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At least 811 records · Page 45Linked to original sources
PubMed2001

Buffer-anion-dependent Ca2+ leaching from horseradish peroxidase at low pH.

Despite highly conserved active-site structures, members of the plant peroxidase superfamily exhibit a wide range of pH optima. Horseradish peroxidase isozyme C (HRPC) is an ideal peroxidase to investigate the structural determinants of pH stability and activity in superfamily members. Conflicting reports exist on the low-pH stability of HRPC and consequently the pKa of the catalytic distal histidine, which is neutral in active peroxidases. Towards resolving such discrepancies, acid-induced changes in HRPC from two popular commercial suppliers were systematically analyzed. Specifically, FTIR v(CO) and Soret-CD spectra of HRPC-CO and Soret absorption of ferric HRPC were recorded to probe time-dependent heme-pocket changes at pH 3.0 in phosphate, citrate and formate buffers, while the FTIR amide I' and far-UV CD spectra were examined to probe changes in secondary structure. Both HRPC-CO samples exhibited identical pH 7.0 v(CO) bands at 1934 and 1905 cm-1. In the pH 3.0 spectrum of sample A, the 1934 cm-1 band was dominant while a broad 1969 cm-1 band appeared in sample B. The intensity of this band, which is assigned to solvent-exposed heme, was greater in citrate than phosphate buffer, but in formate the 1934 cm-1 band remained dominant. Other spectral changes mirrored the v(CO) trends. No time- or buffer-anion-dependent conformation changes were detected in 1 mM CaCl2, revealing that buffer-anion-dependent leaching of stabilizing Ca2+ from HRPC occurs at pH 3.0. Since the N-glycans present in HRPC are of the flexible protein-surface-shielding type, the variation in low-pH conformational stability of the HRPC samples could be attributed to heterogeneous glycosylation, which was detected by SDS-PAGE. It is further proposed that glycosylation patterns may affect the low-pH stability of class II and III plant peroxidases.

Anions↗
PubMed2002

Parvalbumin 3 is an abundant Ca2+ buffer in hair cells.

Ca2+ signaling serves distinct purposes in different parts of a hair cell. The Ca2+ concentration in stereocilia regulates adaptation and, through rapid transduction-channel reclosure, underlies amplification of mechanical signals. In presynaptic active zones, Ca2+ mediates the exocytotic release of afferent neurotransmitter. At efferent synapses, Ca2+ activates the K+ channels that dominate the inhibitory postsynaptic potential. A copious supply of diffusible protein buffer isolates the three signals by restricting the spread of free Ca2+ and limiting the duration of its action. Using cDNA subtraction and a gene expression assay based on in situ hybridization, we detected abundant expression of mRNAs encoding the Ca2+ buffer parvalbumin 3 in bullfrog saccular and chicken cochlear hair cells. We cloned cDNAs encoding this protein from the corresponding inner-ear libraries and raised antisera against recombinant bullfrog parvalbumin 3. Immunohistochemical labeling indicated that parvalbumin 3 is a prominent Ca2+-binding protein in the compact, cylindrical hair cells of the bullfrog's sacculus, and occurs as well in the narrow, peanut-shaped hair cells of that organ. Using quantitative Western blot analysis, we ascertained that the concentration of parvalbumin 3 in saccular hair cells is approximately 3 mM. Parvalbumin 3 is therefore a significant mobile Ca2+ buffer, and perhaps the dominant buffer, in many types of hair cell. Moreover, parvalbumin 3 provides an early marker for developing hair cells in the frog, chicken, and zebrafish.

Amino Acid Sequence↗
PubMed1979

Bicarbonate-buffered peritoneal dialysis. An effective adjunct in the treatment of lactic acidosis.

Severe lactic acidosis is associated with poor prognosis. Usually, the patient is treated with massive amounts of intravenous sodium bicarbonate, which in itself carries many undesirable consequences such as fluid overload and hypernatremia. We have successfully used peritoneal dialysis with a bicarbonate-buffered dialysate in the management of severe acidosis. Bicarbonate-buffered peritoneal dialysis provided an unlimited supply of physiologic buffer over a prolonged period without causing hypervolemia or hypernatremia. Furthermore, significant amounts of lactate were removed by dialysis. We, therefore, recommend the use of bicarbonate-buffered peritoneal dialysis as an adjunct in the treatment of severe lactic acidosis.

Acidosis↗
PubMed1990

Potentiometric measurements of hydrogen and cyanide ions in buffered media.

Potentiometric measurements of hydrogen and cyanide levels were studied in different buffered media that are used in biological experiments. The addition of 1-10mM NaCN increased the pH (from 7.2-7.5 up to 9.5) of either bicarbonate/phosphate-buffered (artificial cerebrospinal fluid (ACSF), sera, and whole blood) or Hepes-buffered solutions in a concentration-dependent manner. While in aerated ACSF this increase was transient (half-relaxation time less than 4 min), the increase in pH was sustained in Hepes buffer. A mathematical model that predicts the maximum cyanide-induced increases in pH was derived. When NaCN was added to either ACSF or Hepes-buffered solutions, a Nernst relation was obtained, but deviations from Nernstian responses resulted when NaCN was added to sera or whole blood. These responses, however, differed substantially from the theoretical results that were based on the relative amounts of ionized cyanide present in the various pH environments. In addition, attempts were made to indirectly examine the formation and escape of HCN by calculating the time constants of decay (tau d) of the cyanide-induced potentiometric responses. With progressively higher NaCN concentrations in ACSF, the values of tau d did not decrease, but were constant. As predicted, however, the values of tau d were larger when solutions were covered or had higher initial pH values. These results suggest that in the solutions tested, the cyanide electrode used in the present study measured the total amount rather than the ionized portion of dissolved cyanide and that the values of tau d do not correspond to formation/escape rates of HCN.

Buffers↗
PubMed1990

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.

Bicarbonates↗
PubMed1991

A test to evaluate the effect of individual components of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffers on enzymatic activity.

A simple dilution test for evaluating the individual effect on enzymatic activity of [Ca2+], [EGTA], or [Ca.EGTA] variations in Ca-EGTA buffers is presented. We verified that a 50-fold dilution of the buffer (25-0.5 mM) at constant pH did not affect [Ca2+] (measured with fura-2), whereas [EGTA] and [Ca.EGTA] varied. Therefore the test can be applied to evaluate the proper effect of Ca2+ in a Ca-EGTA buffer on enzyme activity because such an effect is expected to remain unchanged upon dilution of the buffer. Applications of the test are shown for three enzymes apparently sensitive to Ca2+ but found to be effectively influenced only by Ca.EGTA (liver glucose-6-phosphatase), EGTA (intestinal mucosa phosphatase), or indeed Ca2+ (brain cyclic nucleotide phosphodiesterase).

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

Bound and determined: a computer program for making buffers of defined ion concentrations.

A computer program that allows the preparation of buffers containing known concentrations of metal-ligand complexes at defined pH values and temperatures is described. Ligands are defined as compounds that bind metals and may include AMP, ADP, ATP, GMP, GDP, GTP, EGTA, EDTA, BAPTA, phosphate, sulfate, chloride, monocarboxylic acids, dicarboxylic acids, organophosphates, and/or citric acid. Metals may include sodium, potassium, magnesium, calcium, and/or manganese. The program uses association constants corrected for temperature and ionic strength so that solutions between 0 and 40 degrees C and between pH values of 4 and 10 can be defined. The program can perform the following: (i) calculate the concentration of all metal-ligand complexes when total metal and total ligand concentrations are known, (ii) calculate the concentration of metal ion required to make a solution of known free metal ion concentration when total ligand concentrations are known, (iii) calculate the concentration of ligand required to make a solution of known free metal ion concentration when total metal concentrations are known, and (iv) calculate the total concentrations of metal and ligand required to make a buffer of known metal-ligand concentration. Options i-iii are useful for making buffers of defined free metal ion concentrations; option iv is useful for making buffers of defined metal-nucleotide concentrations.

Adenosine Triphosphate↗
PubMed1990

The approximal bone height and intrabony defects in young adults, related to the salivary buffering capacity and counts of Streptococcus mutans and Lactobacilli.

Using a computerized technique the bone height and prevalence of approximal periodontal intrabony defects were assessed on posterior bite-wing radiographs from 151 young adults. The results were related to the buffering capacity and counts of Streptococcus mutans and lactobacilli in whole stimulated saliva. The mean distance from the cement-enamel junction to the alveolar bone crest was greater in the high buffering group than in the low buffering group (p less than 0.05), and particularly in non-smokers (p less than 0.01). Intrabony defects were more common in the low buffering group (p less than 0.05) and in women (p less than 0.001).

Adult↗
PubMed1993

Effect of buffers and osmolality on anion uniport across the mitochondrial inner membrane.

The effects of buffers and osmolality of the suspending medium on the pH-dependent anion uniport across the inner membrane of isolated rat liver mitochondria have been studied using the light scattering technique to measure passive osmotic swelling. In contrast to some other transport processes the rates of entry of chloride and other anions via the anion-conducting channel decreased steeply with increasing solute concentration. This effect appears to be a result of increased osmolality or decreased matrix volume rather than inhibition by the anion since it was also produced by increasing the osmolality by addition of non-penetrant solutes. The effects of some pH buffers on the mitochondrial anion-conducting channel were also investigated. Some zwitterionic buffers had little effect other than that produced by increasing osmolality but Tricine, Popso and Caps produced marked additional inhibition of anion uniport and several other zwitterionic buffers were also inhibitory. The correlation between increased anion conductivity and increased matrix volume supports the proposal that this channel functions in regulation of the volume of the mitochondrial matrix.

Alkanesulfonates↗
PubMed1980

Calcium buffering in presynaptic nerve terminals. Free calcium levels measured with arsenazo III.

The particulate fraction from osmotically shocked synaptosomes ('synaptosomal membrances') sequesters Ca when incubated with ATP]containing solutions. This net accumulation of Ca can reduce the free [Ca2+] of the bathing medium to sub-micromolar levels (measured with arsenazo III). Two distinct types of Ca sequestration site are responsible for the Ca2+ buffering. One site, presumed to be smooth endoplasmic reticulum, operates at low [Ca2+] (less than 1 microM), and has a relatively small capacity. Ca sequestration at this site is prevented by the Ca2+ ionophore, A-23187, but not by mitochondrial poisons. The secone (mitochondrial) site, in contrast, is blocked by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and oligomycin. Since the intraterminal organelles can buffer [Ca2+] to about 0.3-0.5 microM, this may be an upper limit to the normal resting level of [Ca2+]i in nerve terminals. In the steady state, total cell Ca and [Ca2+]i will be governed principally be Ca transport mechanisms in the plasmalemma; the intracellular organelle transport systems then operate in equilibrium with this [Ca2+]. During activity, however, Ca rapidly enters the terminals and [Ca2+]i rises. The intracellular buffering mechanisms then come into play and help to return [Ca2+]i toward the resting level; the non-mitochondrial Ca sequestration mechanism probably plays the major role in this Ca buffering.

Adenosine Triphosphate↗
PubMed1986

Free protons do not substitute for sodium ions in buffer-mediated phosphorylation of (Na+ + K+)-ATPase.

In view of our recent finding of imidazole-activation of the phosphorylation of (Na+ + K+)-ATPase and the suggestion by others of an activating role of protons, in lieu of sodium ions, in the overall hydrolytic and phosphorylation processes of the enzyme, we have investigated the effect of pH on the phosphorylation process. No indication of proton activation is found. Rather, phosphorylation at low pH in the absence of Na+ is dependent on the buffer concentration. Imidazole-H+ stimulated phosphorylation at pH 5 reaches the same maximal steady-state level as Na+-stimulated phosphorylation. Low pH also elicits Tris-H+ stimulated phosphorylation, but due to a simultaneous inhibitory effect of this buffer the maximal steady-state level is no more than 50% of the Na+-stimulated phosphorylation level. Protons inhibit rather than activate phosphorylation. Upon decreasing the pH from 7 to 5, we observe for all ligands, whether activating or inhibiting phosphorylation (ATP, Na+, protonated imidazole, Mg2+ and K+), a decrease in affinity (largest for Mg2+) and a decrease in the maximal steady-state phosphorylation capacity. The effects of Na+ and imidazole-H+ on the phosphorylation step have been compared with those on the E2----E1 conformational change, which leads to the phosphorylation step. The different pH-dependence of the affinities for Na+ and protonated buffer in the E2----E1 transition suggests that there are separate activation sites with different pK values for Na+ and the buffer cation. The above findings rule out a role of free protons as a substitution for Na+ in the phosphorylation process.

Adenosine Triphosphate↗
PubMed1986

Sodium and buffer cations inhibit dephosphorylation of (Na+ + K+)-ATPase.

Effects of various cations on the dephosphorylation of (Na+ + K+)-ATPase, phosphorylated by ATP in 50 mM imidazole buffer (pH 7.0) at 22 degrees C without added Na+, have been studied. The dephosphorylation in imidazole buffer without added K+ is extremely sensitive to K+-activation (Km K+ = 1 microM), less sensitive to Mg2+-activation (Km Mg2+ = 0.1 mM) and Na+-activation (Km Na+ = 63 mM). Imidazole and Na+ effectively inhibit K+-activated dephosphorylation in linear competitive fashion (Ki imidazole 7.5 mM, Ki Na+ 4.6 mM). The Ki for Na+ is independent of the imidazole concentration, indicating different and non-interacting inhibitory sites for Na+ and imidazole. Imidazole inhibits Mg2+-activated dephosphorylation just as effective as K+-activated dephosphorylation, as judged from the Ki values for imidazole in the two processes. Tris buffer and choline chloride, like imidazole, inhibit dephosphorylation in the presence of residual K+ (less than 1 microM), but less effectively in terms of I50 values and extent of inhibition. Tris inhibits to the same extent as choline. This indicates different inhibitory sites for Tris or choline and for imidazole. These findings indicate that high steady-state phosphorylation levels in Na+-free imidazole buffer are due to the induction of a phosphorylating enzyme conformation and to the inhibition of (K+ + Mg2+)-stimulated dephosphorylation.

Animals↗
PubMed1978

The effect of different buffers on terminal deoxynucleotidyl transferase activity.

The polymerization of dATP, dCTP, and dGTP onto the defined length initiator, d(pA)10, has been carried out in four buffers. The relative effectiveness of the buffers for the polymerization of each deoxynucleoside triphosphate decreased in the order: cacodylate, 2(N-morpholino)ethane sulfonic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and Tris. With the poorer buffers, activity could be increased by the addition of KCl: this effect is not primarily due to an increase in ionic strength. With dGTP as the substrate, but not with dATP or dCTP, activity increased when the concentration of the more active buffers was raised beyond 0.2 M.

Alkanesulfonates↗
PubMed1988

Fictive locomotion in the lamprey; a comparison using bicarbonate and HEPES buffers.

Fictive locomotion in the lamprey, elicited by N-methyl-DL-aspartate or D-glutamate, was compared in two buffer solutions, one using bicarbonate, the other using HEPES. No differences were observed in burst proportion, cycle length, or phase lag per segment between these buffers solutions. These data indicate that HEPES-buffered artificial cerebrospinal fluid, which does not require gassing with carbon dioxide to maintain pH, may be a suitable alternative to bicarbonate-buffered artificial cerebrospinal fluid.

Animals↗
PubMed1996

Intracellular pH and intrinsic H+ buffering capacity in normal and hypertrophied right ventricle of ferret heart.

OBJECTIVE: To answer the questions: (a) What is the effect of hypertrophy on the intracellular pH (pHi) and buffering power of cardiac muscle, and (b) How does hypertrophy affect the ability of cardiac muscle to recover from intracellular acidosis induced by hypoxia. METHODS: In nominally HCO3(-)-free, HEPES-buffered Tyrode solution (35 degrees C), pHi and the intrinsic buffering power (beta i, measured in the presence of amiloride) was investigated using pH-sensitive microelectrodes. RESULTS: beta i was similar in both preparations (25 mM/pH unit at pHi 7.04). beta i was inversely related to pHi but the relationship was not significantly modified by hypertrophy. In the absence of amiloride, the time constant of pHi recovery (tau r) on removal of NH+4, was similar in normal (4.0 +/- 0.2 min, n = 5) and in hypertrophied muscles (4.3 +/- 0.3 min, n = 4; n.s.). In both preparations, net acid extrusion (JH) was similarly increased at lower values of pHi. Lowering temperature from 35 degrees to 22 degrees caused an alkalinization (0.15 pH units) of pHi. At 22 degrees C the mean values of pHi, beta i, tau r and JH were similar in normal and in hypertrophied muscles. At both temperatures and in both groups of preparations, recovery of pHi following hypoxia is approximately exponential. The time constant of recovery of pHi following hypoxia (tau rh) at 22 degrees C was not significantly different in hypertrophied muscles (7.2 +/- 0.9 min, n = 8) compared to controls (10.6 +/- 1.8 min, n = 13). However, at 35 degrees C, there was a significant difference in the mean values of tau rh which was smaller for hypertrophied muscles (3.9 +/- 0.3 min, n = 7) than for normal (7.1 +/- 1.1 min, n = 4, P < 0.005). For pHi 6.8-7.0, net acid extrusion in hypertrophied preparations was increased by a factor of 4 compared to normal. CONCLUSIONS: The intracellular buffering capacity and the pHi regulating capacity via Na+/H+ exchange are not significantly modified by right ventricular hypertrophy in ferret heart. The faster pHi recovery from hypoxia-induced acidification can be interpreted in terms of the role of lactate efflux in pHi control. The possible role of energy compartmentalization, its influence on the Na+ gradient and thus on pHi control after hypoxia, is discussed.

Amiloride↗
PubMed1980

Influence of inorganic phosphate on the activity determination of isoenzymes of alkaline phosphatase in various buffer systems.

The inhibitory effect of inorganic phosphate on the activity determination of isoenzymes of alkaline phosphatase (AP) in diethanolamine (DEA), glycine and 2-amino-2-methyl-1,3-propandiol (AMPD) buffer was studied. This effect depends on the buffer used and isoenzyme investigated. Especially the placental isoenzyme is inhibited; the inhibitory effect in DEA buffer is stronger than in the other buffers used. The requirement of purity for 4-nitrophenylphosphate with respect to its content of inorganic phosphate and conclusions for using control sera enriched with AP isoenzymes are discussed.

4-Nitrophenylphosphatase↗
PubMed1986

Reversible induction of actin rods in mouse C3H-2K cells by incubation in salt buffers and by treatment with non-ionic detergents.

Incubating conditions which induced actin paracrystal-like intracellular structures (actin rods) were investigated by using several cell lines. We have found that an incubation of cells of a mouse fibroblastic cell line, C3H-2K, in an isotonic solution of NaCl containing 1 mM MgCl2, 1 mM CaCl2 and 10 mM MES, pH 6.5, induced disintegration of stress fibers and formation of actin rods in the cytoplasm. Actin rods were induced also by incubating in salt buffers in which Na+ of the above solution was substituted by most cations except K+ or Rb+. When the actin rod-forming cells were transferred back to DMEM containing 10% FBS, actin rods disappeared and stress fibers subsequently re-formed within 1 h at 37 degrees C. Although the induction was observed in NaCl buffer at a wide range of pH values (5.5-10), the optimal pH was 6.5. Formation of actin rods is dependent upon cellular metabolism, as it was inhibited at 4 degrees C, or by metabolic inhibitors. Incubation in NaCl buffer induced actin rods in HeLa, L, NRK, BALB/c 3T3 and Swiss 3T3 cells, but not in CEF or MEF cells. A decrease in cell volume was observed parallel with the induction of actin rods, except for CEF and MEF cells. Alterations in intracellular concentrations of Na, K or Ca were not correlated with the induction, however. Actin rods were also induced in C3H-2K cells by a brief treatment with non-ionic detergents. Tween 80 at concentrations as low as 0.003% was effective for the induction, but did not increase the passive membrane transport of p-nitrophenylphosphate. In contrast to the induction by NaCl buffer, treatment with Tween 80 induced numerous tiny actin rods at 4 degrees C, which became larger when further incubated at 37 degrees C. Double immunofluorescence staining with anti-actin antibody and anti-vinculin antibody showed that vinculin plaques remained at least in an early stage of the actin rod formation. We discuss the mechanism for the induction of actin rods based upon the present findings.

Actins↗
PubMed1991

Optimization of resolution in capillary zone electrophoresis: combined effect of applied voltage and buffer concentration.

Expressions are formulated for the prediction of solute migration time and resolution as a function applied voltage and buffer concentration in capillary zone electrophoresis. The resolution equation assumes that solute diffusion is the only operative zone-broadening mechanism. A resolution surface in applied voltage and buffer concentration space is presented featuring isochrones that are used to predict the behavior of resolution under constant analysis time. In the resolution-voltage planes the resolution increases continuously with increasing voltage. At the high-voltage border, the resolution decreases continuously with increasing concentration, however, at the low-voltage border the resolution passes through a shallow maximum as the buffer concentration is increased. At constant analysis time, resolution is optimized by simultaneously increasing the voltage and the buffer concentration. In comparison, this theoretical approach, which predicts resolution from solute migration times only, gives values that are consistently about 40-50% higher than experimentally determined resolution.

Acetates↗