31P nuclear magnetic resonance studies of isolated rat liver cells

Cohen, S. M.; Ogawa, Satoshi; Rottenberg, Hagai; Glynn, Paul; Yamane, T; Brown, Truman R.; Shulman, R. G.; Williamson, John

doi:10.1038/273554a0

Cited by 142 publications

(46 citation statements)

References 13 publications

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“…These two internal compartments have been assigned to represent the cytoplasm and the vacuole. The resonance in the lower pH region cannot originate from mitochondrial P~, because the respiring mitochondrion is expected to have a higher pH than the cytoplasm (Cohen et al 1978).…”

Section: Discussionmentioning

confidence: 99%

“…Although the NMR method can provide information about intracellular compartmentation, the number of systems in which this phenomenon has been demonstrated by 31p NMR is limited (Cohen et al 1978;Busby et al 1978;. Navon et al (1979) and Gillies et al (1981) have reported the presence of two 31p NMR resonances from intracellular inorganic phosphate in the yeast Saccharomyces cerevisiae.…”

Section: Offprint Requests To : K Nicolaymentioning

confidence: 99%

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

et al. 1982

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Abstract. 31p 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 3 ~p 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 3 ~p 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.

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Section: Discussionmentioning

confidence: 99%

Section: Offprint Requests To : K Nicolaymentioning

confidence: 99%

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

et al. 1982

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“…However, three narrow resonances are clearly visible. By analogy with 3~P-NMR data obtained for hepatocytes [15], we can assign these resonances as follows: the largest peak has the chemical shift position of inorganic phosphate, and the low-field peak the position of a phosphate monoester. The origin of the highfield peak is unknown.…”

Section: Microso Ruesmentioning

confidence: 99%

31P-NMR studies on membrane phospholipids in microsomes, rat liver slices and intact perfused rat liver

Kruijff

Rietveld

Cullis

1980

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Summary1. The 36.4 and 81 MHz 31P-NMR spectra of isolated rat liver microsomes, rat liver slices and perfused rat liver have been recorded in the 4--40°C temperature range.2. In isolated microsomes at 37°C the majority of the phospholipids undergo isotropic motion, whereas at 4°C most of the phospholipids give rise to typical 'bilayer' spectra.3. Isolated hydrated rat liver microsomal phosphatidylethanolamine is organised in the hexagonal HH phase above 7°C.4. The Mn 2÷ permeability of the microsomal membrane is strongly temperature dependent. At 37°C Mn 2÷ addition eliminates the entire 31P-NMR spectrum, demonstrating that all phospholipids interact with Mn ~÷. At 4°C a 43% reduction in signal intensity is observed, indicating that at this temperature 43% of the phospholipids are located in the outer monolayer of a lipid bilayer.5. In liver slices incubated in oxygenated Krebs-Ringer buffer at 4 ° C there is a rapid decrease in ATP level such that within 20 min almost all ATP is degraded. In these ATP-depleted liver slices at 4°C, virtually all phospholipids in all membranes have 3~P-NMR spectra indicating bilayer structure. At 37°C approx. 14% of the phospholipids undergo isotropic motion.6. Preliminary experiments on perfused rat liver show stable ATP levels for 4 h at 37°C. The spectra, furthermore, suggest similar behaviour for the membrane phospholipids to that observed in the liver slices.

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“…High-resolution 31P nuclear magnetic resonance (NMR) provides a rapid, accurate, and noninvasive method for the study of phosphorylated metabolites in vivo (7)(8)(9). Furthermore, 31P NMR measurements possess the capability to distinguish among nucleoside phosphate resonances arising from different subcellular compartments when these compartments maintain differences in pH, divalent cation concentration, or both.…”

mentioning

confidence: 99%

Adenine nucleotide storage and secretion in platelets as studied by 31P nuclear magnetic resonance.

Uğurbil¹,

Holmsen²,

Shulman³

1979

Proc. Natl. Acad. Sci. U.S.A.

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Suspensions of human and pig blood platelets have been studied by 31p NMR at 145.7 MHz and by chemical and radiochemical determination of nucleotide levels. In both types of platelets the cytoplasmic nucleotide pool, which was prelabeled by incubation with 1j4CJadenine, was selectively reduced by addition of H202/NaN3 or 2-deoxyglucose/antimycin A. After the reduction of cytoplasmic ATP in human platelets, the 31P NMR spectra showed an almost complete loss of the nucleoside di-and triphosphate resonances at temperatures examined (4-50'C), indicating that only the cytoplasmic nucleotides had been observed, with no detectable contributions from the granular ATP, ADP, and pyrophosphate. Slow tumbling of the granular nucleotides, possibly due to aggregation, is the probable explanation of their undetectability at 145.7 MHz. Similar experiments showed that, in pig platelets, granular ATP and ADP were not detected by 31P NMR at 4VC but were observed at higher temperatures, indicating that aggregation may be occurring at the lower temperatures. Upon thrombin stimulation of human platelets, the NMR spectra and the chemical and radioactivity analyses showed that the granular adenylates and pyrophosphate were secreted, and that cytoplasmic ATP levels were appreciably reduced.Blood platelets are free-floating anucleate cells that play a fundamental role in hemostasis and the maintenance of vascular integrity. These functions are mediated, in part, by adenine nucleotides, which are segregated into two distinct compartments (1). One of the two compartments is the membraneenclosed dense granules, in which as much as 65% of the total cellular ATP and ADP is sequestered together with serotonin, pyrophosphate, and Ca2+ in human platelets (2) and with serotonin and Mg2+ in pig (3) platelets. The cytoplasmic pool contains the remaining adenylates with an ATP/ADP ratio of 7-10; this pool participates in the energy metabolism of the cell and, unlike the granular pool, is affected by metabolic inhibitors (4, 5). During platelet secretion at 370C the granular pool is extruded to the extracellular volume, whereas the cytoplasmic ATP is retained intracellularly, and is partially converted into hypoxanthine (1). When platelets are incubated with radioactive nucleotide precursors such as l14C]adenine, the cytoplasmic pool is labeled rapidly (half time~40 sec); in contrast, the granules incorporate the label slowly (half time -18 hr) (6).High-resolution 31P nuclear magnetic resonance (NMR) provides a rapid, accurate, and noninvasive method for the study of phosphorylated metabolites in vivo (7-9). Furthermore, 31P NMR measurements possess the capability to distinguish among nucleoside phosphate resonances arising from different subcellular compartments when these compartments maintain differences in pH, divalent cation concentration, or both. With this in mind, we have examined the 31P NMR spectra of resting platelets, of cells challenged with thrombin to induce secretion, and of cells metabolically perturbed with H202 plus NaN3 or...

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31P nuclear magnetic resonance studies of isolated rat liver cells

Cited by 142 publications

References 13 publications

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

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

31P-NMR studies on membrane phospholipids in microsomes, rat liver slices and intact perfused rat liver

Adenine nucleotide storage and secretion in platelets as studied by 31P nuclear magnetic resonance.

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