Phosphorus-31 nuclear magnetic resonance detection of unexpected phosphodiesters in muscle

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-

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

³¹ P nuclear magnetic resonance studies of HeLa cells

Evans

Kaplan

1977

“…Two home-built modifications to the probe of this pulsed spectrometer were made so that satisfactory results could be obtained on biological samples in which phosphate metabolites are in the mM range-namely: (i) because field-frequency drift is known to have a deleterious effect on the results of the CPMG method, we installed an external deuterium lock system in the pulse probe, which was capable of holding the 31p field-frequency stability within 1 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.…”

Section: Methodsmentioning

confidence: 99%

“…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°.…”

mentioning

confidence: 99%

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

Cohen¹,

Burt²

1977

ABSTRACT31P nuclear magnetic relaxation rates for phosphocreatine in intact frog gastrocnemius were compared with those observed in model solutions at 40, a temperature at which muscle maintains its physiological state for at least 5 hr. Both nuclear Overhauser effect and spin-lattice relaxation rate(1/Tj) experiments indicate that dirle-dipole interactions form the dominant relaxation path for P in intact muscle and model solutions, independent of phosphocreatine and Mg concentrations. 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°. The spin-lattice relaxation time (TI) and the spin-spin relaxation time (T2) of PCr in intact muscle are compared with those observed in model solutions at 4°. Both relaxation rates are valuable as dynamic probes of the internal environment in muscle because T2 can be sensitive to processes that do not affect T, at all. These relaxation studies also enable us to assign a value for the free intracellular Mg concentration in intact gastrocnemius via an intrinsically nonintrusive method.It is now clear that metal ions are of central importance in many biological reactions and knowledge of free Mg levels has particular significance. For (10). Spinning 10-mm samples were used; the field was locked either to a capillary of D20 (muscles and PCr/H20 solutions) or D20 solvent. Typical conditions included a 900 pulse of ca 20 lisec, 32-128 scans of 4000 data points each, sweep width of 1 kHz, exponential multiplication introducing 0.6-Hz line broadening, and 'H irradiation.T, measurements were made with the following pulse sequence: 900 pulse, spoil field homogeneity, wait delay time r, 90°pulse, sample free-induction decay, and then spoil field homogeneity. This sequence (11), which was repeated n times, afforded a considerable saving of time over the inversion-recovery method in the T, regimen of 31P in PCr, thus making it possible to carry out T, measurements on muscle during the interval in which there was no more than a 10% change in PCr.The Carr-Purcell-Meiboom-Gill (CPMG) (12) T2 measurements were performed on a Bruker B-KR pulsed spectrometer operating in conjunction with the same Nicolet data system and a 21.14 kG magnet used for high-resolution spectra. Two home-built modifications to the probe of this pulsed spectrometer were made so that satisfactory results ...

“…Peak positions of 31P resonances were expressed in parts per million (ppm) from an external reference of 85% HaPO4. Recently high-resolution 31P nuclear magnetic resonance (NMR) technique has been used to study metabolic processes in various cells (1), tissues (2,3), cell organelles (4), and even in perfused animal organs (5,6). -The technique provides considerable information on various phosphate metabolites in tvvo.…”

mentioning

confidence: 99%

High-resolution 31P nuclear magnetic resonance study of rat liver mitochondria.

Ogawa¹,

Rottenberg²,

Tr³

et al. 1978

Intact mitochondria were studied by highresolution 31P nuclear magnetic resonance. Observable internal phosphate compounds included inorganic phosphate (Pi), ADP, and ATP. The internal pH was determined by the chemical shift of the internal Pi, the pI( (6.7) of which was measured in uncoupled mitochondria. The observed equilibrium relation between the internal and the external Pi was consistent with the exchange equilibrium through the H2R4-/OH-carrier. RESULTS Observable phosphate compoundsA typical 31p NMR spectrum of a freshly prepared suspension of rat liver mitochondria at 00C is shown in Fig. 1A. Peaks Al and A2 are in the spectral region of phosphomonoesters, and peaks Bi and B2 are internal and external orthophosphate (Pi) resonances. Peaks Cl and C2 are in the phosphodiester region and pH insensitive. They coincide with the positions of glycerophospho(3)ethanolamine and glycerophospho(3)choline, respectively. These two peaks do not disappear after repeated washing and differential centrifugation and, therefore, most likely originate inside the mitochondria. The peak of externally added glycerophospho(3)choline coincides with peak C2, showing that the external and internal glycerophospho(3)choline are in the same magnetic environmes4 Peak D is from the terminal phosphates of ADP and/or ATP prsent inside mitochondria. The broad absorption peak E includi a phosphates of ATP and ADP, and diphosphate groups of NAt), NADP, and other diphosphodiesters. There were slight variations in relative intensities of those peaks among various' samples of similar conditions, but repeated washing did not alter the spectra appreciably. Except for peak B2 (external Pi), all peaks can be assigned to internal phosphate compounds. Fig. 1B shows the NMR spectrum of a perchloric acid extract of the mitochondrial suspension from which Fig. 1A was obtained. In this extract most of the divalent metal ions were chelated by added EDTA. The spectrum was taken at pH 8.6. The peak assignments in the extract spectrum were made by measuring the pH dependence of the chemical shifts and, most positively, by adding the likely candidates for assignment into the extract solution. The assignments in the phosphomonoester region (group a peaks) are: al, glycerol(3)phosphate; a2, not assigned; a3, AMP; a4, 2'-phosphate of NADP+ and NADPH;