The effects of elevated pH and high salt concentrations on tubulin

The effects of incubating phosphocellulose-purified bovine tubulin at 4 degrees C in nucleotide-free buffers at alkaline pH or at high concentrations of NaCl, KCl, (NH4)2SO4, or NH4Cl have been studied. At pH greater than or equal to 7.5 or at NaCl concentrations greater than or equal to 0.7 M, tubulin releases bound nucleotides irreversibly and loses, with apparent first-order kinetics, the ability to assemble into microtubules. In 0.1 M 1,4-piperazinediethanesulfonic acid buffer, pH 6.9, in the presence of 1.3 M NH4Cl, tubulin undergoes more rapid loss of capacity to assemble than it does in NaCl and KCl, but 1.3 M (NH4)2SO4 causes no detectable change in tubulin after 1-h incubation. Incubation at high pH or at high neutral salt concentrations also causes an apparently irreversible change in the ultraviolet difference spectrum and in the sedimentation velocity profile of tubulin. At elevated salt concentrations a decrease of approximately 10% in the molar ellipticity within the wavelength range 220-260 nm is observed. The changes that occur during 1-h exposure to pH 8.0 can be completely prevented by including 1 mM guanosine 5'-triphosphate (GTP) or 4 M glycerol in the buffer, but those which occur at pH 9.0 cannot be prevented by these additions. In 1 M NaCl when the ratio of bound guanine nucleotide to tubulin reaches approximately 1.0, tubulin loses the abilities to assemble into microtubules and to bind colchicine. The rate of loss of nucleotide in 2 M NaCl is decreased in the presence of 1 mM GTP, and tubulin is protected almost completely from 1 M NaCl-induced loss of GTP (and retains the ability to exchange [3H]GTP as well) in the presence of bound colchicine. Investigators who anticipate exposing tubulin to buffers of elevated pH or high concentrations of chaotropic salts should be extremely cautious in interpreting the resulting data unless they can demonstrate that irreversible alteration of the protein has not occurred.     

Real-time measurements of the interaction between single cells of Listeria monocytogenes and nisin on a solid surface

A method to obtain real-time measurements of the interactions between nisin and single cells of Listeria monocytogenes on a solid surface was developed. This method was based on fluorescence ratio-imaging microscopy and measurements of changes in the intracellular pH (pH(i)) of carboxyfluorescein succinimidyl ester-stained cells during exposure to nisin. Immobilized cells were placed in a chamber mounted on a microscope and attached to a high-precision peristaltic pump which allowed rapid changes in the nisin concentration. In the absence of nisin, the pH(i) of L. monocytogenes was almost constant (approximately pH 8.0) and independent of the external pH in the pH range from 5.0 to 9.0. In the presence of nisin, dissipation of the pH gradient (DeltapH) was observed, and this dissipation was both time and nisin concentration dependent. The dissipation of DeltapH resulted in cell death, as determined by the number of CFU. In the model system which we used the immobilized cells were significantly more resistant to nisin than the planktonic cells. The kinetics of DeltapH dissipation for single cells revealed a variable lag phase depending on the nisin concentration, which was followed by a very rapid decrease in pH(i) within 1 to 2 min. The differences in nisin sensitivity between single cells in a L. monocytogenes population were insignificant for cells grown to the stationary phase in a liquid laboratory substrate, but differences were observed for cells grown on an agar medium under similar conditions, which resulted in some cells having increased resistance to nisin.     

The apoplastic pH of the substomatal cavity of Vicia faba leaves and its regulation responding to different stress factors.

The apoplastic pH of the substomatal cavity is an essential determinant of stomatal movement. In detached leaves of Vicia faba substomatal apoplastic pH and its dependence on external (stress) factors was investigated using a non-invasive approach: pH-microsensors were inserted into open stomata, and upon contact with the apoplastic fluid, pH was measured continuously, as apoplastic pH was challenged by changed conditions of light, atmosphere (NH(3), CO(2)), and xylem sap (abscisic acid, cyanide, fusicoccin, pH, inorganic salts). Apoplastic pH proved extremely sensitive to infiltration and local flooding, which rapidly increased the apoplastic pH by more than 1.5 pH units. Recovery from infiltration took several hours, during which light effects on the apoplastic pH were strongly impeded. This indicates that pH tests carried out under such conditions may not be representative of the undisturbed leaf. NH(3), flushed across the stomata, yielded a rapid apoplastic alkalinization from which an apoplastic buffer capacity of 2-3 mM per pH unit was calculated. Fusicoccin, fed into the xylem sap acidified the apoplast, whereas cyanide alkalized it, thus underscoring the importance of the plasma membrane H(+) pump for apoplastic pH regulation. To address the question to what extent pH was a drought signal, the effect of iso-osmotic pH changes, fed into the xylem through the petiole were tested. It is demonstrated that the apoplastic response remained below 0.1 pH per pH unit imposed, regardless of the buffer capacity. An increase in the osmolarity of the bath solution (harbouring the cut petiole) using KCl, NaCl, CaCl(2) or sorbitol alkalized the substomatal apoplast. It is suggested that pH may only act as drought signal when accompanied by elevated osmolarity.     

Buffer power and intracellular pH of frog sartorius muscle.

Intracellular pH (pHi) and buffer power of frog muscle were measured using pH-sensitive microelectrodes under conditions used previously in energy balance experiments because pH strongly influences the molar enthalpy change for phosphocreatine splitting, the major net reaction during brief contractions. The extracellular pH (pHe) of HEPES buffered Ringer's solution influenced pHi, but change in pHi developed slowly. Addition or removal of CO2 or NH3 from the extracellular solution caused a rapid change in pHi. The mean buffer power measured with CO2 was 38.4 mmol.l-1.pH unit-1 (+/- SEM 2.1, n = 49) and with NH3 was 36.2 (+/- SEM 5.5, n = 4) at 20-22 degrees C. At 5 degrees C, in experiments with CO2 the mean buffer power was 40.3 (+/- SEM 2.6, n = 3). For pHi values above approximately 7.0, the observed buffer power was greater than that expected from the values in the literature for the histidine content of intracellular proteins, carnosine and inorganic phosphate in the sarcoplasm. The measured pHi values were similar to those assumed in energy balance calculations, but the high measured buffer power suggests that other buffering reactions occur in addition to those included in energy balance calculations.     

Impaired intestinal NHE3 activity in the PDK1 hypomorphic mouse

In vitro experiments have demonstrated the stimulating effect of serum- and glucocorticoid-inducible kinase (SGK)1 on the activity of the Na+/H+ exchanger (NHE3). SGK1 requires activation by phosphoinositide-dependent kinase (PDK)1, which may thus similarly play a role in the regulation of NHE3-dependent epithelial electrolyte transport. The present study was performed to explore the role of PDK1 in the regulation of NHE3 activity. Because mice completely lacking functional PDK1 are not viable, hypomorphic mice expressing approximately 20% of PDK1 (pdk1(hm)) were compared with their wild-type littermates (pdk1(wt)). NHE3 activity in the intestine and PDK1-overexpressing HEK-293 cells was estimated by utilizing 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence for the determination of intracellular pH. NHE activity was reflected by the Na+-dependent pH recovery from an ammonium prepulse (DeltapH(NHE)). The pH changes after an ammonium pulse allowed the calculation of cellular buffer capacity, which was not significantly different between pdk1(hm) and pdk1(wt) mice. DeltapH(NHE) was in pdk1(hm) mice, only 30 +/- 6% of the value obtained in pdk1(wt) mice. Conversely, DeltapH(NHE) was 32 +/- 7% larger in PDK1-overexpressing HEK-293 cells than in HEK-293 cells expressing the empty vector. The difference between pdk1(hm) and pdk1(wt) mice and between PDK1-overexpressing and empty vector-transfected HEK cells, respectively, was completely abolished in the presence of the NHE3 inhibitor S3226 (10 microM). In conclusion, defective PDK1 expression leads to significant impairment of NHE3 activity in the intestine, pointing to a role of PDK1-dependent signaling in the regulation of NHE-mediated electrolyte transport.     

Ammonium ions enhance the flow through the pyruvate dehydrogenase in Ehrlich ascites tumor cells

The flow through pyruvate dehydrogenase was assayed in glycolysing cells by the evolution of 14CO2 from [1-14C] pyruvate. Parallel incubations were carried out in high bicarbonate buffer (25 mM) and in bicarbonate-free buffer. The activation of the complex by NH+4 was only observed in high bicarbonate buffer, because the dilution of labelled CO2 in the presence of an excess of bicarbonate enables the quantitative determination of labelled CO2 evolved from pyruvate in the decarboxylase step. In the bicarbonate-free buffer the activation of the complex was not observed, because the 14CO2 evolved from pyruvate was consumed by biosynthetic processes inside the cell. On the contrary in isolated hepatocytes the NH+4 activation of the pyruvate dehydrogenase was observed in both buffers. In Ehrlich ascites cells, in common with other mammalian tissues, pyruvate dehydrogenase activity was found to be inversely correlated to the intramitochondrial ATP/ADP ratio.     

Protective effect of sucrose and sodium chloride for Lactococcus lactis during sublethal and lethal high-pressure treatments

The bactericidal effect of hydrostatic pressure is reduced when bacteria are suspended in media with high osmolarity. To elucidate mechanisms responsible for the baroprotective effect of ionic and nonionic solutes, Lactococcus lactis was treated with pressures ranging from 200 to 600 MPa in a low-osmolarity buffer or with buffer containing 0.5 M sucrose or 4 M NaCl. Pressure-treated cells were characterized in order to determine viability, the transmembrane difference in pH (DeltapH), and multiple-drug-resistance (MDR) transport activity. Furthermore, pressure effects on the intracellular pH and the fluidity of the membrane were determined during pressure treatment. In the presence of external sucrose and NaCl, high intracellular levels of sucrose and lactose, respectively, were accumulated by L. lactis; 4 M NaCl and, to a lesser extent, 0.5 M sucrose provided protection against pressure-induced cell death. The transmembrane DeltapH was reversibly dissipated during pressure treatment in any buffer system. Sucrose but not NaCl prevented the irreversible inactivation of enzymes involved in pH homeostasis and MDR transport activity. In the presence 0.5 M sucrose or 4 M NaCl, the fluidity of the cytoplasmic membrane was maintained even at low temperatures and high pressure. These results indicate that disaccharides protect microorganisms against pressure-induced inactivation of vital cellular components. The protective effect of ionic solutes relies on the intracellular accumulation of compatible solutes as a response to the osmotic stress. Thus, ionic solutes provide only asymmetric protection, and baroprotection with ionic solutes requires higher concentrations of the osmolytes than of disaccharides.     

CO2 enhanced peroxidase activity of SOD1: the effects of pH

At pH 7.4, CO2, rather than HCO3-, markedly enhances the oxidation of diverse substrates by SOD1 plus H2O2. Since the concentration of CO2 would fall with rising pH in HCO3- buffers, it was of interest to explore the effects of pH on the peroxidase activity of SOD1 in the presence and in the absence of HCO3-. The rate of NADPH peroxidation in the HCO3- buffer was minimally affected by pH in the range of 8-10.5; in a pyrophosphate buffer, the rate increased markedly, such that at pH 10.5 the rates in the two buffers were nearly identical. Similar results were obtained when urate was used as the peroxidizeable substrate. These results are explicable on the basis of an increase in the rate with pH due to the ionization of H2O2 to the effective HO2- coupled with a decrease in [CO2] due to the ionizations of H2CO3, which displaces the hydration equilibrium to the right. These two opposing effects counteract in the HCO3(-)-buffered reaction mixtures; in the pyrophosphate buffer, only the effect of increasing [H02-] was seen.     

Experimental generation and computational modeling of intracellular pH gradients in cardiac myocytes

It is often assumed that pH(i) is spatially uniform within cells. A double-barreled microperfusion system was used to apply solutions of weak acid (acetic acid, CO(2)) or base (ammonia) to localized regions of an isolated ventricular myocyte (guinea pig). A stable, longitudinal pH(i) gradient (up to 1 pH(i) unit) was observed (using confocal imaging of SNARF-1 fluorescence). Changing the fractional exposure of the cell to weak acid/base altered the gradient, as did changing the concentration and type of weak acid/base applied. A diffusion-reaction computational model accurately simulated this behavior of pH(i). The model assumes that H(i)(+) movement occurs via diffusive shuttling on mobile buffers, with little free H(+) diffusion. The average diffusion constant for mobile buffer was estimated as 33 x 10(-7) cm(2)/s, consistent with an apparent H(i)(+) diffusion coefficient, D(H)(app), of 14.4 x 10(-7) cm(2)/s (at pH(i) 7.07), a value two orders of magnitude lower than for H(+) ions in water but similar to that estimated recently from local acid injection via a cell-attached glass micropipette. We conclude that, because H(i)(+) mobility is so low, an extracellular concentration gradient of permeant weak acid readily induces pH(i) nonuniformity. Similar concentration gradients for weak acid (e.g., CO(2)) occur across border zones during regional myocardial ischemia, raising the possibility of steep pH(i) gradients within the heart under some pathophysiological conditions.     

Real-time monitoring of nasal mucosal pH during carbon dioxide stimulation: implications for stimulus dynamics

Carbon dioxide is a commonly employed irritant test compound in nasal chemesthetic studies because it is essentially free of olfactory stimulus properties. CO(2) is thought to act via hydration to H(2)CO(3) and dissociation to H(+) in nasal mucus, with resulting activation of acid sensors. However, transient changes in nasal mucosal pH have not been documented during CO(2) stimulation in humans. We placed a small pH probe on the floor of the right anterior nasal cavity during CO(2) stimulation in eight human subjects with historically high (>30%) and low (< or =20%) CO(2) detection thresholds. Three second pulses of CO(2) (15-45% v/v) paired with air in random order (12-15 s inter-stimulus interval; 60 s inter-trial interval) were administered by nasal cannula at 5 l/min. in an ascending series. For each subject, both a CO(2) detection threshold and suprathreshold psychophysical ratings [psi; labeled magnitude scale] were generated. All subjects showed phasic drops in pH associated with CO(2) stimulation (DeltapH). For all subjects combined, a positive correlation was apparent between applied [CO(2)] and both DeltapH and psi, as well as between DeltapH and psi themselves (P < 0.0001 for each comparison). Subjects with historically low CO(2) thresholds showed steeper dose-response curves for psi as a function of both applied [CO(2)] and DeltapH, but not for DeltapH as a function of applied [CO(2)]. For the six of eight subjects with measurable pH changes at threshold, DeltapH was positively related to log [CO(2) threshold] (P < 0.01). These data imply that variability in CO(2) detection thresholds and suprathreshold rating may derive from intrinsic differences in neural sensitivity, rather than differences in stimulus activation to hydrogen ion.     

pH and buffering in the bicinchoninic acid (4,4'-dicarboxy-2,2'-biquinoline) protein assay

The HCO3/CO3(2-) buffer used in the bicinchoninic acid (BCA) protein assay has only weak buffering capacity at the recommended pH (11.25). Consequently the assay is rather sensitive to interference from effectively acid or alkaline samples, particularly in the micro method. Adjustment of pH in these alkaline solutions of high [Na+] is complicated by Na+ errors on the pH electrode. Hence it is recommended to prepare the buffers from known amounts of NaHCO3 and Na2CO3, and to reduce the pH to around 10.7; this offers much better buffering capacity with only a limited reduction in color development.     

Divalent metal ion effect on helix-coil transition of high molecular weight DNA in neutral and alkaline solutions

Effect of Mg(2+), Ca(2+), Ni(2+) and Cd(2+) ions on parameters of DNA helix-coil transition in sodium cacodylate (pH 6.5), Tris (pH 8.5) and sodium tetraborate (pH 9.0) buffers have been studied by differential UV-visible spectroscopy and by thermal denaturation. Anomalous behavior of the melting temperature T(m) and the melting interval ΔT in the presence of MgCl(2) was observed in Tris, but not in cacodylate or tetraborate buffers. Changes in the buffer type and pH did not influence T(m) and ΔT dependence on Ca(2+) and Cd(2+) concentrations. Decrease of the T(m) and ΔT of DNA in the presence of Ni(2+) and Cd(2+) was caused by preferential ion interaction with N7 of guanine. This type of interaction was also found for Mg(2+) in Tris buffer. The anomalous decrease in the T(m) and ΔT values was connected to formation of complexes between metal ions and Tris molecules. Transition of DNA single-stranded regions into a compact form with the effective radius of the particles of 300±100 ? was induced by Mg(2+) ions in Tris buffer.     

High-activity biocatalysts in organic media: solid-state buffers as the immobilisation matrix for protein-coated microcrystals

Recently, we reported a new high-activity biocatalyst for use in organic media termed protein-coated microcrystals (PCMC) (Kreiner et al. [2001] Chem Commun 12:1096-1097). These novel particles consist of water-soluble micron-sized crystalline particles coated with the given biocatalyst(s) and are prepared in a one-step rapid dehydration process. In this study we extended the choice of immobilisation matrix from a simple inorganic salt, K(2)SO(4), to other compounds, both inorganic and zwitterionic, that act as solid-state buffers for biocatalysis in organic media. The catalytic activity of serine proteases subtilisin Carlsberg (SC) and alpha-chymotrypsin (CT) were significantly increased when coated onto the surface of solid-state buffers, as measured in acetonitrile/1wt% H(2)O. SC-PCMC with both organic and inorganic buffer carriers (Na-AMPSO, Na(2)CO(3), and NaHCO(3)) showed a 3-fold greater activity than that observed when using the unbuffered system (PCMC-SC/K(2)SO(4)). In comparison with freeze-dried preparations, this represents an approximately 3,000-fold increase in catalytic activity. Importantly, there is no improvement in catalytic activity upon external addition of any of the solid-state buffers to the reaction mixture. When acting in a solid-state buffer capacity, good buffering capacity was observed with SC-PCMC (3 wt% protein loading) prepared from a 1:1 mixture of AMPSO and AMPSO-Na. Alternatively, increasing the amount of solid-state buffer in the system allows improvement of the buffering. This can be achieved either by decreasing the protein loading of the SC/Na-AMPSO-PCMC or by addition of further external solid-state buffer to the reaction mixture. The catalytic activity of lipase-PCMC prepared from solid-state buffers was found less responsive to immobilisation.     

Modulation of rat chorda tympani NaCl responses and intracellular Na+ activity in polarized taste receptor cells by pH

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH (pH(i)) and Na(+) activity ([Na(+)](i)) in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the lingual surface with NaCl solutions adjusted to pHs ranging between 2.0 and 10.3 increased the magnitude of NaCl CT responses linearly with increasing external pH (pH(o)). At pH 7.0, the epithelial sodium channel (ENaC) blocker, benzamil, decreased NaCl CT responses and inhibited further changes in CT responses induced by varying pH(o) to 2.0 or 10.3. At constant pH(o), buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HCO(3)(-)/CO(2) inhibited NaCl CT responses relative to CT responses obtained with NaCl solutions buffered with HEPES. The carbonic anhydrase blockers, MK-507 and MK-417, attenuated the inhibition of NaCl CT responses in HCO(3)(-)/CO(2) buffer, suggesting a regulatory role for pH(i). In polarized TRCs step changes in apical pH(o) from 10.3 to 2.0 induced a linear decrease in pH(i) that remained within the physiological range (slope = 0.035; r(2) = 0.98). At constant pH(o), perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO(3)(-)/CO(2) decreased resting TRC pH(i), and MK-507 or MK-417 attenuated the decrease in pH(i) in TRCs perfused with HCO(3)(-)/CO(2) buffer. In parallel experiments, TRC [Na(+)](i) decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical Na(+), and (d) acid loading the cells with NH(4)Cl or sodium acetate at constant pH(o). Diethylpyrocarbonate and Zn(2+), modification reagents for histidine residues in proteins, attenuated the CO(2)-induced inhibition of NaCl CT responses and the pH(i)-induced inhibition of apical Na(+) influx in TRCs. We conclude that TRC pH(i) regulates Na(+)-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures.     

Inhibition of bovine spermatozoa by caudal epididymal fluid: II. Interaction of pH and a quiescence factor

Previous studies (Carr and Acott , 1984) indicate that bovine sperm are maintained in a quiescent state in the caudal epididymis (CE) by a pH-dependent inhibitory factor. Here, we have determined that the pH of bovine CE fluid and of CE semen is approximately 5.8, and that the motility of CE sperm in undiluted CE fluid increases as the pH is elevated. Therefore, the acidity of CE fluid may play a physiological role in the maintenance of sperm quiescence. The changes in sperm motility, in response to changes in the pH of CE fluid, are reversible and rapid. Dilution of CE fluid with buffers at either pH 5.5 or 7.6 produces a much slower initiation of motility. In buffer a significantly lower pH is required to inhibit sperm motility than is required in CE fluid. The apparent pKs for inhibition are 5.3 in buffer and 6.6 in CE fluid. However, the motility of sperm in buffers that contain lactate, shows a pH dependence similar to sperm in CE fluid. That is, lactate inactivates sperm in buffer at pH 5.5 but not at pH 7.6. Lactate, and several other permeant weak acids, have previously been shown to reduce the intracellular pH of bovine sperm and many other types of cells. We show that these permeant weak acids, but not impermeant weak acids, reversibly reduce CE sperm motility in buffer at pH 5.5 but not at pH 7.6. Weak bases, which have previously been shown to elevate intracellular pH, initiate sperm motility in CE fluid. These results suggest that intracellular pH can regulate CE sperm motility and may be the intracellular messenger for the pH-dependent quiescence factor. Although sperm cyclic AMP levels have been previously correlated with motility stimulation, cyclic AMP levels do not change when the pH of CE fluid is elevated, even though full motility is initiated.     

Basolateral membrane Na/base cotransport is dependent on CO2/HCO3 in the proximal convoluted tubule

The mechanism of basolateral membrane base transport was examined in the in vitro microperfused rabbit proximal convoluted tubule (PCT) in the absence and presence of ambient CO2/HCO3- by means of the microfluorometric measurement of cell pH. The buffer capacity of the cells measured using rapid NH3 washout was 42.8 +/- 5.6 mmol.liter-1.pH unit-1 in the absence and 84.6 +/- 7.3 mmol.liter-1.pH unit-1 in the presence of CO2/HCO3-. In the presence of CO2/HCO3-, lowering peritubular pH from 7.4 to 6.8 acidified the cell by 0.30 pH units and lowering peritubular Na from 147 to 0 mM acidified the cell by 0.25 pH units. Both effects were inhibited by peritubular 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS). In the absence of exogenous CO2/HCO3-, lowering peritubular pH from 7.4 to 6.8 acidified the cell by 0.25 pH units and lowering peritubular Na from 147 to 0 mM decreased cell pH by 0.20 pH units. Lowering bath pH from 7.4 to 6.8 induced a proton flux of 643 +/- 51 pmol.mm-1.min-1 in the presence of exogenous CO2/HCO3- and 223 +/- 27 pmol.mm-1.min-1 in its absence. Lowering bath Na from 147 to 0 mM induced proton fluxes of 596 +/- 77 pmol.mm-1.min-1 in its absence. The cell acidification induced by lowering bath pH or bath Na in the absence of CO2/HCO3- was inhibited by peritubular SITS or by acetazolamide, whereas peritubular amiloride had no effect. In the absence of exogenous CO2/HCO3-, cyanide blocked the cell acidification induced by bath Na removal, but was without effect in the presence of exogenous CO2/HCO3-. We reached the following conclusions. (a) The basolateral Na/base n greater than 1 cotransporter in the rabbit PCT has an absolute requirement for CO2/HCO3-. (b) In spite of this CO2 dependence, in the absence of exogenous CO2/HCO3-, metabolically produced CO2/HCO3- is sufficient to keep the transporter running at 30% of its control rate in the presence of ambient CO2/HCO3-. (c) There is no apparent amiloride-sensitive Na/H antiporter on the basolateral membrane of the rabbit PCT.     

Effect of ethanesulfonic acid buffers and pH on the accumulation of a nervous system-specific protein (S-100) and a glial-enriched enzyme in a clonal line of rat astrocytes (C6).

Stationary phase cultures of a clonal line of rat astrocytes (C6) were maintained at pH values ranging from 6.0 to 8.4 using media buffered with various combinations of organic buffers or graded concentrations of bicarbonate ion at a constant CO2 tension. The accumulation of a soluble acidic protein unique to the nervous system (S-100) in media buffered with organic buffers was optimal in the pH range 6.4 to 6.8, significantly more acid than that optimal for cell growth (pH 7.0 to 7.8). Cells maintained in CO2-bicarbonate-buffered media exhibited a higher and less marked pH optimum for S-100 protein accumulation and a lower efficiency of accumulation of the protein. These data suggest that the organic buffer ions themselves, apart from their function as buffers, are influencing the accumulation of S-100. The specific activity (assayed at the enzymatic pH optimum) of a membrane-bound enzyme enriched in glial cells and myelin, 2',3'-cyclic nucleotide 3'-phosphohydrolase, was markedly pH-dependent. The optimal pH range was 6.4 to 6.7 in organic buffer controlled media. In CO2-bicarbonate controlled media the optimal pH range was only slightly higher (pH 6.6 to 7.0), but the specific activities were reduced relative to organic buffer-grown cells. The structural relationship of some of the aminoethanesulfonic acid buffers used in these experiments to certain compounds of neurochemical interest (such as taurine and alpha-flupenthixol) is noted.     

Buffer dependence of CO2 hydration catalyzed by human carbonic anhydrase I.

The steady-state kinetics of CO2 hydration catalyzed by human carbonic anhydrase I (carbonate hydro-lyase, EC 4.2.1.1) has been investigated at three pH values corresponding to different parts of the pH-rate profile. Two buffer systems with similar pKa values were used at each pH. The results show that the catalyzed rates depend on the buffer concentration but also on the chemical nature of the buffer. For example, at pH 8.8 the buffer 1,2-dimethylimidazole behaves formally as a second substrate in a 'ping-pong' mechanism yielding a maximal kcat value of 2.2 x 10(5) s-1, whereas much lower rates were obtained with Taps buffers. Similarly, at pH 7.3 1-methylimidazole yields higher rates than Mops and at pH 6.3 3,5-lutidine is more efficient than Mes. Non-Michaelis-Menten kinetics were observed with all buffers except 1,2-dimethylimidazole. In addition, while the apparent buffer activation by 1,2-dimethylimidazole can be described by a single Km value of 26 mM, the Mes concentration dependence is consistent with the presence of two components of similar magnitudes with Km values of 45 mM and 0.15 mM. These results are interpreted within the framework of the 'zinc-hydroxide' mechanism in terms of multiple pathways for the rate-contributing transfer of a proton from the zinc-bound water molecule, formed during CO2/HCO3- interconversion, to the reaction medium, thus, regenerating zinc-bound OH-.     

Mechanistic studies of carboxypeptidase Y from Saccharomyces cerevisiae. pH and pD profiles and inactivation at low pH (pD) values.

Steady-state kinetics of carboxypeptidase Y, a proteinase from yeast, were studied by using the reaction of 4-nitrophenyl trimethylacetate as a probe. The pH profile of kcat. is sigmoidal in H2O-based buffers for the carboxypeptidase Y-catalysed hydrolysis of this ester (kcat. referring to the rate of deacylation of trimethylacetyl-carboxypeptidase Y). The corresponding pD profile in 2H2O is doubly sigmoidal, with inflexions at pD approximately 3.8 and approximately 6.8. The ionization of pKDapp. approximately 3.8 is caused by a rapid inactivation in 2H2O media by a process that is only slowly reversed on transfer to pH 7.00 phosphate buffer in H2O. The corresponding inactivation in H2O-based buffers of low pH is considerably slower (approximately 30-fold), follows a first-order rate-dependence and is very strongly pH-dependent, indicating some form of co-operative change in enzyme tertiary structure.     

Intracellular alkalinization leads to Ca2+ mobilization from agonist-sensitive pools in bovine aortic endothelial cells

Receptor-stimulated phosphoinositide turnover leads to activation of Na+/H+ exchange and subsequent intracellular alkalinization. To probe the effect of increased intracellular pH (pHi) on Ca2+ homeostasis in cultured bovine aortic endothelial cells (BAEC), we studied the effect of weak bases, ammonium chloride (NH4Cl) and methylamine (agents which increase pHi by direct passive diffusion), on resting and ATP (purinergic receptor agonist)-induced Ca2+ fluxes. Changes in cytosolic free Ca2+ ([Ca2+]i) or pHi were monitored in BAEC monolayers using the fluorescent dyes, fura-2 or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively. NH4Cl-induced, dose-dependent (5-20 mM) increases in [Ca2+]i (maximum change = 195 +/- 26 nM) which were temporally similar to the NH4Cl-induced pHi increases. Methylamine (20 mM) induced a more sustained pHi increase and also stimulated a prolonged [Ca2+]i increase. When BAEC were bathed in HCO3- buffer, removal of extracellular CO2/bicarbonate caused pHi to increase and also induced [Ca2+]i to increase transiently. Extracellular Ca2+ removal did not abolish the rapid NH4Cl-induced rise in [Ca2+]i, although the response was blunted and more transient. NH4Cl addition to BAEC cultures resulted in an increase in 45Ca efflux and decrease in total cell 45Ca content. BAEC treatment with ATP (100 microM) to deplete inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools completely blocked the NH4Cl (20 mM)-induced rise in [Ca2+]i. Likewise, prior NH4Cl addition partially inhibited ATP-induced increases in [Ca2+]i, as well as slowed the frequency of repetitive [Ca2+]i spikes in single endothelial cells due to agonist. NH4Cl augmented the rate of [Ca2+]i increase that occurs in response to the depletion of agonist-sensitive intracellular Ca2+ pools. However, the internal Ca2+ store remained depleted during the continued presence of NH4Cl, as indicated by a decreased [Ca2+]i response to ATP in Ca2(+)-free medium. Finally, NH4Cl exerted these actions without affecting basal or ATP-stimulated IP3 formation. These observations provide direct evidence that increased pHi leads to Ca2+ mobilization from an agonist-sensitive pool and impairs Ca2+ pool(s) refilling mechanisms without altering cellular IP3 levels.