P-31 AND C-13 NUCLEAR MAGNETIC-RESONANCE STUDIES OF ANAEROBIC GLUCOSE-METABOLISM AND LACTATE TRANSPORT IN STAPHYLOCOCCUS-AUREUS CELLS
P-31 AND C-13 NUCLEAR MAGNETIC-RESONANCE STUDIES OF ANAEROBIC GLUCOSE-METABOLISM AND LACTATE TRANSPORT IN STAPHYLOCOCCUS-AUREUS CELLS
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DOI：
10.1021/bi00285a020
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发表时间：
1983-01-01
期刊：
影响因子：
2.9
通讯作者：
RUSSELL, AF
中科院分区：
文献类型：
--
作者：
EZRA, FS;LUCAS, DS;RUSSELL, AF
High-resolution Fourier transform 31P and 13C NMR was used to probe several aspects of glucose metabolism and lactate transport in S. aureus. The 31P NMR spectra show resonances due to intracellular (Piin) and extracellular orthophosphate (Piex), sugar phosphate and nucleoside di- and triphosphates. A peak due to teichoic acid was also identified. Its appearance indicates a relatively high degree of mobility in the backbone of this cell wall polymer. The intracellular pH was estimated from the chemical shift of the Piin resonance and depended on the pH of the external medium. A prominent feature of the 31P NMR spectra is the progressive broadening and downfield shift of the Piin resonance that occur when the cells are maintained in an anaerobic environment. Oxygenation causes a narrowing and an upfield shift of the Piin resonance, thereby reversing the trends observed under anaerobic conditions. These line width and chemical shift vriations are attributed mainly to a binding of the Pi to paramagnetic ions accumulated by the cells during growth. The ESR spectrum of a perchloric acid extract shows a sextet characteristic of Mn(II) hexaquo ions. The Mn may be involved in oxygen metabolism. 13C NMR spectra of S. aureus cells that were incubated anaerobically with [1-13C]- or [6-13C]glucose show resonances due to fructose 1,6-bisphosphate as an intermediary metabolite and mannitol, lactate and ethanol as the major end products of glucose metabolism. The identity of mannitol is determined from the 13C NMR spectrum of a perchloric acid extract. The pH of the external medium affects the glycolytic rate and the distribution of end products. When the pH is raised from 6.0 to 7.5, the rate of glucose consumption is enhanced and the amount of mannitol produced relative to lactate is drastically reduced. The latter effect is explained in terms of the regulation of phosphofructokinase activity by the intracellular pH. The intra- and extracellular lactates appear as 2 well-resolved resonances due primarily to the presence of Mn2+ inside the cells. The results is a downfield shift and broadening of the intracellular resonance which depend on the oxygenation state of the cells and resemble the trends observed in the 31P NMR spectra. The chemical shift inequivalence of the 2 lactate resonances allows the distribution and transport of this metabolite to be measured, with the internal and external components being monitored independently. During anaerobic glycolysis, a lactate concentration gradient favoring the cytoplasmic compartment is established. The final intracellular concentration is estimated to be 2-5 times greater than that in the external medium. In the presence of O2, lactate is transported into the cells. A rapid efflux occurs as the cells revert to an anaerobic state. Treatment with a fatty acid antimicrobial agent, octanoate, results in a concentration-dependent reduction of the transmembrane pH gradient and a loss of lactate from the cells during glycolysis. The uptake of lactate during oxygenation is also completely inhibited.
