Observation of the ^(31)P signal from various intracellular phosphates can provide a convenient, nondestructive technique for determining intracellular conditions such as pH. This procedure has been explored with particular reference to the erythrocyte. Both the chemical shift of intracellular inorganic phosphate relative to that of serum phosphate and the positions of, and more especially the difference between, the chemical shifts of 2,3-diphosphoglycerate have been used to monitor intracellular pH of erythrocytes whose hemoglobin has been liganded with carbon monoxide.
Measurements have been made of cytoplasmic pH, (pHi) and free Mg2+ concentration, ( [Mg2+]i), in pig and mouse lymphocytes. pHi was measured in four ways: by a digitonin null-point technique; by direct measurement of the pH of freeze-thawed cell pellets; from the 31P nuclear magnetic resonance (NMR) spectrum of intracellular inorganic phosphate; and by the use of a newly synthesized, intracellularly- trappable fluorescent pH indicator. In HEPES buffered physiological saline with pH 7.4 at 37 degrees C, pHi was close to 7.0. Addition of physiological levels of HCO3- and CO2 transiently acidified the cells by approximately 0.1 U. Mitogenic concentrations of concanavalin A (Con A) had no measurable effect on pH in the first hour. [Mg2+]i was assessed in three ways: (a) from the external Mg2+ null-point at which the ionophore A23187 produced no net movement of Mg2+ or H+; (b) by Mg- sensitive electrode measurements in freeze-thawed pellets; and (c) from the 31P nuclear magnetic resonance spectrum of the gamma-phosphate of intracellular ATP. Total cell Mg2+ was approximately 12 mmol per liter cell water. The NMR data indicated [Mg2+]i greater than 0.5 mM. The null-point method gave [Mg2+]i approximately 0.9 nM. The electrode measurements gave 1.35 mM, which was thought to be an overestimate. Exposure to mitogenic doses of Con A for 1 h gave no detectable change in total or free Mg2+.
The intracellular pH (pHi) of squid giant axons has been measured using glass pH microelectrodes. Resting pHi in artificial seawater (ASW) (pH 7.6-7.8) at 23 degrees C was 7.32 +/- 0.02 (7.28 if corrected for liquid junction potential). Exposure of the axon to 5% CO2 at constant external pH caused a sharp decrease in pHi, while the subsequent removal of the gas caused pHi to overshoot its initial value. If the exposure to CO2 was prolonged, two additional effects were noted: (a) during the exposure, the rapid initial fall in pHi was followed by a slow rise, and (b) after the exposure, the overshoot was greatly exaggerated. Application of external NH4Cl caused pHi to rise sharply; return to normal ASW caused pHi to return to a value below its initial one. If the exposure to NH4Cl was prolonged, two additional effects were noted: (a) during the exposure, the rapid initial rise in pHi was followed by a slow fall, and (b) after the exposure, the undershoot was greatly exaggerated. Exposure to several weak acid metabolic inhibitors caused a fall in pHi whose reversibility depended upon length of exposure. Inverting the electrochemical gradient for H+ with 100 mM K- ASW had no effect on pHi changes resulting from short-term exposure to azide. A mathematical model explains the pHi changes caused by NH4Cl on the basis of passive movements of both NH3 and NH4+. The simultaneous passive movements of CO2 and HCO3-cannot explain the results of the CO2 experiments; these data require the postulation of an active proton extrusion and/or sequestration mechanism.
The N(2)O : N(2) product ratio of denitrification is negatively correlated with soil pH, but the mechanisms involved are not clear. We compared soils from field experiments where the pH had been maintained at different levels (pH 4.0-8.0) by liming (> or = 20 years), and quantified functional gene pools (nirS, nirK and nosZ), their transcription and gas kinetics (NO, N(2)O and N(2)) of denitrification as induced by anoxic incubation with and without a carbon substrate (glutamate). Denitrification in unamended soil appeared to be based largely on the activation of a pre-existing denitrification proteome, because constant rates of N(2) and N(2)O production were observed, and the transcription of functional genes was below the detection level. In contrast, glutamate-amended soils showed sharp peaks in the transcripts of nirS and nosZ, increasing the rates of denitrification and pH-dependent transient accumulation of N(2)O. The results indicate that the high N(2)O : N(2) product ratio at low pH is a post-transcriptional phenomenon, because the transcription rate of nosZ relative to that of nirS was higher at pH 6.1 than at pH 8.0. The most plausible explanation is that the translation/assembly of N(2)O reductase is more sensitive to low pH than that of the other reductases involved in denitrification.