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 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.
M. Rostkowski, M. Olsson, Chresten R. Søndergaard
et al.
BackgroundCharge states of ionizable residues in proteins determine their pH-dependent properties through their pKa values. Thus, various theoretical methods to determine ionization constants of residues in biological systems have been developed. One of the more widely used approaches for predicting pKa values in proteins is the PROPKA program, which provides convenient structural rationalization of the predicted pKa values without any additional calculations.ResultsThe PROPKA Graphical User Interface (GUI) is a new tool for studying the pH-dependent properties of proteins such as charge and stabilization energy. It facilitates a quantitative analysis of pKa values of ionizable residues together with their structural determinants by providing a direct link between the pKa data, predicted by the PROPKA calculations, and the structure via the Visual Molecular Dynamics (VMD) program. The GUI also calculates contributions to the pH-dependent unfolding free energy at a given pH for each ionizable group in the protein. Moreover, the PROPKA-computed pKa values or energy contributions of the ionizable residues in question can be displayed interactively. The PROPKA GUI can also be used for comparing pH-dependent properties of more than one structure at the same time.ConclusionsThe GUI considerably extends the analysis and validation possibilities of the PROPKA approach. The PROPKA GUI can conveniently be used to investigate ionizable groups, and their interactions, of residues with significantly perturbed pKa values or residues that contribute to the stabilization energy the most. Charge-dependent properties can be studied either for a single protein or simultaneously with other homologous structures, which makes it a helpful tool, for instance, in protein design studies or structure-based function predictions. The GUI is implemented as a Tcl/Tk plug-in for VMD, and can be obtained online at http://propka.ki.ku.dk/~luca/wiki/index.php/GUI_Web.