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

Abstract

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.

31 P nuclear magnetic resonance studies of HeLa cells

“…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). We report here the application of 31P NMR at 40 MHz to the analysis of human cancer cells (HeLa) grown from tissue culture.…”

31P Nuclear‐Magnetic‐Resonance Studies of Energy Metabolism in Tissue from the Marine Invertebrate Tapes watlingi

“…Applications have included the observation and identification of tissue constituents, the measurement of intracellular pH values, and quantitative studies of metabolic changes associated with tissue aging under various conditions. Studies have been carried out on intact blood cells [3 -61, yeast [7], Escherichia coli [8,9], hepatocytes [lo], developing embryos [ l l ] tumor cells [ 12,131, mammalian organs [ 14 -161 and organelles [17,18], and whole mice [19]. Of particular importance to us are a number of studies on intact vertebrate skeletal muscle [20 -241.…”

31P nuclear magnetic relaxation studies of phosphocreatine in intact muscle: determination of intracellular free magnesium.

“…Spin-spin relaxation rates (1/T2) measured by modified Carr-Purcell-Melboom-Gill spin-echo experiments suggest the importance of scalar coupling modulated by chemical exchange with free Mg. From these results, we estimate the free intracellular Mg in intact muscle as 4.4 mM and demonstrate that 31p T2 experiments can be used as a tool for studying free Mg levels with minimum disturbance of the intact cell. 31P nuclear magnetic resonance (NMR) studies of intact cells to date have concentrated on high-resolution spectra of metabolites in intact muscle (1,2), erythrocytes (3), yeast (4), and Escherichia coli and Ehrlich ascites tumor cells (5,6). We report here on the feasibility of measuring the relaxation parameters of phosphocreatine (PCr) in intact frog muscle at 4°.…”