Klaas Nicolay

Phosphorus-31 nuclear magnetic resonance studies of intracellular pH, phosphate compartmentation and phosphate transport in yeasts

Abstract31P NMR spectra were obtained from suspensions of Candida utilis, Saccharomyces cerevisiae and Zygosaccharomyces bailii grown aerobically on glucose. Direct introduction of substrate into the cell suspension, without interruption of the measurements, revealed rapid changes in pH upon addition of the energy source. All 31P NMR spectra of the yeasts studied indicated the presence of two major intracellular inorganic phosphate pools at different pH environments. The pool at the higher pH was assigned to cytoplasmic phosphate from its response to glucose addition and iodoacetate inhibition of glycolysis. After addition of substrate the pH in the compartment containing the second phosphate pool decreased. A parallel response was observed for a significant fraction of the terminal and penultimate phosphates of the polyphosphate observed by 31P NMR. This suggested that the inorganic phosphate fraction at the lower pH and the polyphosphates originated from the same intracellular compartment, most probably the vacuole. In this vacuolar compartment, pH is sensitive to metabolic conditions. In the presence of energy source a pH gradient as large as 0.8 to 1.5 units could be generated across the vacuolar membrane. Under certain conditions net transport of inorganic phosphate across the vacuolar membrane was observed during glycolysis: to the cytoplasm when the cytoplasmic phosphate concentration had become very low due to sugar phosphorylation, and into the vacuole when the former concentration had become high again after glucose exhaustion.

In vivo 31P NMR studies on the role of the vacuole in phosphate metabolism in yeasts

Abstract31P NMR was used to study the dynamics of phosphate pools during substrate utilization by aerobic and anaerobic suspensions of the yeast Candida utilis and by aerobic suspensions of the yeast Brettanomyces intermedius. In both yeast, the cytoplasmic pH was monitored; in C. utilis also the vacuolar pH could be measured. When glucose was used as a substrate for C. utilis, the vacuolar store of inorganic phosphorus (both orthophosphate and polyphosphate) was mobilized to replenish cytoplasmic phosphate which had become very low due to the build-up of high sugar phosphate levels. The hydrolysis of polyphosphate was glucose-dependent; it did not occur with ethanol as the substrate. After glucose depletion resynthesis of polyphosphate occurred only under aerobic conditions; anaerobic C. utilis cells continued to hydrolyze vacuolar polyphosphate. This difference between the aerobic and anaerobic suspension could be related to differences in cellular ATP levels. When ethanol was employed as a substrate, both Candida utilis and Brettanomyces intermedius exhibited a substantial increase in polyphosphate levels. These observations suggested a dual role for polyphosphate in yeasts both as a phosphate and an energy store. The cytoplasmic pH in C. utilis displayed characteristic responses to metabolic changes during glucose degradation. B. intermedius experienced a strong cytoplasmic acidification upon ethanol utilization due to acetic acid formation. The mechanism of transport of Pi across the vacuolar membrane in C. utilis appeared to be different from that reported for the plasma membrane.

Quantitative agreement between the values for the light-induced ΔpH in Rhodopseudomonas sphaeroides measured with automated flow-dialysis and 31P-NMR

Quantitative determination of transmembrane pH and electrical potential gradients is a prerequisite for a further refinement of the concepts of the chemiosmotic hypothesis [ 11. Several methods are available for the measurements of ApH and A

Energetics of smooth muscle taenia caecum of guinea‐pig: a 31P‐NMR study

(review [21), of which the ‘spectroscopic’ and ‘distribution’ methods are most widely used. The outcome of the various methods, however, does show significant variations and it has been established by stringent comparisons, that the optical methods overestimate the magnitude of the transmembrane gradients [3-61. The distribution methods [2,7] (except flow-dialysis [S]) have the disadvantage that leakage of the probe molecules may occur during the separation step. Furthermore, in any distribution method uncertainties remain concerning: (i) The homogeneity of the internal aqueous phase and the absence of subcellular compartments; (ii) The ‘ideal behaviour’ [2] of the indicator probe; (iii) The activity coefficient of the probe molecules in the internal aqueous phase. With the application of 3’P NMR to biological systems [9-l l] a powerful and independent method has become available for the quantitation of ApH. This technique makes use of the pH dependence of 3’P NMR chemical shifts of phosphate metabolites. It can be used only if calibration curves of the chemical shift

The electrochemical H+ gradient in the yeast Rhodotorula glutinis

Smooth muscle cell energetics of taenia caeci during relaxation, activity and maximal contraction were investigated using 31P‐NMR. In relaxed muscle obtained in calcium‐free medium, [ATP], [phosphocreatine] and [sugar phosphate] were 4.4 mM, 7.7 mM and 2.8 mM, respectively. There was only a small difference in the energetics of spontaneously active and maximally contracted muscles, but under both conditions substantial changes occurred as compared with relaxed muscles. The internal pH in relaxed muscle was found to be 7.05, which acidified to 6.5 during contraction. The level of sugar phosphates was found to be not a limiting factor in energetics.

31P NMR studies of pH homeostasis in intact adult Fasciola hepatica

AbstractThe electrochemical gradient of protons,

Carbon-13 nuclear magnetic resonance studies of acetate metabolism in intact cells of Rhodopseudomonas sphaeroides

\Delta \tilde \mu _{H + }

Laser photo-CIDNP as a surface probe for proteins in solution

, was estimated in the obligatory aerobic yeastRhodotorula glutinis in the pH0 range from 3 to 8.5. The membrane potential, ΔΩ, was measured by steady-state distribution of the hydrophobic ions, tetraphenylphosphonium (TPP+) for negative ΔΩ above pH0 4.5, and thiocyanate (SCN−) for positive ΔΩ below pH0 4.5. The chemical gradient of H+ was determined by measuring the chemical shift of intracellular Pi by31P-NMR at given pH0 values. The values of pHi increased almost linearly from 7.3 at pH0 3 to 7.8 at pH0 8.5. In the physiological pH0 range from 3.5 to 6,