphate uptake instantly but only slightly affected K⁺ uptake. Collapse of the proton gradient across the membrane by (p-trifluoromethoxy) carbonyl cyanide phenylhydrazone resulted in immediate inhibition of K⁺ uptake but only later inhibited phosphate uptake. Changing the pH of the absorption solution had opposite effects on K⁺ and phosphate uptake. In addition, a 4-hour washing of corn root tissue induced a 5-fold increase in the rate of K⁺ uptake with little or no lag, but only a 2- to 3-fold increase in phosphate uptake with a 30- to 45-minute lag. Collectively these differences strongly support the coupling of an electrogenic H⁺/K⁺ -exchanging ATPase to an OH⁻/Pi antiporter in corn root tissue.     

Sucrose and proton cotransport in Ricinus cotyledons

In Ricinus cotyledons, evidence for proton extrusion came from observation of direct acidification of the medium in the presence of potassium salts. Increasing K⁺ influx with increasing pH suggested a link between K⁺ influx and H⁺ efflux by an H⁺ pump. The kinetics of K⁺ influx and H⁺ efflux were consistent with a 1:1 stoichiometry K⁺:H⁺, which may indicate either electrical coupling or carrier mediated exchange. The results were consistent with an H⁺ pump setting up an electrochemical potential gradient which provides the driving force for an H⁺-sucrose cotransport and the movement of K⁺. With reference to this, a model for phloem loading is suggested.     

Membrane H+ Conductance of Clostridium thermoaceticum and Clostridium acetobutylicum: Evidence for Electrogenic Na+/H+ Antiport in Clostridium thermoaceticum

H⁺ conductance in de-energized cells of Clostridium thermoaceticum and Clostridium acetobutylicum was determined from the rate of realkalinization of the medium after an acid pulse. In both organisms, cell membrane proton permeability was increased by fermentation end products and ionophores. In C. thermoaceticum, H⁺ conductance was increased by Na⁺ ions compared with K⁺ as counterions. In these cells, addition of Na⁺, but not K⁺, elicited efflux of H⁺; H⁺ efflux was stimulated by SCN⁻ and decreased by various ionophores. We concluded that C. thermoaceticum possesses an electrogenic Na⁺/H⁺ antiporter. In contrast, C. acetobutylicum cells did not have an electrogenic Na⁺/H⁺ antiporter.     

Anaerobic ammonia-oxidation coupled with Fe3+ reduction by an anaerobic culture from a piggery wastewater acclimated to NH4 +/Fe3+ medium

The oxidation of ammonia coupled with the reduction of iron is a unique pathway mostly reported in soils and sediments. An anaerobic sludge from a piggery wastewater treatment plant had been acclimated to an NH⁺/Fe³⁺-rich environment to secure an enrichment culture and investigate an anaerobic ammonia oxidation coupled with an iron reduction. The enrichment culture showed an average pH of 6.8 and the concentration of mixed liquor volatile suspended solid was measured as 1,120 mg/L. The mol ratio of oxidized NH₄ ⁺ and reduced Fe³⁺ was 0.33 mol NH₄ ⁺/mol Fe³⁺. It was suggested that the culture acclimated to NH₄ ⁺/Fe³⁺ contained the anaerobic ammonia oxidizing bacteria as well and thus NH₄ ⁺ was fully oxidized to NO₃ ⁻ by the bacterial consortia. In a batch experiment using the culture, the oxidation of NH₄ ⁺ was increased as the initial concentration increased. However, it was suspected from the experimental results that other iron reducing bacteria had grown under the environment applied for the enrichment culture. As a result, it was observed that heterotrophic and autotrophic iron reducers were competing for Fe³⁺.     

Design,synthesis and biological evaluation of negatively charged 111In-DTPA-octreotide derivatives

Our previous studies indicated that ¹¹¹In-diethylenetriaminepentaacetic acid (¹¹¹In-DTPA)-octreotide derivatives with an additional negative charge by replacing N-terminal d-phenylalanine (d-Phe) with an acidic amino acid such as l-aspartic acid (Asp) or its derivative exhibited low renal radioactivity levels when compared with ¹¹¹In-DTPA-d-Phe¹-octreotide. On the basis of the findings, we designed, synthesized and evaluated two Asp-modified ¹¹¹In-DTPA-conjugated octreotide derivatives, ¹¹¹In-DTPA-Asp¹-octreotide and ¹¹¹In-DTPA-Asp⁰-d-Phe¹-octreotide. While ¹¹¹In-DTPA-Asp¹-octreotide showed negligible AR42J cell uptake, ¹¹¹In-DTPA-Asp⁰-d-Phe¹-octreotide exhibited AR42J cell uptake similar to that of ¹¹¹In-DTPA-d-Phe¹-octreotide. When administered to AR42J tumor-bearing mice, ¹¹¹In-DTPA-Asp⁰-d-Phe¹-octreotide exhibited renal radioactivity levels significantly lower than did ¹¹¹In-DTPA-d-Phe¹-octreotide at 1 and 3 h post-injection. No significant differences were observed in tumor accumulation between ¹¹¹In-DTPA-Asp⁰-d-Phe¹-octreotide and ¹¹¹In-DTPA-d-Phe¹-octreotide after 1 and 3 h injection. The findings in this study suggested that an interposition of an Asp at an appropriate position in ¹¹¹In-DTPA-d-Phe¹-octreotide would constitute a useful strategy to develop ¹¹¹In-DTPA-d-Phe¹-octreotide derivatives of low renal radioactivity levels while preserving tumor accumulation.     

Proton-pumping adenosine triphosphatase in membrane vesicles of tobacco callus: Sensitivity to vanadate and K+

H⁺-pumping adenosinetriphosphatases (ATPases, EC 3.6.1.3) were demonstrated in sealed microsomal vesicles of tobacco callus. Quinacrine fluorescence quenching was induced specifically by MgATP and stimulated by EGTA and Cl^?. Fluorescence quenching reflected a relative measure of pH gradient formation (inside acid), as it could be reversed by gramicidin (an H⁺/cation conductor) or 10 mM NH₄Cl (an uncoupler). H⁺ pumping was inhibited by tributyltin (an ATPase inhibitor) and sodium vanadate, but it was insensitive to oligomycin or fusicoccin. The vanadate concentration required to inhibit pH gradient formation was similar to that needed to inhibit KCl-stimulated Mg²⁺-ATPase activity and generation of a membrane potential (measured by ATP-dependent ³⁵SCN^? uptake). About 45% of all three activities (ATPase, pH gradient, membrane potential generation) were vanadate-insensitive, supporting the idea that non-mitochondrial membranes of plants have at least two types of electrogenic H⁺ pump.A vanadate-insensitive, H⁺-pumping ATPase previously shown by methylamine accumulation was characterized to be anion-sensitive and possibly enriched in vacuolar membranes (Churchill, K.A. and Sze, H. (1983) Plant Physiol. 71, 610–617). Yet, pH gradient formation determined by quinacrine fluorescence quenching was decreased by monovalent cations with a sequence K⁺, Rb⁺, Na⁺ > Cs⁺,Li⁺> choline, bisTris-propane. Since K⁺ stimulated ATPase activity more than Bistris-propane, K⁺ appeared to collapse formation of the pH gradient by an H⁺/K⁺ countertransport. The sensitivity to vanadate and K⁺ provides evidence that the plasma-membrane ATPase is an electrogenic H⁺ pump.     

Bacteriophage T4 transfer RNA. I. Isolation and characterization of two-phage-coded nonsense suppressors

Two phage-coded nonsense suppressors, psuf_a⁺ and psu_b⁺, have been isolated and characterized. Both were isolated as pseudo-wild type revertants of phage strains which carry multiple amber mutations. psu_a⁺ is an amber suppressor which occurs at a frequency of 10⁻¹¹ to 10⁻¹² and is indistinguishable from wild type phage in its growth on both B and K strains of Escherichia coli bacteria. psu_b⁺ may be either an amber or an ochre suppressor, which occurs at a frequency of 10⁻⁷ to 10⁻¹⁰ and makes small plaques on B strains, but grows very poorly or not at all on K strains. Phage with the characteristics of psu_a⁺ occur in populations of psu_b⁺ phage at a frequency of 10⁻⁴. Both suppressors insert serine in response to the amber codon at an efficiency of about 45%.     

SR 140333, a Novel, Selective, and Potent Nonpeptide Antagonist of the NK1 Tachykinin Receptor: Characterization on the U373MG Cell Line

The effects of a novel nonpeptide NK1 tachy-kinin receptor antagonist, SR 140333, on the functional consequences of NK1 receptor activation in a human astrocytoma cell line, U373MG, were investigated. Radioligand binding conducted with ¹²⁵l-Bolton-Hunter substance P revealed a competitive inhibition by SR 140333 and its R enantiomer SR 140603 with K_i values of 0.74 and 7.40 nM, respectively. The NK1-selective agonist, [Sar⁹,Met(O₂)¹¹]-substance P, stimulated the formation of inositol phosphates with an EC₅₀ of 3.8 × 10^?9M. SR 140333 blocked the stimulatory effect of this agonist (10^?7M) with an IC₅₀ of 1.6 × 10^?9M,whereas the effect of another NK1 agonist, septide (EC₅₀= 1.5 × 10^?8M)was antagonized with an IC₅₀ of 2.1 × 10^?10M.Enhancement of [³H]taurine release by [Sar⁹,Met(O₂)¹¹]-substance P (EC₅₀= 7.4 × 10^?9M) was also inhibited by SR 140333 with an IC₅₀ of 1.8 × 10^?9 M. SR 140603 was 10-fold less potent than SR 140333 in inhibiting inositol monophosphate formation and [³H]taurine release. The calcium mobilization induced by [Sar⁹,Met(O₂)¹¹]-substance P (10^?8M) was totally prevented by 10^?8MSR 140333. Patchclamp experiments showed that SR 140333 depressed the outward current evoked by 5 × 10^?8M [Sar⁹, Met(O₂)¹¹]-substance P with an IC₅₀ of 1.3 × 10^?9M. The expression of c-fos was stimulated by [Sar⁹,Met(O₂)¹¹]-substance P with an EC₅₀ of 2.5 × 10^?10M, an effect that was also inhibited by SR 140333 with an IC₅₀ of 1.1 × 10^?9M. The present results illustrate the sequential events of the response elicited by NK1 agonists, which were antagonized by SR 140333, demonstrating its powerful NK1 antagonist activity on a functional basis.     

Effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) upon membrane ionic exchanges in sea urchin eggs

The effect of TPA (12-O-tetradecanoylphorbol-13-acetate) upon ionic exchanges was investigated in eggs of the sea urchin Arbacia lixula. Ouabain-sensitive ⁸⁶Rb uptake and amiloride-sensitive ²⁴Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na⁺/H⁺ and Na⁺/K⁺ exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na⁺/H⁺ activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na⁺/H⁺ exchange at the same rate as fertilization, probably because of an absence of calcium-dependent events. Further fertilization of TPA-pretreated eggs triggered an enhancement of sodium pump activity when the TPA treatment duration did not exceed 10 min. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na⁺ pump, Na⁺/H⁺ exchange) without eliciting corresponding regulatory mechanisms (Na⁺ stat, pH stat).     

Interrelation between Ca2+ and K+ ions in the flocculation of two Brewers yeast strains

Summary Flocculation in two strains of Saccharomyces uvarum appears to be governed by the ratio of K⁺ and Ca²⁺ concentrations ([K⁺]/[Ca²⁺]). When the ratio is diminished either by an increased [Ca²⁺] or by a decreased [K⁺], the intensity and rate of flocculation are increased.K⁺ seems to be an antagonist of Ca²⁺ in the flocculation mechanism since enhancement of [K⁺] in the medium decreases uptake of Ca²⁺. Conversely an increase in [Ca²⁺] decreases the uptake of K⁺.     

Purification and characterization of the ouabain-sensitive H/K-ATPase from guinea-pig distal colon

Distal colon absorbs K⁺ through a Na⁺-independent, ouabain-sensitive H⁺/K⁺-exchange, associated to an apical ouabain-sensitive H⁺/K⁺-ATPase. Expression of HKα2, gene associated with this ATPase, induces K⁺-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensitive and ouabain-insensitive K⁺-ATPase activities have been described in colonocytes. However, native H⁺/K⁺-ATPases have not been identified as unique biochemical entities. Herein, a procedure to purify ouabain-sensitive H⁺/K⁺-ATPase from guinea-pig distal colon is described. H⁺/K⁺-ATPase is Mg²⁺-dependent and activated by K⁺, Cs⁺ and NH₄⁺ but not by Na⁺ or Li⁺, independently of K⁺-accompanying anion. H⁺/K⁺-ATPase was inhibited by ouabain and vanadate but insensitive to SCH-28080 and bafilomycin-A. Enzyme was phosphorylated from [³²P]-γ-ATP, forming an acyl-phosphate bond, in an Mg²⁺-dependent, vanadate-sensitive process. K⁺ inhibited phosphorylation, effect blocked by ouabain. H⁺/K⁺-ATPase is an α/β-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKα2 and Na⁺/K⁺-ATPase β1-subunit, respectively. Thus, colonic ouabain-sensitive H⁺/K⁺-ATPase is a distinctive P-type ATPase.     

Presence of a Na+/H+ exchanger in brush border membranes isolated from the kidney of the spiny dogfish,Squalus acanthias

Summary A membrane fraction, rich in brush border membranes, was prepared from renal proximal tubules of the spiny dogfish,Squalus acanthias, and the sodium-proton exchange mechanism in these membrane vesicles was investigated by both a rapid filtration technique and the fluorescence quenching of acridine organe.²²Na⁺ uptake was stimulated by an outwardly directed H⁺ gradient, and was inhibited by amiloride at a single inhibitory site with an apparentK _i of approximately 1.7×10^–5 M. In the presence of an H _i ⁺ >H _o ⁺ gradient, the of the Na⁺/H⁺ exchanger were 9.7±0.8 mM and 48.0±12.0 nmol·mg protein^–1·min^–1, respectively. The uptake of Na⁺ was electroneutral in the presence of a H⁺ gradient, indicating a stoichiometry of 1. In the fluorescence studies, quenching of acridine orange occurred in the presence of an outwardly directed Na⁺ gradient which was inhibited by amiloride. Thus, an electroneutral Na⁺/H⁺ exchanger with properties similar to those found in the mammalian kidney is also present in the spiny dogfish and may contribute to the urinary acidification of this marine animal.     

Sodium-Dependent Antiporters in Choroid Plexus Epithelial Cultures from Rabbit

Abstract: The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na⁺-dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO₃^? plus 95% O₂/5% CO₂. Intracellular pH (pH_i) was determined from the steady-state distribution of [¹⁴C]benzoate. Recovery after acidification with NH₄Cl was rapid (t_1/2= 5 min) and was dependent on external Na⁺ (EC₅₀= 12 mM). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na⁺/H⁺ antiport, blocked 80% of recovery when [Na⁺] was 5 mM with IC₅₀ values of 100 nM. However, neither drug blocked recovery in normal [Na⁺]. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of Cl^?/HCO₃^? antiports, blocked recovery of pH_i in a dose-related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na⁺ channel, nor with dimethylbenzamil, an inhibitor of Na⁺/Ca²⁺ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH-clamped with nigericin and KCl, the initial rate of ²²Na⁺ uptake was very rapid (227 pmol/μg of DNA/min at pH 6.2), was dependent on external [Na⁺] with an EC₅₀ value of 8 mM, and was inversely related to the pH of the medium. The maximal inhibition of ²²Na⁺ uptake by hexamethyleneamiloride was 60% with an IC₅₀ value of 76 nM. We conclude that both the Na⁺/H⁺ antiport and a DIDS-sensitive bicarbonate-dependent antiport are important mechanisms of regulation of the internal pH of rabbit CPE under acidifying conditions. Further, our data suggest that the rabbit choroid plexus Na⁺/H⁺ exchanger can be classified as amiloride insensitive, suggesting that this antiport may play a greater role in controlling transport mechanisms than does the pH of the CNS.     

SALINITY ADAPTATION BY DUNALIELLA TERTIOLECTA. I. INCREASES IN CARBONIC ANHYDRASE ACTIVITY AND EVIDENCE FOR A LIGHT-DEPENDENT Na+/H+ EXCHANGE1,2

When Dunaliella tertiolecta, previously adapted to medium containing 0.5 M NaCl, is transferred to higher salinities, there is a lag in growth, suggesting an adaptation period. Since there is no significant difference in the Na⁺ content of cells grown between 0.5 and 3.5 M NaCl, a mechanism for Na⁺ extrusion or exclusion is indicated. Increasing the salinity of cell suspensions stimulates an incorporation of H⁺ by the cells, suggesting an H⁺/Na⁺ exchange. Cells adapted to higher salinities have, increased carbonic anhydrase activity, suggesting that increased CO₂ or HCO₃^? transport may be required at higher salinities. Growth, of D. tertiolecta at salinities above 2.5 M requires continuous illumination; therefore a light-driven H⁺/Na⁺ exchange accompanied by a HCO₃^? influx is proposed.     

Kinetics and ion specificity of Na/Ca exchange mediated by the reconstituted beef heart mitochondrial Na/Ca antiporter

The Na⁺/Ca²⁺ antiporter was purified from beef heart mitochondria and reconstituted into liposomes containing fluorescent probes selective for Na⁺ or Ca²⁺. Na⁺/Ca²⁺ exchange was strongly inhibited at alkaline pH, a property that is relevant to rapid Ca²⁺ oscillations in mitochondria. The effect of pH was mediated entirely via an effect on the K_m for Ca²⁺. When present on the same side as Ca²⁺, K⁺ activated exchange by lowering the K_m for Ca²⁺ from 2  to 0.9 μM. The K_m for Na⁺ was 8 mM. In the absence of Ca²⁺, the exchanger catalyzed high rates of Na⁺/Li⁺ and Na⁺/K⁺ exchange. Diltiazem and tetraphenylphosphonium cation inhibited both Na⁺/Ca²⁺ and Na⁺/K⁺ exchange with IC₅₀ values of 10 and 0.6 μM, respectively. The V_max for Na⁺/Ca²⁺ exchange was increased about fourfold by bovine serum albumin, an effect that may reflect unmasking of an autoregulatory domain in the carrier protein.     

H+/Ca2+ exchange in rabbit renal cortical endosomes

Summary We have examined the effect of second messengers on ATP-driven H⁺ transport in an H⁺ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1mm) had no effect on H⁺ transport. Acridine orange fluorescence in the presence of 0.5mm Ca²⁺ (+1mm EGTA) was 19±6% of control. Inhibition of ATP-driven H⁺ transport by Ca²⁺ was concentration dependent; 0.25 and 0.5mm Ca²⁺ (+1mm EGTA) inhibited acridine orange fluorescence by 50 and 80%, respectively. Ca²⁺ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca²⁺ did not affect ATP hydrolysis. ATP-dependent Br^– uptake was virtually unchanged in the presence of 0.5mm Ca²⁺ (+1mm EGTA). These vesicles were also shown to transport Ca²⁺ in an ATP-dependent mode. Inositol 1, 4, 5-trisphosphate had no effect on ATP-dependent Ca²⁺ uptake. These results are consistent with the co-existence of an H⁺ ATPase and an H⁺/Ca²⁺ exchanger on these endosomes, the latter transport system using the H⁺ gradient to energize Ca²⁺ uptake. Attempts to demonstrate an H⁺/Ca²⁺ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca²⁺ uptake could be demonstrated. A Ca²⁺-dependent increase in H⁺ permeability and an ATP-dependent Ca²⁺ uptake might have important implications for the regulation of vacuolar H⁺ ATPase activity as well as the homeostasis of cytosolic Ca²⁺ concentration.     

Early biochemical events in lymphocyte activation. I. Investigations on the nature and significance of early calcium fluxes observed in mitogen-induced T and B lymphocytes.

⁴⁵Ca²⁺ uptake was detected within minutes following addition of T- and B-cell² mitogens to mouse lymphocytes. The T-cell mitogens (Con A and PHA) gave an ~twofold increase in ⁴⁵Ca²⁺ uptake (representing an influx of ~ 130 amol per lymphocyte, corresponding to an increase in average cellular Ca²⁺ of ~0.95 mM). B-cell mitogens which gave the largest ⁴⁵Ca²⁺ uptake (~twofold) were purified LPS preparations from Salmonella minnesota R595 and Escherichia coli 0111:2125. The ⁴⁵Ca²⁺ uptake by rabbit splenocytes using specific anti-b4 allotype antiserum was comparable to that obtained with the two purified LPS preparations. A23187, in low nontoxic doses, gave an ~sixfold increase in ⁴⁵Ca²⁺ uptake with mouse T cells. The ⁴⁵Ca²⁺ uptake was modulated by cyclic nucleotides showing a “yin-yang” effect. The results suggest a possible entry of ⁴⁵Ca²⁺ from the extracellular medium through “gated Ca²⁺ channels” in the plasma membrane into the cytosol by passive diffusion. The Ca²⁺ may be sequestered in the mitochondria, and the excess Ca²⁺ is later effluxed into the extracellular medium. The fact that ⁴⁵Ca²⁺ uptake appears to be one of the earliest events occurring after ligand binding to the cell, together with the demonstration of a Ca²⁺-dependent glucose uptake and a requirement for extracellular Ca²⁺ for DNA synthesis, suggest that, as it is now known to function in many other cellular responses, Ca²⁺ may operate as a second messenger for lymphocyte activation.     

Acidosis and Ca2+ distribution in myocardial tissue of flounder and rat

Summary The effect of acidosis on the myocardial Ca²⁺ distribution was examined at 15°C in ventricular strips of the flounder (Platichthys flesus) and at 30°C in atrial strips of the rat (Rattus norvegicus).Lowering the Ringer pH from 7.6 to 6.9 by increasing its CO₂ (flounder 2% to 12%, rat 4% to 14%), resulted in an elevated Ca²⁺ efflux in resting strips as well as in strips stimulated (12/min) to contraction. A decrease in pH of the Ringer used for the flounder myocardium by a lowering of bicarbonate (30 mM to 5 mM) also resulted in an elevation of the Ca²⁺ efflux, but the effect was smaller than that produced by an increased CO₂.With 11 mM Ca²⁺ and 10 mM EGTA added to the Ringer to reduce the amount of⁴⁵Ca²⁺ bound to extracellular sites, an increased CO₂ with a concomitant drop in Ringer pH resulted in an increased Ca²⁺ efflux in both myocardia. The Ca²⁺ efflux was only marginally elevated in the flounder myocardium and unchanged in that of rat when the same drop in Ringer pH was produced with a lowering in bicarbonate.In a nominally Ca²⁺-free Ringer with 0.1 mM EGTA the⁴⁵Ca²⁺ efflux was stimulated for both myocardia by an increase in CO₂.The flounder myocardium was exposed to high CO₂ in a nominally Na⁺, Ca²⁺-free Ringer and again the⁴⁵Ca²⁺ efflux increased. After a return to Na, Ca and low CO₂ in the Ringer, a higher efflux persisted in the strips being subjected to a high CO₂ than in the controls.The Ca²⁺ uptake rate was about the same at high and low CO₂ for both myocardia.Based on these results the measured increase in Ca efflux following an increase in CO₂ or a decrease in bicarbonate probably results from an elevated cytoplasmatic Ca²⁺ activity. It seems unlikely that an increased uptake rate of Ca²⁺ or a direct stimulation of Ca²⁺ transporting mechanisms in the cell membrane are responsible for the change.     

Molecular Recognition of Adenosine Deaminase: 15N NMR Studies

The elucidation of the molecular recognition of adenosine deaminase (ADA), the interpretation of the catalytic mechanism, and the design of novel inhibitors are based mostly on data obtained for the crystalline state of the enzyme. To obtain evidence for molecular recognition of the physiologically relevant soluble enzyme, we studied its interactions with the in situ formed inhibitor, 6-OH-purine riboside (HDPR), by 1D-¹⁵N- and 2D-(¹H-¹⁵N)- NMR using the labeled primary inhibitor [¹⁵N₄]-PR. We synthesized both [¹⁵N₄]-PR and an [¹⁵N₄]-HDPR model, from relatively inexpensive ¹⁵N sources. The [¹⁵N₄]-HDPR model was used to simulate H-bonding and possible Zn²⁺-coordination of HDPR with ADA. We also explored possible ionic interactions between PR and ADA by ¹⁵N-NMR monitored pH-titrations of [¹⁵N₄]-PR. Finally, we investigated the [¹⁵N₄]-PR-ADA 1:1 complex by 2D-(¹H-¹⁵N) NMR. We found that HDPR recognition determinants in ADA do not include any ionic-interactions. HDPR N1 H is an H-bond acceptor, and not an H-bond donor. Despite the proximity of N7 to the Zn²⁺-ion, no coordination occurs; instead, N7 is an H-bond acceptor. We found an overall agreement between the crystallographic data for the crystallized ADA:HDPR complex and the ¹⁵N-NMR signals for the corresponding soluble complex. This finding justifies the use of ADA's crystallographic data for the design of novel inhibitors.