Changes In Buffer Concentration Research Articles

Structural differences between foot-and-mouth disease and poliomyelitis viruses influence their inactivation by aziridines

Inactivation of foot-and-mouth disease virus (FMDV) and poliovirus by ethyleneimine (EI) and N-acetylethyleneimine (AEI) has been studied at 25° and at 37°C and in different ionic conditions. FMDV is inactivated rapidly in 100 mM Tris pH 7.6 by each reagent at both temperatures. Poliovirus is also inactivated rapidly in 100 mM Tris by EI at both temperatures and by AEI at 37°C. However, it is inactivated much more slowly by AEI at 25°C; but if the virus is first incubated overnight at 2°C with AEI before transferring to 25°C inactivation then proceeds rapidly. Moreover, the rate of inactivation at 25°C is markedly increased if the virus is suspended in 1 mM Tris. We had interpreted these differences as being due to the greater penetrability of poliovirus (i) in 100 mM Tris at 37°C compared with 25°C and (ii) at lower ionic strength. This interpretation has been confirmed by electron microscopy of FMDV and poliovirus particles stained with phosphotungstic acid. At the elevated temperature, poliovirus had an average diameter of 34±0.21 nm and the stain outlined the nucleic acid core and the individual subunits, whereas at 25°C it averaged 28±0.13 nm and the stain did not penetrate the particle. This study also showed that the particle diameter alters with changes in buffer concentration, being 28±0.13 nm in 100 mM Tris, 31±0.16 nm in 10 mM Tris and 34±0.21 nm in 1 mM Tris. The changes in poliovirus are reversible as addition of 1/10 volume of 1 M Tris to the virus in 1 mM Tris resulted in the return of the diameter to 28±0.13 nm. FMDV, on the other hand, was less sensitive to osmotic differences as its particle diameter only varied by 7% over the 100-fold change in buffer concentration compared with the 22% change observed for poliovirus.

Calcium buffer injections delay cleavage in Xenopus laevis blastomeres.

Microinjection of calcium buffers into the two-cell Xenopus laevis embryo delays cell division in a dose-dependent manner. Four calcium buffers in the BAPTA series with different affinities for calcium were used to distinguish between a localized calcium gradient regulating cleavage and the global calcium concentration regulating this event. DibromoBAPTA (Kd = 1.5 microM) was found to delay cleavage at the lowest intracellular concentration (1.3 mM) of the four buffers tested. The effectiveness of the calcium buffers was dependent upon the buffer dissociation constant but not in a linear fashion. The concentration of buffer required to delay cleavage increased as the buffer's dissociation constant shifted above or below that of the optimum buffer, dibromoBAPTA. This relationship between a calcium buffer's effectiveness at delaying cleavage and its calcium affinity provides support for the hypothesis that a calcium concentration gradient is required for normal cell cycle progression (Speksnijder, J. E., A. L. Miller, M. H. Weisenseel, T.-H. Chen, and L. F. Jaffe. 1989. Proc. Natl. Acad. Sci. USA. 86:6607-6611). DibromoBAPTA was also injected with two different amounts of coinjected calcium to test the possibility that the free calcium concentration of the buffer solution is the important parameter for delaying cleavage. However, we found that changes in buffer concentration have a much stronger effect than changes in the free calcium concentration. This observation supports the hypothesis that BAPTA-type buffers exert their effect by shuttling calcium from regions of high concentration to those of lower concentration, reducing any calcium concentration gradients present in the Xenopus embryo.

155 THE ROLE OF LACTATE IN MEETING THE ENERGY REQUIREMENTS OF THE LATE FETAL HEART

An isolated perfused working heart preparation was used to investigate the role of lactate in the energy metabolism of the fetal heart. Hearts were perfused under working conditions with Krebs-Henseleit buffer containing either glucose 5mM or glucose 5mM and lactate 5mM combined. Insulin 100 μU/ml was present in all perfusions. Pressure development in both great vessels was maintained at 55 mmHg and mean atrial pressure was adjusted to 6 mmHg. Glucose uptake was measured by production of 3H2O from 23H glucose and lactate uptake by measuring changes in buffer concentration. With glucose as the sole exogenous substrate its utilization(3.48 ± .29 μmol/min/g dry), corrected for lactate production by the heart(3.06 ± μmol/min/g dry)could account for only 56 ± 9% of the total oxygen consumption of the heart(20.7 ± μmol/min/g dry). When both glucose and lactate were present in the buffer, glucose uptake was inhibited(0.71 μmol/min/g dry)and lactate- uptake(6.55 μmol/min/g dry)accounted for approximately 91 ± 12% of the total oxygen consumption(22.3 ± 1.8 μmol/min/g dry). These findings suggest that lactate oxidation is the primary source of metabolic energy for the fetal heart in late gestation, since glucose alone does not satisfy the oxidative needs of the fetal heart and since the presence of lactate profoundly suppresses glucose uptake. Lactate is the primary metabolic substrate for the fetal heart when present in concentrations similar to those observed in vivo, much as lipids are the primary metabolic substrate in more mature hearts.

Role of luminal buffers in renal tubular acidification.

The acidification of kinetics of artificial solutions containing buffers of different permeancy were studied in rat proximal tubules by means of stationary microperfusion techniques. Luminal pH changes were measured by antimony microelectrodes and used to calculate net rates of acidification and the approach to steady-state pH levels. For most buffer species, tracer efflux out of the lumen was compared with changes in buffer concentration as derived from calculations based on the Henderson Hasselbalch equation. Steady-state luminal pH was similar for most buffer systems studied. However, secretory hydrogen ion fluxes into the lumen were significantly higher for permeant than for less permeant buffers. The most likely explanation is that permeant buffers behave as "open" systems maintaining constant low diffusible acid levels in the lumen, whereas impermeant buffers behave as "closed" systems in which non-ionized acid levels are maintained at higher levels. A behavior consistent with this thesis was directly demonstrated for glycodiazine and, to a lesser degree, for DMO. In contrast, phosphate and creatinine behave like buffers in a "closed" system. Characteristics of proximal tubular acidification, of buffer reabsorption, and the effect thereupon of carbonic anhydrase inhibitors are satisfactorily explained by an essential role of (1) hydrogen ion secretion, (2) pK differences, and (3) different permeance of the non-ionized buffer species. However, specific transport mechanisms may, in addition, also contribute to differences in transepithelial buffer movement.

Regulation by salt and by krebs cycle metabolites of citrate synthase from an osmoregulator, white shrimp, Penaeus setiferus, and from a non-osmoregulator, sea anemone, Bunedosoma cavernata

1. 1. Citrate synthase of the white shrimp, Penaeus setiferus, and of the sea anemone, Bunedosoma cavernata, was partially purified. 2. 2. When reacted in various buffer concentrations, Penaeus citrate synthase demonstrated a distinct peak in catalytic activity in 75 mM potassium phosphate or 100 mM Tris-Cl (pH 7·5). Bunedosoma citrate synthase demonstrated a broad, almost flat profile under these conditions. 3. 3. The apparent Michaelis constant for acetyl coenzyme A was increased about fiftyfold in the Penaeus citrate synthase in the presence of 100 mM potassium phosphate buffer as compared to 20 mM buffer. The K m of Bunedosoma citrate synthase was not markedly affected by changes in buffer concentration. The K m 's for OAA were not greatly affected. 4. 4. Both citrate synthases were inhibited by ATP, NADH and α-KG, and an increase in AcSCoA reduced the inhibitions. 5. 5. The molecular weight of P. setiferus and B. cavernata citrate synthases was estimated by gel filtration to be 100,000 Daltons.

The stacked-disk structure of tobacco mosaic virus protein

The protein of TMV polymerizes slowly in alkaline solution (pH 8–10.5) to form rod-shaped particles which have the stacked-disk structure. The polymerization process has been followed by electron microscopy and analytical centrifugation; possible mechanisms are discussed. The particles are resistant to changes in buffer concentration and pH, and are not in equilibrium with their precursors. They are degraded only at a pH above 12 or below 2 and are resistant to proteolytic enzymes. Electrophoresis indicates that the particles consist of more than one structural type.

ELECTROPHILIC DISPLACEMENT REACTIONS: XV. KINETICS AND MECHANISM OF THE BASE-CATALYZED PROTODEBORONATION OF ARENEBORONIC ACIDS

An investigation of the kinetics of the protodeboronation of benzeneboronic acid in water in the pH range 2.0 to 6.7 is described. In addition to the acid-catalyzed reaction studied earlier a reaction whose rate is independent of pH and one whose rate increases linearly with hydroxide ion concentration have been observed. The effect of malonate buffer concentration at low pH confirms the earlier observations of general acid catalysis. Changes in buffer concentration at pH 6.70 have no effect on rate indicating specific hydroxide ion catalysis. Effect of substituents in the ortho, meta, and para position of the benzene ring on the rate of protodeboronation have been examined. Ortho–para ratios for this reaction are high; possible reasons for this are discussed. The Hammett equation using σ correlates the rates for meta and para substituents.