PH-dependence of detergent-induced hemolysis and vesiculation of erythrocytes

The influence of pH of the medium on the parameters of detergent-induced fast hemolysis and vesiculation of human erythrocytes was studied. In the range of pH 6.3-7.2 neither the extent nor the rate of the vesiculation induced by 25 microM sodium dodecyl sulfate (SDS) changed. However, a decrease of pH from 8.0 to 5.8 strongly modified both the extent and the rate of the hemolysis induced by SDS. Within the range of pH 8.0-6.4, the effect can be ascribed to the increase of the positive charge of the membrane. This could lead to the accumulation of the membrane-bound anion detergent and, hence, to the change of the hemolysis parameters. Non-charged detergent Triton X-100 did not display any pH-dependence. At pH between 6.4 and 5.8 the extent and rate of hemolysis changed in a complicated manner. The kinetic curves of hemolysis could be approximated by a single exponential within the pH range between 8.0 and 7.2. Upon further reduction of pH, a second exponential component, with a larger time constant, appeared in the kinetic curves. At 5.8 < pH < 7.2, the contribution of the "fast" hemolysis dropped virtually to zero, with pK about 6.0. This points to a structural transition of the membrane, possibly involving histidine. We suggest that the parameters of the detergent-induced hemolysis are sensitive to the changes of the charge and structural state of erythrocyte membrane.     

Heamolysis during in vitro CO2 removal from human blood using a membrane lung

Haemolysis of human blood has been examined in vitro as a function of pH in the range 7.2–8.0. The hydrogen ion concentration of freshly donated blood from 11 donors was manipulated in 42 experiments, entirely by altering the carbon dioxide fraction of air with which the blood was equilibrated using a membrane lung. In contrast to the known alkalaemic haemolysis which occurs in canine blood, we observed no correlation between plasma haemoglobin concentrations and blood pH. We conclude that alkalaemic haemolysis is unlikely to complicate the clinical application of extracorporeal carbon dioxide removal in the management of acute respiratory failure.     

Some properties of glutamine synthetase and glutamate synthase from Derxia gummosa

The incorporation of ¹⁵N into washed cells of Derxia gummosa from labelled-(NH₄)₂SO₄ and -KNO₃ respectively was inhibited by both L-methionine-DL-sulphoximine and azaserine. Glutamine synthetase purified to homogeneity from this bacterium had a molecular weight of 708 000 and was composed of 12 similar subunits each of 59 000. The enzyme assayed by γ-glutamyltransferase method had K_m values for L-glutamine and hydroxylamine of 12.5 and 1.2 mM, respectively. Optimal pH values for adenylylated and deadenylylated forms were pH 7.0 and pH 8.0, respectively. The adenylylated enzyme was deadenylylated by treatment with snake venom phosphodiesterase. The inhibitions by both glutamate and ammonia were competitive. The activity was markedly inhibited by L-methionine-DL-sulphoximine, alanine, glycine and serine and to a lesser extent by aspartate, phenylalanine and lysine. Various tri-, di- and mono-phosphate nucleotides, organic acids (pyruvate, oxalate and oxaloacetate) were also inhibitory. Glutamate synthase purified 167-fold had specific requirements for NADH, L-glutamine and 2-ketoglutarate. The K_m values for NADH, glutamine and 2-ketoglutarate were 9.6, 270 and 24 μM respectively. Optimal pH range was 7.2–8.2. The enzyme was inhibited by azaserine, methionine, aspartate, AMP, ADP and ATP.     

Effects of Soil pH on Reproduction of Pratylenchus penetrans and Forage Yield of Alfalfa

''Vernal'' alfalfa was grown for 30 weeks in nematode-free soil and in soil infested with Pratylenchus penetrans. Charlottetown fine sandy loam soil was used at its pH of 4.4 and at adjusted reactions of 5.2, 6.4 and 7.3. Nematode reproduction was significantly greater at pit 5.2 and 6.4 and was not related to alfalfa root production over the full pH range studied. A significant nematode infestation X soil pit interaction on forage yield was recorded. Nematode infestation significantly decreased forage yields at ptt 5.2 and 6.4 but not at pH 4.4 and 7.3.     

Arginine uptake by rabbit spermatozoa

The transport of l-arginine by rabbit spermatozoa was found to proceed by saturable, chemically specific mechanisms. Kinetic analysis of initial rates of transport at substrate concentrations from 1.0 μm to 1.0 μm indicate the presence of two saturable transport components. A low-affinity component has an apparent K_m of 0.64 μm and an apparent V of 43.4 nmol/10⁸ cells/30 s. A second, high-affinity component has an apparent K_m of 4.0 μm and an apparent V of 425 pmol/10⁸ cells/30 s. Rabbit spermatozoa actively transported l-arginine in a range of pH values from 6.5 to 10.5 with a pH optimum for the low-affinity component of 7.2–7.6 and a pH optimum for the high-affinity component of 7.8–8.0. Inhibitor studies indicate that the energization for transport may be dependent on ATP rather than on a pH gradient or transmembrane potential. Competition experiments with arginine analogs and amino acids suggest that the high- and low-affinity components may recognize the terminal guanidino group.     

Effects of internal ph on the nonselective cation channel from the mouse collecting tubule

We investigated the effects of internal pH on Ca-activated, nucleotide-inhibited nonselective cation channels in the basolateral membranes of mouse collecting tubules, using the inside-out variant of the patch clamp technique. pH modulated the channel open probability (P _o ), giving a bell-shaped curve peaking at pH 6.8/7.0: P _o at pH 6.0 was 11±6% of P _o at pH 7.2 and 32 ±7% at pH 8.0. The open and closed time distributions, best fitted to the sum of two exponentials, were differently sensitive to acid and alkaline conditions. Low pH reduced the short and long open times to 38 and 24% of their pH 7.2 values, while high pH produced a 4-fold increase in the long closed time. As previously reported, 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS) induced a quasi-permanent opening of the channel. The inhibition of the channel produced by high pH disappeared in the presence of SITS, while the inhibition produced by low pH was unaffected. These results suggest that the pH dependence of the channel is due to two separate mechanisms. pH was without effect on the ATP-evoked inhibition of the channel, while high pH profoundly reduced the steepness of the AMP inhibition curve, without altering the half-maximal inhibitory AMP concentration.     

The buffering capacity of the internal phase of thylakoids and the magnitude of the pH changes inside under flashing light

The buffering capacity inside thylakoids is determined and the magnitude of flash-induced pH changes inside is calibrated in the pH range from 6.4 to 8.1. The work is based on flash-induced absorption changes of neutral red in a chloroplast suspension in which the outer phase is strongly buffered by bovine serum albumin. It is shown that neutral red is bound inside thylakoids. The binding can be described by a simple isotherm with an apparent K_m = 4 μM and saturation at 1 neutral red per 17 chlorophylls. The apparent pK of neutral red is shifted from 6.6 in solution to 7.25 when bound inside. It is demonstrated that neutral red is a clean indicator of pH changes inside, i.e. when properly used it shows no response to other events. Although bound it reports pH changes which occur in the internal osmolar (aqueous) volume of thylakoids. This is obvious from the influence of chemically very different buffers on the magnitude of the absorption changes of neutral red. These act in a manner proportional to their calculated buffering capacity in aqueous solution. The intrinsic buffering capacity of the internal phase is determined with the aid of these buffers, at pH 7.2 it is between 0.8 and 1 mM (at 60 mosM). The absence of large variations in the buffering capacity in the range from pH 6.4 to 8.1 suggests that proteinaceous groups are involved in addition to the lipids which may dominate the buffering capacity at lower pH. The magnitude of the internal pH change is approx. 0.6 (at pH 7.3) under stimulation of both photosystems with a short xenon flash of light.     

The influence of pH on the kinetics of dissimilatory nitrite reduction in Paracoccus denitrificans

An almost stoichiometric conversion of nitrite to nitrous oxide was observed during the nitrite reduction by Paracoccus denitrificans cells in a medium of pH 6.4. The N₂O accumulated in the reaction medium and was decomposed only after nitrite had been consumed; when the pH of the medium was higher than 7.3–7.4, nitrous oxide did not accumulate. The activity of N₂O reductase was, in the whole range of pH 6.4–9.2, higher than the activity of NO⁻₂ reductase, both activities showing the maximum at the pH higher than 8.0. Using an artificial donor, TMPD plus ascorbate, the maximum activity of NO⁻₂ reductase, but not N₂O reductase was shifted by about two pH units to acidic region. The activity of nitrite reductase declined in the presence of N₂O only at higher pH values. Cytochrome c, as a common electron donor for both N₂O and NO⁻₂ reductase, was more oxidized at pH < 7.3 in the presence of NO⁻₂ than in the presence of N₂O, the opposite being true at pH > 7.3. The increased flux of electrons to cytochrome c has for a constant pH value (6.4) no effect on their distribution over NO⁻₂ and N₂O. The results indicate that the distribution of electrons in the terminal part is determined by the different pH optima for NO⁻₂ reductase and N₂O reductase, and by a mutual dependence of activities of the two reductases due to the competition for redox equivalents from a substrate.     

Purification and properties of chalcone isomerase from cell suspension cultures of Phaseolus vulgaris

Chalcone isomerase (EC 5.5.1.6) from cell suspension cultures of Phaseolus vulgaris has been purified about 400-fold. The molecular weight, as estimated by gel-filtration and SDS-polyacrylamide gel electrophoresis, is approx. 28 000. No isoenzymic forms are observed. The enzyme, which appears to require no cofactors, catalyses the isomerisation of both 6′-hydroxy and 6′-deoxy chalcones to the corresponding flavones. Likewise, a range of both 5-hydroxy and 5-deoxy flavonoids and isoflavonoids act as competitive inhibitors. The most potent inhibitors include the naturally occurring antimicrobial comcpounds kievitone (K_i 9.2 μM) and coumestrol (K_i 2.5 μM). The kinetics of the isomerisation of 2′,4,4′-trihydroxychalcone to the flavanone liquiritigenin have been investigated at a range of pH values. The pH optimum was around 8.0 and K_m changed with pH in a manner consistent with control by groups which ionise with pK_a values of 7.05 and 8.7 respectively. At pH 8.0, the energy of activation was 17.56 kJ/mol in the range 25–40°C. The role of the enzyme in the induced accumulation of flavonoid/isoflavonoid derivatives inthe Frech bean in discussed.     

Time resolved spectroscgpy of tryptopkyl fluorescence of yeast 3-phosphoglycerate kinase

The tryptophyl fluorescence emission of yeast 3-phosphoglycerate kinase decreases from pH 3.9 to pH 7.2 following a normal titration curve with an apparent pK of 4.7. The fluorescence decays have been determined at both extreme pH by photocounting pulse fluorimetry and have been found to vary with the emission wavelength. A quantitative analysis of these results according to a previously described method allows to determine the emission characteristics of the two tryptophan residues present in the protein molecule. At pH 3.9, one of the tryptophan residues is responsible for only 13% of the total fluorescence emission. This first residue has a lifetime τ₁= 0.6 ns and a maximum fluorescence wavelength λ_2max = 332 nm. The second tryptophan residue exhibits two lifetimes τ₂₁= 3.1 ns and τ₂₂= 7.0 ns (λ_2max= 338 nm). In agreement with the attribution of τ₂₁and τ₃₂ to the same tryptophan residue, the ratio β = C₂₁/C₂₂ of the normalized amplitudes is constant along the fluorescence emission spectrum. At pH 7.2, the two tryptophan residues contribute almost equally tc the protein fluorescence. The decay time of tryptophan 1 is 0.4 ns. The other emission parameters are the same as those determined at pH 3.9. We conclude that the fluorescence quenching in the range pH 3.9 to pH 8.0 comes essentially from the formation of a non emitting internal ground state complex between the tryptophan having the longest decay times and a neighbouring protein chemical group. The intrinsic pK of this group and the equilibrium constant of the irternal complex can be estimated. The quenching group is thought to be a carboxylate anion. Excitation transfers between the two tryptophyl residues of the protein molecule appear to have a small efficiency.     

Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes

Rats were injected with⁵⁹Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2–7.4 in the former case and 6.4–6.7 in the latter. Red cells were then centrifuged (5) and approximately 7–10% of the packed cells from the top and 7–10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2–7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4–6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4–6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.     

Purification and Properties of Phospholipase D from Actinomadura sp. Strain No. 362

An extracellular phospholipase D from Actinomadura sp. Strain No. 362 was purified about 430-fold from the culture filtrate. The purified enzyme preparation was judged to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point of the enzyme were estimated to be about 50,000—60,000 and 6.4, respectively. The enzyme was most active at pH 5.5 and 50°C in the presence of Triton X-100, but showed the highest activity at pH 7.0 and 60 — 70°C in its absence. The enzyme was stable up to 30°C at pH 7.2 and also stable in the pH range of 4.0 to 8.0 on 2 hr incubation at 25°C. With regard to substrate specificity, this enzyme hydrolysed lecithin best among the phospholipids tested. It was activated by Fe^{3 +}, Al³⁺, Mn^{2 +}, Ca^{2 +}, diethyl ether, sodium deoxycholate and Triton X-100, but was inhibited by cetyl pyridinium chloride and dodecylsulfate.     

Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP⁺, and Mg²⁺ were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K_m = 58 micromolar), but also inhibition by high substrate (K_i = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K_m = 120 micromolar). At both pH conditions, activity response to NADP⁺ exhibited Michaelis-Menten behavior with K_m values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg²⁺ suggested the presence of at least two Mg²⁺ - binding sites with different affinity. The K_a values for Mg²⁺ obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg²⁺ levels could be important for the physiological regulation of NADP-malic enzyme.     

Inhibitory Effects of Phenolic Ecotoxicants on Photobacteria at Various pH Values

Kinetic characteristics of light emission by intact cells of photobacteria Photobacterium phosphoreum and Vibrio harveyi were studied (at pH 5.5, 7.0, and 8.0), as well as inhibitory effects of 2,4-di- and 2,4,5-triphenoxyacetic acids (2,4-D and 2,4,5-T), pentachlorophenol (PCP), and 2,6-dimethylphenol (2,6-DMP) (at the same pH values). The emission kinetics lacked a steady state, irrespective of pH. At pH 5.5, luminescence decayed exponentially in the 60-s range; at pH 7.0 and 8.0, a 5-min luminescence activation was observed. The respiratory activity of the cells decreased by more than an order of magnitude at pH 5.5 (compared to the levels observed at pH 7.0 and 8.0). The inhibitory effects of 2,4-D, 2,4,5-T, and PCP differed by one to two orders of magnitude, depending on pH. Maximum cell sensitivity to these compounds appeared at pH 5.5; minimum sensitivity, at pH 8.0. The effect of 2,6-DMP was pH-independent. The inhibitory effect was determined by the hydrophobicity of the molecule and pK values of the toxicants. At all pH values, substrate-depleted cells of photobacteria were more sensitive to chlorophenolic compounds than cells supplied with energy.     

Activity and adsorption of lipase from Nigella sativa seeds on Celite at different pH values

Lipase from Nigella sativa seeds was immobilized by adsorption on Celite 535 from phosphate buffer solutions varying pH values of 5.0–8.0 at 25?°C. Langmuir isotherms described the adsorption equilibria well for lipase adsorption at all pH range. The saturation capacity for adsorption of lipase increased from 14.5 to 24.3 mg g^?1 Celite as the adsorption pH was reduced from 8 to 5, but the adsorption equilibrium constant remained constant and was determined to be 1.92 × 10⁵ M^?1. The adsorbed enzymes showed different activity values depending on the pH of the adsorption medium. The immobilized enzymes prepared at pH 6 displayed the highest activity values.     

Effects of pH and phosphate on the production of struvite by Myxococcus xanthus

We studied the influence of pH and the phosphate content of the culture medium on the precipitation of struvite by Myxococcus xanthus, a bacterium that undergoes autolysis at the end of its exponential growth phase in liquid cultures. The best results were obtained with pH values between 7.2 and 8.0 and with a phosphate concentration of 10 mM. Our studies reveal for the first time that the precipitation of struvite always begins at the onset of autolysis and that culture conditions favoring the early occurrence of autolysis also enhance struvite production.     

Proton Gradients in Intact Cyanobacteria

The internal pH values of two unicellular cyanobacterial strains were determined with electron spin resonance probes, over an external pH range of 6 to 9, in the light and in the dark. The slow growing, thylakoid-lacking Gloeobacter violaceus was found to have a low capacity for maintaining a constant internal pH. The distribution pattern of weak acid and amine nitroxide spin probes across the cell membranes of this organism, in the light and in the dark, was consistent with the assumption that it contains a single intracellular compartment. At an external pH of 7.0, intracellular pH was 6.8 in the dark and 7.2 in the light. The cells of Agmenellum quadruplicatum, a marine species, were found to contain two separate compartments; in the dark, the pH of the cytoplasmic and the intrathylakoid spaces were calculated to be 7.2 and 5.5, respectively. Upon illumination, the former increased and the latter decreased by about 0.5 pH units.     

Molecular weight and pH aspects of the efficacy of oligochitosan against methicillin-resistant Staphylococcus aureus (MRSA)

Oligochitosan samples varying in molecular weight (M_w) and having narrow polydispersities were prepared by means of depolymerization of chitosan in hydrochloric acid, and their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) was measured at pH values 5.5-8.0. The antibacterial testing of oligochitosans obtained showed that oligochitosans having M_w in the range of 0.73-20.0 kDa could be used both at slightly acidic and neutral pH values, and that the activity against MRSA remained moderate for oligochitosan samples having M_w about 3-5 kDa even at slightly basic pH values. The self-assembling behavior of oligochitosan macromolecules in the dilute solution at various pH values as a function of chain length was investigated. At first it was shown that oligochitosans formed supramolecular aggregates in dilute solutions below the critical pH value 6.5. Despite the aggregation phenomenon, the formation of nano-sized aggregates did not prevent oligochitosan from demonstrating the bactiostatic activity.     

Effect of External pH on the Internal pH of Chlorella saccharophila

The overall internal pH of the acid-tolerant green alga, Chlorella saccharophila, was determined in the light and in the dark by the distribution of 5,5-dimethyl-2-[¹⁴C]oxazolidine-2,4-dione ([¹⁴C]DMO) or [¹⁴C]benzoic acid ([¹⁴C]BA) between the cells and the surrounding medium. [¹⁴C]DMO was used at external pH of 5.0 to 7.5 while [¹⁴C]BA was used in the range pH 3.0 to pH 5.5. Neither compound was metabolized by the algal cells and intracellular binding was minimal. The internal pH of the algae obtained with the two compounds at external pH values of 5.0 and 5.5 were in good agreement. The internal pH of C. saccharophila remained relatively constant at pH 7.3 over the external pH range of pH 5.0 to 7.5. Below pH 5.0, however, there was a gradual decrease in the internal pH to 6.4 at an external pH of 3.0. The maintenance of a constant internal pH requires energy and the downward drift of internal pH with a drop in external pH may be a mechanism to conserve energy and allow growth at acid pH.     

The Hexose-Proton Cotransport System of Chlorella : pH-Dependent Change in Km Values and Translocation Constants of the Uptake System

The proton concentration in the medium affects the maximal velocity of sugar uptake with a K_m of 0.3 mM (high affinity uptake). By decreasing the proton concentration a decrease in high affinity sugar uptake is observed, in parallel the activity of a low affinity uptake system (K_m of 50 mM) rises. Both systems add up to 100%. The existence of the carrier in two conformational states (protonated and unprotonated) has been proposed therefore, the protonated form with high affinity to 6-deoxyglucose, the unprotonated form with low affinity. A plot of extrapolated V_max values at low substrate concentration versus proton concentration results in a K_m for protons of 0.14 µM, i.e. half-maximal protonation of the carrier is achieved at pH 6.85. The stoichiometry of protons cotransported per 6-deoxyglucose is close to 1 at pH 6.0–6.5. At higher pH values the stoichiometry continuously decreases; at pH 8.0 only one proton is cotransported per four molecules of sugar. Whereas the translocation of the protonated carrier is strictly dependent on sugar this coupling is less strict for the unprotonated form. Therefore at alkaline pH a considerable net efflux of accumulated sugar can occur. The dependence of sugar accumulation on pH has been measured. The decrease in accumulation with higher pH values can quantitatively be explained by the decrease in the amount of protonated carrier. The properties of the unprotonated carrier resemble strikingly the properties of carrier at the inner side of the membrane. The inside pH of Chlorella was measured with the weak acid 5,5-dimethyl-2, 4-oxazolidinedion (DMO). At an outside pH of 6.5 the internal pH was found to be 7.2. To explain the extent of sugar accumulation it has to be assumed that the membrane potential also contributes to active sugar transport in this alga.