Phosphorus-31 nuclear magnetic resonance studies on intact erythrocytes. Determination of intracellular pH and time course changes in phosphorus metabolites

A method to determine the intracellular pH of intact erythrocytes using phosphorus-31 nuclear magnetic resonance spectroscopy is described. Changes in phosphorus metabolites due to the alkalization of intracellular pH were also examined. The normal erythrocytes gave signals of phosphate groups corresponding to 2,3-bisphosphoglycerate, inorganic phosphate, ATP, and NAD. Among them, the separation between alpha and gamma peaks of ATP was shown to be a good indicator of the intracellular pH free from the perturbation caused by hemoglobin. This method enabled us to determine the intracellular pH of the erythrocytes without any pretreatment. The separation between alpha and gamma peaks of ATP was also dependent on the degree of complexation with Mg2+, and was consistent with approximately 80% of total ATP complexing with Mg2+ in the samples investigated here. The pKa value of ATP in the erythrocytes was estimated to be 6.1 at 23 degrees C, which is lower than the value of 6.5 obtained for the Mg2+-free ATP solution. In the alkalized erythrocytes, fructose 1,6-bisphosphate and dihydroxyacetone phosphate were observed in addition to the metabolites found in the normal erythrocytes. Time course changes in these phosphorus metabolites were followed along with the intracellular pH monitored from ATP peaks.     

31P and 23Na nuclear magnetic resonance studies of resting and stimulated mast cells

Exocytosis induced by crosslinking the type I receptor for Fc epsilon domains present on rat mucosal mast cells (RBL-2H3-line) requires the influx of Ca2+ ions and is markedly influenced by the concentration of monovalent cations (K+, Na+ and protons) in their medium. We investigated the role of these ions in coupling the immunological stimulus to secretion using NMR spectroscopy to monitor simultaneously intracellular pH, ATP and Na+ concentrations and the secretory response of living adherent mast cells. Using this methodology we observed that: (i) ATP concentration and intracellular pH are highly regulated and no changes could be resolved in them upon stimulation and during exocytosis. (ii) In the absence of potassium ions in the cells' medium, a decrease is observed in the intracellular pH and ATP concentration and an increase in the Na+ concentration. (iii) From the influx of extracellular Na+ following inhibition of the Na+, K(+)-ATPase by ouabain, we estimated the inward Na+ current of resting cells to 5 x 10(7) ions/(cell.s). This value does not vary by more than 10% during exocytosis.     

Regulation of intracellular pH values in higher plant cells. Carbon-13 and phosphorus-31 nuclear magnetic resonance studies.

The regulation of the cytoplasmic and vacuolar pH values (pHc and pHv) in sycamore (Acer pseudoplatanus L.) cells was analyzed using 31P and 13C nuclear magnetic resonance spectroscopy. Suspension-cultured cells were compressed in the NMR tube and perfused with the help of an original arrangement enabling a tight control of the pH (external pH, pHe) of the carefully oxygenated circulating nutrient medium. Intracellular pH values were measured from the chemical shifts of: CH2-linked carboxyl groups of citric acid below pH 5.7; orthophosphate between pH 5.7 and 8.0; 13C-enriched bicarbonate over pH 8.0. pHc and pHv were independent of pHe over the range 4.5-7.5. In contrast intracellular pH values decreased rapidly below pHe 4.5 and increased progressively at pHe over 7.5. There was an acceleration in the rate of O2 consumption accompanied with a decrease in cytoplasmic ATP concentration as pHe decreased. When the rate of O2 consumption was approaching the uncoupled O2 uptake rate, a loss of pHc control was observed. It is concluded that as pHe decreased, the plasma membrane ATPase consumed more and more ATP to reject the invading H+ ions in order to maintain pHc at a constant value. Below pHe 4.5 the efficiency of the H+ pump to react to back leakage of H+ ions became insufficient, leading to an acidification of pHc and to an alkalinization of pHe. On the other hand, over pHe 7.5 a passive influx of OH- ions was observed, and pHc increased proportionally to the increase of pHe. Simultaneously appreciable amounts of organic acids (malate and citrate) were synthesized by cells during the course of the alkalinization of the cytoplasmic compartment. The synthesis of organic acids which partially counteract the alkalinization of the cytoplasmic compartment may result from a marked activation of the cytoplasmic phosphoenolpyruvate carboxylase induced by an increase in cytoplasmic bicarbonate concentration. The fluctuations of pHv followed a similar course to that of pHc. It is concluded that the vacuole, which represents a potentially large H+ ions reservoir, can counteract H+ (or OH-) ion invasion observed at acidic (or alkaline) pHe contributing to the homeostasis of pHc.     

31P nuclear magnetic resonance studies of the phospholipid-protein interface in cell membranes.

Both native and recombined membrane systems from the human erythrocyte membrane and the rabbit sarcoplasmic reticulum have been studied with 31P Nuclear Magnetic Resonance (NMR). We compare intensities of the anisotropic 31P resonance exhibited by these membranes with the intensity expected from the known phospholipid content of the membranous sample. In a recombinant with human erythrocyte glycophorin, a component of the phospholipid is "missing" from the 31P NMR resonance, apparently due to a severe broadening of the resonance of that component. Approximately 29 phospholipid molecules were found immobilized per glycophorin molecule in the membrane, regardless of the phospholipid:protein ratio. Cholesterol may inhibit the immobilization of phospholipids by glycophorin. Recombinants with band three from the human erythrocyte membrane contain an immobilized phospholipid component, analogous to the results with glycophorin. 31P NMR data from the native sarcoplasmic reticulum membrane also revealed an immobilized phospholipid component whose magnitude is independent of temperature between 30 degrees C and 45 degrees C. Extensive papain proteolysis of the membrane completely digests the Ca++ Mg++ ATPase and removes the immobilization of phospholipids noted in the intact membrane. Limited trypsin cleavage, however, does not completely remove the immobilized component; salt reduces the immobilized component.     

31P nuclear magnetic resonance evidence for the regulation of intracellular pH by Ehrlich ascites tumor cells

The phenomenon of intracellular pH (pHin) regulation in cultured Ehrlich ascites cells was investigated using 31P nuclear magnetic resonance (NMR) spectroscopy. Measurements were made with a Bruker WH 360 wide bore NMR spectrometer at a 31P frequency of 145.78 MHz. Samples at a density of 10(8) cells ml-1 were suspended in a final volume of 2 ml of growth medium in 10 mm diameter NMR tubes. Intracellular pH was calculated from the chemical shifts of either intracellular inorganic phosphate (Piin) or intracellular 2- deoxyglucose-6-phosphate (2dG6Pin). The sugar phosphate was used as a pH probe to supplement the Piin measurements, which could not always be observed. When available, the pHin calculated from the Piin peak was identical within experimental error to the pHin calculated from the 2dG6Pin peak. Intracellular pH was measured to be more alkaline than the medium at an external pH (pHex) below 7.1. Typical values were pHin = 7.00 for pHex = 6.50. These measurements were constant for times up to 165 min using well-energized, respiring cells. This pH gradient was seen to collapse immediately upon onset of anaerobic shock. Above a pHex of 7.2 there was no significant difference between pHin and pHex. These results unequivocally demonstrate the steady state nature of the pH regulation and its dependence upon energization.     

2H and31P nuclear magnetic resonance studies of membranes containing bovine rhodopsin

Summary Purified, delipidated rhodopsin is recombined with phospholipid using octyl-glucoside (OG) and preformed vesicles. Normal egg phosphatidylcholine, phosphatidylcholine in which the N-methyl groups are fully deuterated, and dioleoyl phosphatidylcholine labeled with deuterium at carbons 9 and 10 were used.³¹P nuclear magnetic resonance (NMR) and²H NMR measurements were obtained of the pure phospholipids and of the recombined membranes containing rhodopsin.³¹P NMR of the recombined membrane (containing the deuterated phospholipid) showed two overlapping resonances. One resembled a normal phospholipid bilayer, and the other was much broader, representing a motionally restricted phospholipid headgroup environment. The population of phospholipids in the motionally restricted environment can be modulated by conditions in the media.²H NMR spectra of the same recombined membranes showed only one component. These experimental results agree with a theoretical analysis that predicts an insensitivity of²H NMR to lipids bound to membrane proteins. A model containing at least three different phospholipid environments in the presence of the membrane protein rhodopsin is described.Deceased.     

31 P nuclear magnetic resonance studies of the interaction of pyridine nucleotide coenzymes with dehydrogenases.

31P nuclear magnetic resonance spectra of the pyrophosphate group in NAD+ and NADH were recorded in the presence of beef heart lactate dehydrogenase and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. At high lactate dehydrogenase concentrations (60 mg/ml), two NADH resonances are observed: a slowly exchanging peak which is shifted to 1.9 ppm downfield (relative to free NADH) and a rapidly exchanging peak with a downfield shift of 0.5-0.6 ppm. At lover concentrations (15 mg/ml) only the rapidly exchanging peak is observed thus indicating that the peak observed at-1.9 ppm is due to coenzyme bound to an aggregated enzyme species. With NAD+, rapid exchange and downfield shifts are observed at both enzyme and concentrations, with shifts of about 1.5 ppm and 0.6 ppm at 60 and 15 mg/ml, respectively. In the presence of glyceraldehydephosphate dehydrogenase, the results are independent of enzyme concentration, and slow exchange and upfield shifts of 0.4-0.6 ppm occur with each coenzyme. These data indicate that the environment of the pyrophosphate group of oxidized and reduced coenzyme is the same for a given dehydrogenase, but is different in one enzyme from the other. The resonances observed with glyceraldehydephosphate dehydrogenase are broader than those observed with lactate dehydrogenase. This is indicative of either shorter relaxation times with the former enzyme, or the presence of multiple, unresolved resonances.     

Lymphocyte activation and phospholipid pathways. 31P magnetic resonance studies

31P NMR spectra of perfused lymphocytes, embedded in alginate capsules and activated by interleukin-2, were remarkably different from those of control lymphocytes. The main differences were the appearance and gradual increase in phosphodiester signals, glycerophosphocholine and glycerophosphoethanolamine. These metabolic changes also occurred following perfusion with phorbol ester and after incubation with phytohemagglutinin (PHA) and were not dependent on a special growth medium. Nifedipine, a calcium channel blocking drug, inhibited the effects of phytohemagglutinin, but not of interleukin-2. There were no NMR spectral differences between peripheral lymphocytes, stimulated for 3 weeks, and tumor-infiltrating lymphocytes. Thus, sustained accelerated turnover of phosphatidylcholine and phosphatidylethanolamine is an inherent feature of the activation process. 31P NMR spectra of lymphocytes are characterized by a low signal of phosphocholine. Perfusion studies with high concentrations of choline and the use of dapsone, an inhibitor of cytidylyltransferase, indicated that choline kinase plays a key role in regulating phosphaditylcholine synthesis in human lymphocytes.     

31P nuclear magnetic resonance spectroscopy studies of substrate and product binding to fructose-1,6-bisphosphatase.

The enzymatic hydrolysis of fructose 1,6-bisphosphate (Fru-1,6-P2) to fructose 6-phosphate (Fru-6-P) and inorganic phosphate (Pi), which is catalyzed by fructose-1,6-bisphosphatase, has been studied by 31P nuclear magnetic resonance spectroscopy (NMR). At pH 7.5 and 15 degrees C, the equilibrium constant for the central complex K'eq = [E.Fru-6-P.Pi]/[E.Fru-1,6-P2.H2O] is about 2. This observation is in harmony with results obtained with a number of Bi Bi enzyme systems for the determination of K'eq in which a variety of experimental techniques were used (Knowles, J.R. (1980) Annu. Rev. Biochem. 49, 877-919). Significant changes in 31P NMR chemical shifts were observed for both the substrate, Fru-1,6-P2, and the product, Fru-6-P, when bound to the enzyme relative to ligand free in solution. The chemical shifts of the substrate and product were altered further in the presence of Mg2+, the catalytic divalent metal ion. The chemical shifts caused by the addition of metal ion can be reversed in the presence of trans-1,2-diaminocyclohexane- N,N,N',N'-tetraacetic acid (CDTA) or AMP. In the presence of the metal ion chelator or the nucleotide, the substrate had a chemical shift that was about the same as that observed in the absence of metal ion. On the basis of these observations we suggest that AMP and CDTA exhibit similar effects, i.e. they both remove the catalytic metal ion from the enzyme. This finding is supportive of the suggestion (Scheffler, J. E., and Fromm, H.J. (1986) Biochemistry 25, 6659-6665; Liu, F., and Fromm, H.J. (1990) J. Biol. Chem. 265, 7401-7406) that the role of AMP in the regulation of fructose-1,6-bisphosphatase is to prevent binding of the divalent metal activator to the enzyme.     

Estimation of intracellular sugar phosphate concentrations in Saccharomyces cerevisiae using 31P nuclear magnetic resonance spectroscopy

A systematic procedure has been formulated for estimating the relative intracellular concentrations of sugar phosphates in Saccharomyces cerevisiae based upon (31)P nuclear magnetic resonance (NMR) measurements. The sugar phosphate region of the (31)P NMR spectrum is first decomposed by computer analysis, and the decomposition consistency and identification of individual sugar phosphate resonances are established based on in vitro chemical shift calibrations determined in separate experiments. Numerous evaluations of intracellular S. cerevisiae compositions for different strains and different cell environments provide the basis for in vivocorrelations of inorganic phosphate chemical shift with the chemical shifts of 3-phosphoglycerate, beta;-fructose 1,6-diphosphate, fructose 6-phosphate, and glucose 6 phosphate. Relative intracellular sugar phosphate concentrations are obtained by correcting peak areas for partial saturation during transient in vivo experiments. In vivo concentrations estimated by this method agree well with estimates for similar systems based on other techniques. This approach does not require costly la belled compounds, and has the advantage that other important metabolic state variables such-as internal and external pH and intracellular levels of phosphate, ATP, ADP, NAD(H), and polyphosphate may be determined from the same (31)P spectrum. Extension of this strategy to other cellular systems should be straightforward.     

31P nuclear magnetic resonance and saturation transfer studies of the isolated perfused rat kidney

Previous studies using 31P nuclear magnetic resonance (NMR) saturation transfer techniques to quantitate the energy metabolism of the kidney have often resulted in estimates of adenosine triphosphate (ATP) turnover which are much lower than those predicted from the renal oxygen consumption and reasonable values of the P/O ratio. We measured the ATP turnover in isolated perfused kidneys of rats, using 31P NMR saturation transfer and a new procedure for quantitation of the intracellular Pi concentration. The estimated turnover rates of ATP were higher than previously reported. The P/O ratios calculated on the basis of these rates of ATP turnover and rates of renal oxygen consumption reported in the literature were within the range of theoretically possible values. Thus, 31P NMR saturation transfer can be used to quantitate the ATP turnover in the isolated perfused rat kidney.     

Transport and phosphorylation of choline in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies

When sycamore cells were suspended in basal medium containing choline, the latter was taken up by the cells very rapidly. A facilitated diffusion system appertained at low concentrations of choline and exhibited Michaelis-Menten kinetics. At higher choline concentrations simple diffusion appeared to be the principal mode of uptake. Addition of choline to the perfusate of compressed sycamore cells monitored by 31P NMR spectroscopy resulted in a dramatic accumulation of P-choline in the cytoplasmic compartment containing choline kinase and not in the vacuole. The total accumulation of P-choline over a 10-h period exhibited Michaelis-Menten kinetics. During this period, in the absence of Pi in the perfusion medium there was a marked depletion of glucose-6-P, and the cytoplasmic Pi resonance disappeared almost completely. When a threshold of cytoplasmic Pi was attained, the phosphorylation of choline was sustained by the continuous release of Pi from the vacuole although at a much lower rate. However, when 100 microM inorganic phosphate was present in the perfusion medium, externally added Pi was preferentially used to sustain P-choline synthesis. It is clear, therefore, that cytosolic choline kinase associated with a carrier-mediated transport system for choline uptake appeared as effective systems for continuously trapping cytoplasmic Pi including vacuolar Pi entering the cytoplasm.     

31P nuclear magnetic resonance studies of effects of some chlorophenols on Escherichia coli and a pentachlorophenol-degrading bacterium.

A Flavobacterium sp. that mineralizes pentachlorophenol degrades some, but not all, of the other chlorinated phenols. Whole-cell 31P nuclear magnetic resonance was used to compare and observe transmembrane pH gradients and nucleotide pools in the Flavobacterium sp. and Escherichia coli after pentachlorophenol and 3,4,5-trichlorophenol were added to the cell suspensions. The data suggest that those chlorinated phenols which are not degraded by the Flavobacterium sp. may be resistant to degradation because they act as proton dissipators.     

Analysis of 31P nuclear magnetic resonance lineshapes and transversal relaxation of bacteriophage M13 and tobacco mosaic virus.

The experimentally observed 31P lineshapes and transversal relaxation of 15% (wt/wt) M13, 30% M13, and 30% tobacco mosaic virus (TMV) are compared with lineshapes and relaxation curves that are simulated for various types of rotational diffusion using the models discussed previously (Magusin, P. C. M. M., and M. A. Hemminga. 1993. Biophys. J. 64:1851-1860). It is found that isotropic diffusion cannot explain the observed lineshape effects. A rigid rod diffusion model is only successful in describing the experimental data obtained for 15% M13. For 30% M13 the experimental lineshape and relaxation curve cannot be interpreted consistently and the TMV lineshape cannot even be simulated alone, indicating that the rigid rod diffusion model does not generally apply. A combined diffusion model with fast isolated motions of the encapsulated nucleic acid dominating the lineshape and a slow overall rotation of the virion as a whole, which mainly is reflected in the transversal relaxation, is able to provide a consistent picture for the 15 and 30% M13 samples, but not for TMV. Strongly improved lineshape fits for TMV are obtained assuming that there are three binding sites with different mobilities. The presence of three binding sites is consistent with previous models of TMV. The best lineshapes are simulated for a combination of one mobile and two static sites. Although less markedly, the assumption that two fractions of DNA with different mobilities exist within M13 also improves the simulated lineshapes. The possible existence of two 31P fractions in M13 sheds new light on the nonintegral ratio 2.4:1 between the number of nucleotides and protein coat subunits in the phage: 83% of the viral DNA is less mobile, suggesting that the binding of the DNA molecule to the protein coat actually occurs at the integral ratio of two nucleotides per protein subunit.