High-resolution phosphorus-31 nuclear magnetic resonance (31P NMR) spectra of wild-type and mutant strains of Saccharomyces cerevisiae were observed at a frequency of 145.7 MHz. Levels of various phosphorus metabolites were investigated upon addition of glucose under both aerobic and anaerobic conditions. Three mutant strains were isolated and their biochemical defects characterized: pfk lacked phosphofructokinase activity; pgi lacked phosphoglucose isomerase activity; and cif had no glucose catabolite repression of the fructose bisphosphatase activity. Each mutant strain was found to accumulate characteristic sugar phosphates when glucose was added to the cell suspension. In the case of the phosphofructokinase deficient mutant, the appearance of a pentose shunt metabolite was observed. 31P NMR peak assignments were made by a pH titration of the acid extract of the cells. Separate signals for terminal, penultimate, and central phosphorus atoms in intracellular polyphosphates allowed the estimation of their average molecular weight. Signals for glycero(3)phosphochline, glycero(3)phosphoserine, and glycero(3) phosphoethanolamine as well as three types of nucleotide diphosphate sugars could be observed. The intracellular pH in resting and anaerobic cells was in the range 6.5--6.8 and the level of adenosine 5'-triphosphate (ATP) low. Upon introduction of oxygen, the ATP level increased considerably and the intracellular pH reached a value of pH 7.2--7.3, irrespective of the external medium pH, indicating active proton transport in these cells. A new peak representing the inorganic phosphate of one of the cellular organelles, whose pH differed from the cytoplasmic pH, could be detected under appropriate conditions.
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.
ABSTRACT31P nuclear magnetic resonance spectra at 145.7 MHz were obtained of concentrated suspensions of E coli cells.The position of the Pi resonance was used to determine the pH, and in most experiments it was possible to distinguish the intracellular (pHin) and extracellular (pHE.) values. During respiration pHin approached 7.55, while pHex varied from 6.0 to 8.0.With succinate as a carbon source and in a N2 environment, pHin = pHex. Upon (11) and vesicles (12, 13) specific transport systems are coupled to an energized state of the membrane which could be energized either from ATP or oxidation-reduction processes. These typical results suggests that high-resolution 31P NMR studies of E. coli would be valuable because they could give a simultaneous measure both of ApH and the distribution of metabolites. It has been necessary to make measurements rapidly because the life cycle of E. coil is less than 1 hr, and often one wants to obtain a well resolved spectrum with adequate signal to noise in a small fraction of a cell cycle. This has been made possible by the resolution and sensitivity of our Bruker HX360 NMR spectrometer. Another technical detail hindering these measurements was the need to measure the NMR spectra under aerobic conditions in the NMR tube. These conditions have been obtained in two ways, one quite simple, as described below. EXPERIMENTAL E. coli MRE600 were grown in M9 medium with glucose (18 mM), glycerol (45 mM), or succinate (16 mM) as carbon sources. Cells were harvested in exponential phase at a cell density of about 1 X 109/ml, washed once with fresh growth medium, and resuspended at a density of 3 to 5 X 10"1/ml in the same buffer.31P NMR spectra were measured with a Bruker HX360 spectrometer operating in the Fourier Transform mode. Samples were contained in 10-mm diameter tubes and were generally about 1.5 ml in volume. Different methods of bubbling 02 (or N2) were used. In one we took advantage of the large change in the lock signal when the oxygen bubble passed through the solution. By synchronizing the pulse trigger to the level of the lock signal, it was possible to pulse the transmitter when the lock signal returned to its original state, indicating that the bubble had passed. In this way the free induction decay was accumulated when the bubble was not present in the solution and the magnetic field homogeneity was preserved. Also, by introducing a variable delay before the transmitter pulse it was possible to synchronize the NMR measurement with respect to the oxygen pulse. The second method was to bubble oxygen and pulse the transmitter in a nonsynchronous fashion, which gave similar results to the first method except that some line broadening due to magnetic field inhomogeneity was introduced. Fig. 1 shows the alp NMR spectra of E. coli cells grown in glucose. The top spectrum was summed over a 6-min interval between 7 and 1 min before 02 was vigorously introduced. The peaks, designated by letters, are tentatively identified by comparison with known standards and with the ...
We report here the biologically active conformation ofacetylcholine when bound to the high-affinity state of the receptor from Torpedo cakfornica. The acetylcholine conformation was determined in the free and bound states by proton NMR two-dimensional nuclear Overhauser effects. In agreement with x-ray crystallographic data, acetylcholine in solution has an extended conformation with an average distance between the acetyl methyl and choline methyl protons of =5 A. When bound to the acetylcholine receptor, acetylcholine adopts a conformation where the acetyl methyl group is dose (3.3 A) to the methyl groups of the choline moiety. This bent conformation places the oxygens adjacent to one another and allows the methyl groups to form an uninterrupted hydrophobic surface over the rest of the acetylcholine molecule. The significant difference between the fre-and bound-state conformations implies that structure-activity studies based solely on molecular modeling strategies must be approached with caution. To understand the interactions of biologically interesting molecules, it is necessary to know the shape ofthe interaction sites as well as the conformations of the interacting molecules. We report here the biologically active conformation of acetylcholine (AcCho) when bound to the high-affinity (desensitized) state ofthe acetylcholine receptor (AcChoR) from Torpedo californica. The exact location ofthe AcCho binding site on the a-subunits of the AcChoR is still unknown, although it has recently been localized to residues 158-216 by several groups using a variety oftechniques (1-4). In contrast to the uncertainty in the AcCho binding site, crystal structures are available for AcCho and other agonists that also bind to the AcChoR (5-7). However, we show here that the conformation of AcCho in its receptor-bound state is distinctly different from its conformation in solution and in the crystalline solid state. MATERIALS AND METHODSAll experiments used purified AcChoR in asolectin vesicles. The receptor was purified from the electroplax organs of T. californica as described (8). We have shown that under the conditions used in this study, AcCho binds to the AcChoR and that selective proton relaxation measurements are very sensitive to this binding. [Under these conditions the ratio of Kd (acetylcholine)/Kd (nicotine) is 0.14.] Conditions (see text) have been determined to distinguish between the contributions from site-specific binding ofAcCho to the AcChoR and contributions from nonspecific interactions of AcCho both with the lipid and with other areas of the receptor (8).All proton NMR spectra were acquired on a 360-MHz Bruker AM series spectrometer with an Aspect 3000 computer. The two-dimensional nuclear Overhauser effect (NOE) data sets were obtained in the phase-sensitive mode (9) and were processed with Dennis Hare's Fourier transform NMR program. The temperature in all experiments was held at 250C to within PC with the Bruker variable temperature unit by passing heated nitrogen gas through the probe.The conf...
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.
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