Dependence of pepsin conformational states on pH and temperature]

A conformational behaviour of pepsin depending on pH and temperature was studied by circular dichroism, differential UV-spectroscopy, calorimetry and enzymatic hydrolysis kinetics. A subtile conformational transition of the enzyme accompanied by changes in the physico-chemical and enzymatic properties of the protein was observed within the temperature interval of 15--40 degrees and within the pH range of 1,1--5,6. The range of pepsin heat denaturation was studied. A diagram of pepsin conformational states under different values of pH and temperature was built.     

Dependence of the mutagenic effect of N-nitroso-N-methylbiuret on the pH index]

The mutagenic effect of nitrosocompounds is known to be dependent on pH. The effect of N-nitrozo-N-methylbiuret on the conidia of Penicillium chrysogenum was studied within the ranges of pH from 5.0 to 7.0, the role of the buffer and distilled water being also considered. It was found that survival, morphological variation and induction of biochemical mutants depended on the value of pH. The optimal conditions for the culture treatment at the exposures tested were provided at pH 6.0 with the use of a phosphate buffer mixture as a substrate.     

Dependence of erythromycin biosynthesis on the active acidity of the medium]

Dependence of erythromycin biosynthesis on the medium active acidity was studied by the following methods: by changing pH of the initial medium, by changing the concentration of the medium components determining the active acidity of the culture, by using buffer mixtures by automatic control of pH. It was found that pH of the initial medium within 5.7-8.1 had no effect on the culture growth. Biosynthesis of erythromycin markedly decreased at pH 6.3 or lower. The values of pH within 6.6-7.5 (optimal values 6.7-6.9) were favourable for the antibiotic biosynthesis. At pH 6.2-6.3 the antibiotic accumulation was equal to 5-10 per cent of the control.     

Comparison of liposome entrapment parameters by optical and atomic absorption spectrophotometry

Methods for the complete characterization of liposomes prepared by ether-injection are described in detail. The validity of atomic absorption spectrophotometry Ior measuring markers of trapped volume was checked by comparative determinations of markers with established optical spectrophotometrical methods. The favorable results usingl atomic absorption spectrophotometry to quantitate the marker Mn²⁺ are of particular relevance as manganese ion is also the paramagnetic probe in n.m.r, measurements of water permeability of lipo-somes; our results indicate that in such measurements no other marker need be incorporated.     

Induction of tryptophan oxygenase by dexamethasone in isolated hepatocytes. Dependence on composition of medium and pH.

Hepatocytes were isolated from perfused rat livers. 4 x 10-6 cells/ml were incubated at at 37 degrees C in different media in the absence and presence of a steroid hormone, dexamethasone phosphate (2 x 10-5 M). 1. Hormonal enzyme induction occurred in cells suspended in a simple salt medium, devoid of amino acids and macromolecules. This induction was completely blocked by addition of either actinomycin D (2 mu-g/ml) or cycloheximide (50 mu-g/ml). 2. Incubation of cells in media containing defatted albumin did not enhance hormonal enzyme induction, although disintegration of cells during incubation was reduced. Addition of a crude albumin fraction reduced tryptophan oxygenase induction and dextran completely blocked enzyme induction by dexamethasone. 3. An increase of dexamethasone concentration in the presence of albumin to 9 x 10-5 M was unable to raise enzyme induction further, and a still higher concentration of hormone, 3 x 10-4 M, resulted in reduced enzyme induction. 4. The hormonal induction of tryptophan oxygenase was most pronounced when the pH of the medium was between 7.0 and 7.6, with an optium at 7.3. No induction was found when the pH of the medium was either 6.6 or 7.8. The basal tryptophan oxygenase activity was much less influenced by similar pH variations. It is concluded that hepatocytes in suspension are able to carry out hormone-stimulated enzyme synthesis and that factors influencing this process may be studied under controlled conditions in such systems.     

Psoralen-sensitized photohemolysis: Dependence on pH

The effect of pH on psoralen-sensitized photohemolysis (irradiation at 366 nm, 23 W/m²) was investigated. The dose (D) dependence of the rate of photohemolysis (V) fitted V = V₀ + kD^x (where V₀ is the rate of dark hemolysis and k is a coefficient). Variation of pH did not influence the exponent x, which was about 2; however, pH had a strong influence on the k value. The lowest V was observed in the pH interval from 8.0 to 8.4. It nearly doubled as the pH was changed from 8.4 to 9.0 as well as from 8.0 to 7.4. At pH below 7.4 the hemolysis rate increased sharply (by another factor of ～4). Since psoralen does not contain acid/base groups, the effect of pH could hardly be a result of changing the photophysical properties of the sensitizer. The increase in V in the alkaline region could be attributed to more pronounced photooxidation of reduced glutathione as a substrate, while the jump at pH ～7.3 would be ascribed to titration of a psoralen photooxidation product. The latter idea was supported by the HPLC data. Psoralen was preirradiated in ethanol and mixed in the dark with phosphate buffer at different pH, after which HPLC analysis revealed several pH-dependent photoproducts; for one of them the pH titration curve closely resembled the pH profile of psoralen-sensitized hemolysis.     

pH Dependence of Sphingosine Aggregation

Sphingosine and sphingosine 1-phosphate (S1P) are sphingolipid metabolites that act as signaling messengers to activate or inhibit multiple downstream targets to regulate cell growth, differentiation, and apoptosis. The amphiphilic nature of these compounds leads to aggregation above their critical micelle concentrations (CMCs), which may be important for understanding lysosomal glycosphingolipid storage disorders. We investigated the aggregation of sphingosine and S1P over a comprehensive, physiologically relevant range of pH values, ionic strengths, and lipid concentrations by means of dynamic light scattering, titration, and NMR spectroscopy. The results resolve discrepancies in literature reports of CMC and pK_a values. At physiological pH, the nominal CMCs of sphingosine and S1P are 0.99 ± 0.12 μM (pH 7.4) and 14.35 ± 0.08 μM (pH 7.2), respectively. We find that pH strongly affects the aggregation behavior of sphingosine by changing the ionic and hydrogen-bonding states; the nominal critical aggregation concentrations of protonated and deprotonated sphingosine are 1.71 ± 0.24 μM and 0.70 ± 0.02 μM, respectively. NMR measurements revealed that the NH₃⁺-NH₂ transition of sphingosine occurs at pH 6.6, and that there is a structural shift in sphingosine aggregates caused by a transition in the predominant hydrogen-bonding network from intramolecular to intermolecular that occurs between pH 6.7 and 9.9.     

Physiological properties of Saccharomyces cerevisiae during sine-modulated and stepwise changes in the medium pH

The responses of a chemostat Saccharomyces cerevisiae culture (D = 0.1 h-1) to a stepwise increase or decrease in the pH of the medium were shown to be asymmetric. When the pH was lowered from 6.5 to a value above 0.3, the rates of oxygen uptake and carbon dioxide evolution rose for a sort period of time whereas the optical density of the culture fell down. The detected changes in the properties of the culture were identical with those which had been observed in the course of spontaneous undamped oscillations in the physiological parameters of the continuous C. cerevisiae culture. Apparently, in both cases, the energy status of cells changed when the oxidative type of metabolism was substituted by fermentation. When the pH of the medium was elevated within the same range (4.7-6.5), the response of the culture was three times as low and its properties changed in the opposite direction. When the pH of the medium was changed in a cyclic sinusoidal manner, oscillations in the physiological characteristics of the culture, identical with spontaneous oscillations were induced at certain values of the amplitude and the frequency of pH changes.     

Multilayered vesicles prepared by reverse-phase evaporation: liposome structure and optimum solute entrapment

Liposome structure and solute entrapment in multilayered vesicles (MLVs) prepared by reverse-phase evaporation (REV) were studied. MLV-REV vesicles prepared from ether/water emulsions have high entrapment. Entrapment depends on drug, drug concentration, lipid, lipid concentration, and the container used to prepare the vesicles. By use of 300 microL of aqueous phase and 100 mg of phosphatidylcholine (PC), vesicles prepared in a test tube 25 mm X 175 mm have higher entrapment than vesicles prepared in a 100-mL round-bottom or pear-shaped flask. By use of a test tube, 100 mg of PC, and 300 microL of aqueous phase containing sucrose (1-50 mg/mL), greater than 90% sucrose entrapment was obtained. Increasing lipid content to 150 mg of PC decreased entrapment to approximately 80%. Neutral PC MLV-REV vesicles have optimum entrapment. Mixing negatively charged lipids or cholesterol (CH) with PC to make MLV-REV vesicles results in decreased entrapment compared to using only PC. Preparing vesicles with the solid lipid dipalmitoylphosphatidylcholine (DPPC) or DPPC/CH mixtures (0 less than or equal to mol % CH less than or equal to 50) results in approximately 30-40% entrapment when diethyl ether is used to make the MLV-REV emulsion. Substituting diisopropyl ether for diethyl ether and heating the MLV-REV emulsion during vesicle formation generate DPPC/CH vesicles that entrap 60% of added solutes. The high entrapment found for MLV vesicles prepared from water/organic solvent emulsions depends on maintaining a core during the process of liposome formation. A method to calculate the fraction of water residing in the liposomes' core is presented and used to compare multilayered vesicles prepared by different processes. X-ray diffraction data demonstrate that a heterogeneous distribution of lipid may exist in multilayered vesicles prepared by the REV process.     

Dependence on pH of the activity of pig liver esterase

The dependence on pH of the kinetic parameters for the hydrolysis of phenyl acetate catalyzed by pig liver carboxylesterase was examined for purified high-isoelectric point and low-isoelectric point fractions of enzyme that were separated by isoelectric focusing. The values of k_cat are half-maximal at pH 4.3 and 5.1 for the high- and low-isoelectric point forms, respectively, and show a shallow dependence on pH with a value of n = 0.5. The absence of a change in the pH dependence of k_cat for the high-isoelectric point enzyme in the presence of high concentrations of methanol, which reacts with the acetyl-enzyme intermediate to give methyl acetate, provides evidence that the pH dependence is not caused by a change in rate-determining step. This means that if an imidazole group is involved in catalysis its pK must be perturbed downward by 2–3 units. The pH dependence of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ is biphasic with apparent pK values for dissociations of the free enzyme near 7 and 4 for both the high- and low-isoelectric point enzymes. Inhibition by a second molecule of substrate and by methanol are strongest for high-pH forms of the enzyme.     

Effects of a medium pH on the ability of Chinese hamster cells to repair radiation injuries]

In experiments with Chinese hamster cells at exponential and stationary growth phases, it has been shown that the postirradiation incubation of irradiated cells in a medium with low pH (up to 6.0) promotes the recovery of cells from potentially lethal damages; it has also been found that the recovery from sublethal radiation damages does not depend on the medium pH. The long-term incubation of nonirradiated cells with low pHc causes death of part of cells.     

The capacity of growing algal cells to affect the pH and the buffering properties of the medium

Supernatants from cultures of green high-temperature algae,Chlorella 7-11-05 and Stichococcus 6-17-35, were used to obtaintitration curves and to calculate buffer indexes (ß).It was generally observed that the peak of buffering activityin growing cultures shifted to the pH characteristic of thepK of the newly generated buffer(s). Depending on the experimentalconditions and the age of the culture, the buffering capacityat its peak increased up to 4–5 times of the value forthe original medium. Buffering capacity of algal cells was demonstratedfor both strains grown in the media originally buffered eitherwith phosphate or with Tris and for cultures of different agesincluding those of the young synchronized cells. The capacityof growing cells to drastically affect the pH and/or the bufferingcharacteristics of the medium indicates that the role of thebuffer originally employed in a particular medium may oftenextend for a relatively short period of time after which thebuffer system(s) produced by the cells play a more importantpart. (Received May 18, 1971; )     

Effect of the initial pH value of the medium on the growth of Streptococcus lactis and the biosynthesis of nisin]

The effect of the initial pH value of the medium within 4.0 to 6.6 on the growth of Str. lactis and biosynthesis of nicin was studied. It was found that at the initial pH 4.0--4.5 of the medium the growth of the culture was poor, i.e. 11--14% of the control (initiral pH 6.6). With an increase in the value of the initial pH at least to 5.0 the growth of Str. lactis also increased. At the initial pH 4.0 no biosynthesis of nicin was observed. Under the experimental conditions the antibiotic synthesis by tr. lactic started at the initial pH being equal to 4.5 and reached its maximum at pH 6.6.     

Entrapment of ovalbumin into liposomes--factors affecting entrapment efficiency, liposome size, and zeta potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB + 0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential--the factors that are of great importance for the use of liposomes as drug carriers.     

Effect of the pH of the medium on DNA conformation

The hydrodynamical and optical properties of DNA were investigated in the wide-range of pH by the methods of streaming birefringence, viscometry and spectrophotometry for the different ionic strengths of environment. The measurements of the intrinsic viscosity as a function of pH allow us to determine the compactization of protonated DNA without the destruction of double-helical conformation. This transition is accompanied by a decrease in the optical anisotropy of DNA and the coefficient of molar extinction E260 (P). The increase of volume and persistence length of DNA was observed in the alkaline range of pH. Analyses of experimental data lead to an assumption that the predominant cause of these effects is the change of flexibility of DNA as a result of ionization of its bases. The data obtained were compared with those for polycationic molecules.     

Dependence of the intracellular concentrations of univalent ions and hydrogenase activity on the salt composition and pH of the medium in the haloalkaliphilic sulfate-reducing bacterium Desulfonatronum thiodismutans

It has been shown that the intracellular concentrations of Na+, K+, and Cl- ions in Desulfonatronum thiodismutans depend on the extracellular concentration of Na' ions. An increase in the extracellular concentration of Na+ results in the accumulation of K+ ions in cells, which points to the possibility that these ions perform an osmoprotective function. When the concentration of the NaCI added to the medium was increased to 4%, the concentration gradient of Cl- ions changed insignificantly. It was found that D. thiodismutans contains two forms of hydrogenase--periplasmic and cytoplasmic. Both enzymes are capable of functioning in solutions with high ionic force; however they exhibit different sensitivities to Na+, K+, and Li+ salts and pH. The enzymes were found to be resistant to high concentrations of Na+ and K+ chlorides and Na+ bicarbonate. The cytoplasmic hydrogenase differed significantly from the periplasmic one in having much higher salt tolerance and lower pH optimum. The activity of these enzymes depended on the nature of both the cationic and anionic components of the salts. For instance, the inhibitory effect of NaCl was less pronounced than that of LiCl, whereas Na+ and Li+ sulfates inhibited the activity of both hydrogenase types to an equal degree. The highest activity of these enzymes was observed at low Na+ concentrations, close to those typical of cells growing at optimal salt concentrations.