Apomyoglobin stability as dependent on urea concentration and temperature at two pH values

Equilibrium unfolding of apomyoglobin (ApoMb) in the presence of urea was studied as dependent on the temperature (5–2°C) at two pH values (5.7 and 6.2). Thermodynamic parameters of ApoMb transition from the native to the unfolded state were estimated under various conditions. Conformational changes in ApoMb were detected by tryptophan fluorescence and far-UV circular dichroism. The ApoMb stability and the cooperativity of its unfolding at 5°C were considerably lower than at other temperatures at both pH values, where ApoMb is in the native conformation.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 330–335.Original Russian Text Copyright © 2005 by Baryshnikova, Sharapov, Kashparov, Ilyina, Bychkova.     

Differential responses of two wetland graminoids to high ammonium at different pH values

Enhanced soil ammonium () concentrations in wetlands often lead to graminoid dominance, but species composition is highly variable. Although is readily taken up as a nutrient, several wetland species are known to be sensitive to high concentrations or even suffer toxicity, particularly at low soil pH. More knowledge about differential graminoid responses to high availability in relation to soil pH can help to better understand vegetation changes. The responses of two wetland graminoids, Juncus acutiflorus and Carex disticha, to high (2 mmol·l^?1) versus control (20 μmol·l^?1) concentrations were tested in a controlled hydroponic set up, at two pH values (4 and 6). A high concentration did not change total biomass for these species at either pH, but increased C allocation to shoots and increased P uptake, leading to K and Ca limitation, depending on pH treatment. More than 50% of N taken up by C. disticha was invested in N‐rich amino acids with decreasing C:N ratio, but only 10% for J. acutiflorus. Although both species appeared to be well adapted to high loadings in the short term, C. disticha showed higher classic detoxifying responses that are early warning indicators for decreased tolerance in the long term. In general, the efficient aboveground biomass allocation, P uptake and N detoxification explain the competitive strength of wetland graminoids at the expense of overall biodiversity at high loading. In addition, differential responses to enhanced affect interspecific competition among graminoids and lead to a shift in vegetation composition.     

End product profiles of starch hydrolysis by bacterial alpha-amylases at different temperature and pH values

Summary The end product profile of starch hydrolysis by four bacterial alpha-amylases was found to be dependent on the source of the enzyme, and pH and temperature conditions of reaction.     

Microaerophilic metabolism of Pachysolen tannophilus at different pH values

Microaerophilic production of xylitol by Pachysolen tannophilus from detoxified hemicellulose hydrolyzate was optimal between pH values 6.0 to 7.5 when about 90% of xylose was utilized for xylitol production, the rest being fermented to ethanol. At pH values of 3.0 and 12.0, respiration became important, consuming up to 30% of available xylose. A graphic procedure suggests that histamine and cysteine are at the active site of xylose reductase in this yeast.     

Antibiotic susceptibility of Salmonella spp. at different pH values

We have examined the effects of acidic pH, in the range of those prevailing within phagosomes and lysosomes, on the growth and the susceptibility to different antibiotics of several strains of Salmonella spp. The minimal inhibitory concentration and the minimal bactericidal concentration of several beta-lactams were increased considerably during culture at pH 5.2. The extent of the increase was a function of: (1) the beta-lactam structure and, more particularly, the hydrophobicity of the side-chain of the molecule; and (2) the bacterial serotype. This phenotypic resistance at acid pH was not due to beta-lactamase activity or to a lower growth rate. In contrast, rifamycin SV was more active at acidic pH than at neutral pH and chloramphenicol, another highly hydrophobic drug, was equally efficacious at both pH values. Membrane lipopolysaccharide mutants, but not porin mutants, cultivated at an acidic pH were inhibited by lower concentrations of the beta-lactams. This suggests that the increased resistance to beta-lactams, and the increased susceptibility to rifamycin SV, at acidic pH, could have resulted from modified permeability of the outer membrane to antibiotics.     

Comparative effectiveness of nisin and bacteriocin J46 at different pH values

E. HUOT, C. BARRENA-GONZALEZ AND H. PETITDEMANGE. 1996. A Comparative study of the inhibitory activity of nisin, the well-known lantibiotic produced by certain strains of Lactococcus lactis subsp. lactis , and of the bacteriocin produced by L. lactis subsp. cremoris J46, a strain previously isolated from fermented milk, was conducted. For both bacteriocins, the activity against L. lactis subsp. cremoris decreased with increasing pH. In addition, the bacteriocin preparations were more stable at 4 than at 20°C. The influence of the storage temperature was more crucial for nisin. Essentially the same activity was observed for bacteriocin J46 stored for 3 h at 4 or 20°C. More interesting was the observed stability of bacteriocin J46 at pH values between 5.8 and 6.8. For example, about 23% of nisin activity was lost at pH 6.4 whereas no loss of bacteriocin J46 activity was observed.     

Intermonomer flexibility of Ca- and Mg-actin filaments at different pH values.

The fluorescence resonance energy transfer parameter, f, is defined as the efficiency of the energy transfer normalized by the quantum yield of the donor in the presence of acceptor. It is possible to characterize the flexibility of the protein matrix between the appropriate fluorescent probes by monitoring the temperature dependence of f. The intermonomer flexibility of the Ca-actin and Mg-actin filaments was characterized by using this method at pH values of 6.5 and 7.4. The protomers were labeled on Cys374 with donor [N-(((iodoacetyl)amino)ethyl)-5-naphthylamine-1-sulfonate; IAEDANS] or acceptor [5-(iodoacetamido)fluorescein; IAF] molecules. The temperature profile of f suggested that the intermonomer flexibility of actin filaments was larger at pH 7.4 than pH 6.5 in the case of Mg-F-actin while this difference was absent in the case of Ca-F-actin. More rigid intermonomer connection was identified at both pH values between the protomers of Mg-F-actin compared to the Ca-F-actin. The results were further supported by time dependent fluorescence measurements made on IAEDANS and IAF labeled Mg- and Ca-actin filaments at pH 6.5 and 7.4. Our spectroscopic results may suggest that the altered function of muscle following the change of pH within the muscle cells under physiological or pathological conditions might be affected by the modified dynamic properties of the magnesium saturated actin filaments. The change of the intracellular pH does not have an effect on the intermonomer flexibility of the Ca-actin filaments.     

Glycine betaine may have opposite effects on protein stability at high and low pH values

The compatible osmolyte glycine betaine (GB) is the most efficient osmoprotectant and best excluder from the protein surface. It can reverse protein aggregation and correct mutant protein defects and counter the harmful effects of urea and salts in vivo and in vitro. In this study we have investigated the pH dependence of the stabilizing effect of GB on three different proteins, namely, α-lactalbumin (α-LA), lysozyme and ribonuclease-A (RNase-A). We show here that (a) GB stabilizes RNase-A at all pH values, and (b) GB has opposite effects on two proteins at high pH and low pH values, namely, α-LA and lysozyme. This conclusion was reached by determining T_m (midpoint of denaturation), ΔH_m (denaturational enthalpy change at T_m), ΔC_p (constant-pressure heat capacity change) and ΔG_D^o (denaturational Gibbs energy change at 25 °C) of proteins in the presence of different GB concentrations. Another conclusion of this study is that ΔH_m and ΔC_p are not significantly changed in the presence of GB. This study suggests that other methylated glycine osmolytes may also behave in the same manner.     

pH-dependent urea-induced unfolding of stem bromelain: Unusual stability against urea at neutral pH

Equilibrium unfolding of stem bromelain (SB) with urea as a denaturant has been monitored as a function of pH using circular dichroism and fluorescence emission spectroscopy. Urea-induced denaturation studies at pH 4.5 showed that SB unfolds through a two-state mechanism and yields ΔG (free energy difference between the fully folded and unfolded forms) of ∼5.0 kcal/mol and C _m (midpoint of the unfolding transition) of ∼6.5 M at 25°C. Very high concentration of urea (9.5 M) provides unusual stability to the protein with no more structural loss and transition to a completely unfolded state.     

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.     

The content of alpha-helix and beta-structure in proteins crystallized at different pH values

Using the data of X-ray diffraction analysis for 100 three-dimensional structures of 26 proteins uniformly distributed among three main classes of the alpha-helix-beta-structure classification and without potentially polyanion regions, 154 comparisons of the content of alpha-helix and beta-structure content were made for structures obtained at different pH values of the medium, being distributed in the whole among all these proteins in the range from 1.5 to 12.0. No significant influence of pH of the medium on the size and localization of alpha-helices and beta-strands was found. As a consequence, it is suggested for the protein structure in a crystal that the alpha-helical-beta-structural backbone of protein structures does not depend on pH of the medium, except when the whole protein or its part can become a polyion so that the electrostatic interactions would either hinder or favour the formation of regular structures, and the conformational properties of ionizable amino acids are independent of pH of the medium. It is unclear, whether these assumptions can be extended to the case of solution, because the data for the structures in solution have been obtained for one protein only. These results can be used in investigations of protein structure, in protein engineering, and in the creation of specialized data banks of protein structures.     

Two threshold values of low pH block endocytosis at different stages.

The influence of low extracellular pH on endocytosis was studied in baby hamster kidney cells. When the extracellular medium was adjusted to pH 5.7, the intracellular pH decreased within 2 min to pH 6.2 and the endocytosis of horseradish peroxidase (HRP) in the fluid phase dropped to an undetectable level. With an external pH of 6.3, the internal pH dropped to pH 6.8 and HRP was internalized at a normal rate for 5 min but accumulation during longer incubation times did not occur. Morphologically, HRP was visualized in the lumen of a subpopulation of tubular and vesicular endosomes. These observations were confirmed by subcellular fractionation studies using free flow electrophoresis. Low extracellular pH also had an effect on the endocytosis of the membrane-spanning glycoprotein G of vesicular stomatitis virus which was implanted into the plasma membrane. The internalization of G-protein was quantitated by a surface fluoroimmunoassay. The endocytosis of G-protein was not affected when the external pH was dropped to 6.3, but was reduced at an external pH of 5.7. The intracellular ATP was not depleted and the reduction of endocytosis was reversible upon return to physiological pH. Clathrin coated pits were detected by electron microscopy at the plasma membrane of the low-pH-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions.

The objective of this study was to address the question of whether or not urea and guanidine hydrochloride (GdnHCl) give the same estimates of the stability of a particular protein. We previously suspected that the estimates of protein stability from GdnHCl and urea denaturation data might differ depending on the electrostatic interactions stabilizing the proteins. Therefore, 4 coiled-coil analogs were designed, where the number of intrachain and interchain electrostatic attractions (A) were systematically changed to repulsions (R): 20A, 15A5R, 10A10R, and 20R. The GdnHCl denaturation data showed that the 4 coiled-coil analogs, which had electrostatic interactions ranging from 20 attractions to 20 repulsions, had very similar [GdnHCl]1/2 values (average of congruent to 3.5 M) and, as well, their delta delta Gu values were very close to 0 (0.2 kcal/mol). In contrast, urea denaturation showed that the [urea]1/2 values proportionately decreased with the stepwise change from 20 electrostatic attractions to 20 repulsions (20A, 7.4 M; 15A5R, 5.4 M; 10A10R, 3.2 M; and 20R, 1.4 M), and the delta delta Gu values correspondingly increased with the increasing differences in electrostatic interactions (20A-15A5R, 1.5 kcal/mol; 20A-10A10R, 3.7 kcal/mol; and 20A-20R, 5.8 kcal/mol). These results indicate that the ionic nature of GdnHCl masks electrostatic interactions in these model proteins, a phenomenon that was absent when the unchanged urea was used. Thus, GdnHCl and urea denaturations may give vastly different estimates of protein stability, depending on how important electrostatic interactions are to the protein.