Cytoplasmic pH Regulation in Acer pseudoplatanus Cells

glutamate, and (c) efflux of [14C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with I millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyndoxal phosphate-dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H /L-glutamate cotransport is dependent upon GAD activity, (b) the pH-dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H /L-Glu symport may involve both Hz consumption during 4-aminobutyrate production and ATP-driven H+ efflux.

Section: Discussionmentioning

confidence: 99%

“…The cytoplasmic buffering capacity of 14.9 ,umol H' -mL-' cytoplasm pH unit-' is not substantially different from a value of 20 to 40 ,umol H+ -mL-'cytoplasm -pH unit-' found in cultured Acer cells (8). Calculations based on this value, cell volume (4.7 x 10-3 mL.…”

Section: Discussionmentioning

confidence: 99%

Proton/l-Glutamate Symport and the Regulation of Intracellular pH in Isolated Mesophyll Cells

Snedden

Chung²,

Pauls³

et al. 1992

“…The only analogous observations in the plant literature concern the loss of pH control in Acer pseudoplatanus cells that had been exposed to propionic acid (8). Removing the propionic acid, by filtering the cells and washing with an acidfree medium, caused a rapid increase in pHc,, to a value in excess ofthe normal pH.…”

Section: Response To Extreme Values Of Phomentioning

confidence: 95%

“…Clearly, the rapid loss of intracellular propionic acid provided a short-term mechanism for removing H+ from the cytoplasm and the mechanisms that could compensate for this process were unable to respond sufficiently rapidly to prevent an increase in pHr. Subsequently, pHC,, fell below the normal value observed before the propionic acid treatment (8) indicating that the cells had some difficulty in deactivating the regulating mechanisms that were opposing the initial increase in pHcyt caused by the removal of the propionic acid. This effect appears to be analogous to the transient acidifications observed in the oil palm and carrot cells as the tissue recovered from the sudden increase in pHo .…”

Section: Response To Extreme Values Of Phomentioning

confidence: 99%

³¹P NMR Observations on the Effect of the External pH on the Intracellular pH Values in Plant Cell Suspension Cultures

Fox¹,

Ratcliffe²

1990

31P nuclear magnetic resonance (NMR) spectroscopy was used to monitor the response of oil palm (Elaeis guineensis) and carrot (Daucus carota) cell suspensions to changes in the external pH. An airlift system was used to oxygenate the cells during the NMR measurements and a protocol was developed to enable a constant external pH to be maintained in the suspension when required. Phosphonoacetic acid was used as an external pH marker and the intracellular pH values were measured from the chemical shiffs of the cytoplasmic and vacuolar orthophosphate resonances. In contrast to earlier studies the cytoplasmic pH was independent of the extemal pH over the range 5.5 to 8.0 and it was only below pH 5.5 that the cytoplasmic pH varied, falling at a rate of 0.12 pH unit per extemal unit. Loss of pH control was observed in response to sudden increases in extemal pH with the response of the cells depending on the conditions imposed. A notable feature of the recovery from these treatments was the transient acidification of the cytoplasm that occurred in a fraction of the cells and overshoot phenomena of this kind provided direct evidence for the time dependence of the regulatory mechanisms.Many biochemical and biophysical processes are sensitive to pH and if a cell is to function normally, it follows that the intracellular pH values need to be tightly controlled. Regulation of pH in a cellular compartment can be assumed to occur by a combination of several mechanisms, including H+ transport and the metabolic control of the concentration of ionisable groups, and progress in the understanding of these processes in plant cells has been regularly reviewed (2, 5, 1 1,17,18,24). One generalization that has emerged concerns the effect of pHO2 on the internal pH: it appears that pH,,, is relatively insensitive to changes in pH0 (11)

“…The pulse repetition time was 1.2 s and the total acquisition time was usually 10 min. Calibration curves relating pH to Pi chemical shifts were established as described in Guern et al (10). We checked that the presence of sorbitol (0.7 M) had no significant effect on the Pi chemical shift.…”

Section: Protoplast and Vacuole Isolationmentioning

confidence: 99%

Regulation of Vacuolar pH of Plant Cells

Mathieu¹,

Guern²,

Kurkdjian³

et al. 1989

Self Cite

For the first time, the 31P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 x 106 vacuoles per milliliter from the enriched C. roseus cells have an intemal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ApH of 1.56 ± 0.07. Reliable determinations of vacuolar and extemal pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H exchanges at the tonoplast. The 31P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.