Phosphate Metabolism in Intact Human Erythrocytes: Determination by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy

152

High resolution 31P nuclear magnetic resonance (NMR) spectra at 145.7 MHz are presented for intact yeast cells. Several peaks are resolved and assigned. They include the middle phosphate peaks from long chain or cyclic polyphosphates. Our results are consistent with the suggestion that these polyphosphates act as a phosphate store in the cell. We have also been able to measure cytoplasmic pH using the orthophosphate peak inside the cell, as compared with outside the cell. The results show that yeast cells maintain their cytoplasmic pH around 6.3. This value is considerably higher than the acidic extracellular pH at which they normally live. These preliminary results indicate that 31P NMR at 145.7 MHz can be a rapid, informative, and non-invasive method for probing biochemical events within living cells. 0.5 g of MgSO4; 0.5 g of CaC12; and 50 g of glucose. The pH was adjusted to 5 at the beginning of growth. The cells were grown to stationary phase. Cells grown on intermediate-and low-phosphate media had the same initial composition as above except for the amount of KH2PO4 added (intermediate = 10-2 g and low = 10-4 g). When the stationary phase of growth (determined by the number of cells per ml) was reached, the cultures of yeast cells were harvested by lowspeed centrifugation at 40 in a Sorvall high-speed centrifuge. The cells, always in the cold, were packed to about 250% by volume and brought to room temperature when the extracellular pH was measured. D20 was added to these cells to about a 10% level and the sample was placed in the NMR sample tube. When the extracellular pH was changed from the normal acidic level to more alkaline values NaOH was used. The sodium tripolyphosphate used for titration in these experiments was obtained as a gift from Monsanto in New Jersey.The 31P NMR spectra were measured on a Bruker HX-360 spectrometer at 145.7 MHz (84.5 kG) although one spectrum taken at 40.5 MHz on a Varian XL-100 NMR spectrometer is presented for comparison. In the XL-100 12 mm diameter tubes were used, whereas in the HX-360 10 mm tubes were used and both samples were 1.0 cm high. Unless otherwise specified, spectra were measured at 250. The pulsed Fourier transform technique was used on both instruments. In the HX-360 the field homogeneity was adjusted on 10% D20 added to the cell suspensions and the field was locked on that signal. In some cases, D20 in a 4 mm capillary at the center of a 10 mm sample tube was used for the field lock. For the spectrum observed with the XL-100 the field was locked on the H20 signal. In our experience with yeast and other cells, spinning the sample did not measurably sharpen most of the 31P lines observed, although it did sometimes sharpen the narrow lines observed from extracellular orthophosphate (see below). Nevertheless, sample spinning was usually employed. All spectra are reported relative to 85% phosphoric acid at 0 parts per million ppm.

“…6 shows results from such measurements using data from Figs. 2 Fig. 4, a well-resolved line is seen at a higher field whose position is consistent with the measured extracellular pH.…”

supporting

confidence: 55%

mentioning

confidence: 99%

High resolution 31P nuclear magnetic resonance studies of intact yeast cells.

Salhany¹,

Yamane²,

Shulman³

et al. 1975

152

“…Intracellular phosphorus compounds were first observed by NMR in erythrocytes (1,2). This enabled the measurement of intracellular pH (1) as well as of concentration changes of several metabolites including 2,3-diphosphoglycerate and ATP (2). The technique has been applied to various tissues (3)(4)(5)(6)(7)(8)(9)(10) and cells (11)(12)(13) and recently to Ehrlich ascites tumor cells (11).…”

mentioning

confidence: 99%

“…The technique has recently been applied to the study of cells and tissues. Intracellular phosphorus compounds were first observed by NMR in erythrocytes (1,2). This enabled the measurement of intracellular pH (1) as well as of concentration changes of several metabolites including 2,3-diphosphoglycerate and ATP (2).…”

mentioning

confidence: 99%

³¹ P nuclear magnetic resonance studies of HeLa cells

Evans

Kaplan

1977

A survey of phosphorus compounds present in HeLa cells and their acid extracts-has been carried out by 31p nuclear magnetic resonance spectroscopy at 40 MHz. The proton decoupled 31p spectrum of the neutralized extract had resolution adequate to enable the identification of the main phosphate compounds. The spectral intensities were converted to concentrations. The lower detection limit with extensive signal averaging was 0.02 Mmol for the extract. The composition, listed in order of decreasing concentration, was: inorganic phosphate, ATP, phosphorylcholine, creatine phosphate, UTP, NAD+, glucose 6-phosphate, fl-D-fructose 1,6-bisphosphate, a-

“…In addition to the dominant peaks of fructose 1,6-bisphosphate (Fru-P2) labeled D and E from the P anomer, we see in peak A and probably in peak G contributions from the a anomer. The decomposition products of Fru-P2 are dihydroxyacetone phosphate (DHAP), whose two forms are responsible for peaks B and F, and glyceraldehyde 3-phosphate (GAP), which is assigned to peak C. Peaks J and K are assigned to glycero-3-phosphorylethanolamine and glycero-3-phosphorylcholine, respectively, on the basis of their chemical shifts and the fact that they sharpen upon proton decoupling and look like 1 (2). The most unexpected assignment is that of the strong peak H to phosphorylcholine.…”

mentioning

confidence: 99%

31P nuclear magnetic resonance studies of Ehrlich ascites tumor cells.

Navon¹,

Ogawa²,

Shulman³

et al. 1977

167

High-resolution 31P nuclear magnetic resonance spectra at 145.7 MHz are reported for intact Ehrlich ascites tumor cells and their perchloric acid extracts. In the extracts it was possible to assign resonances to fructose 1,6-bisphosphates, dihydroxyacetone phosphate, ATP, ADP, AMP, Pi, NAD+, phosphorylcholine, glycero-3-phosphorylcholine, glycero-3-phosphorylethanolamine, and glyceraldehyde 3-phosphate from their chemical shifts, pH behavior, and spin couplings. cine)-phosphate buffer described previously (6), but containing 10 mM phosphate instead of 4 mM.Extracts were prepared by rapidly mixing the cell suspension in the NMR tube with 0.2 volume of 35% (wt/vol) perchloric acid. After centrifuging the supernatant was neutralized with K2C03 and centrifuged again to remove the precipitated KCI04.NMR spectra were obtained at a frequency of 145.