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

Abstract

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;

31P NMR studies of photophosphorylation in intact cells ofChromatium vinosum

“…The maximal values for the phosphorylation potential in Chr. vinosum reported here are similar to alp NMR results obtained from respiring mitochondria [24,25] and glycolizing, anaerobic Escherichia coli [26]. These values, however, are low compared to those published for a suspension of chromatophores.…”

Phosphorus‐31 nuclear magnetic resonance studies of photosynthesizing Chlorella

“…As mentioned earlier, the increase in ATP under illumination was also observed at the same time. In the present work it is shown that 31P-NMR will be a potent method to elucidate the bioenergetics in photophosphorylation as well as in oxydative phosphorylation [14]. On the other hand, the decrease in cytoplasmic pH was induced by illumination, and was not inhibited by DCMU ( fig.2,4).…”

Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A 31P-NMR study

“…The 4~C spectrum consists of a strong phospholipid component with a well-defined low-field shoulder in the region of --20 ppm, characteristic of phospholipids in a liquid crystalline bilayer configuration. Superimposed on this broad bilayer spectrum are sIP-NMR signals arising from small phosphorus~containing molecules, which can be assigned to sugar phosphates, Pi and ADP [19]. This system was stable for times of the order of hours at 4°C.…”