Measurement of intracellular (compartmental) pH by P-31 NMR in Aspergillusniger

P-31 nuclear magnetic resonance (P-31 NMR) was used to monitor cytoplasmic and vacuolar pH values in the filamentous fungus Aspergillus niger. To obta in a homogeneous cell sample and to be able to perform long term in vivo NM R measurements A. niger mycelium was kept in a setup that allows perfusion of the cell plug within the NMR tube. Mycelial samples, however, became rap idly clogged during perfusion leading to (partial) anaerobiosis of the plug with subsequent acidification of the cytoplasm. As a result, only short-te rm NMR measurements (5-10 min) were possible using free mycelium. To increa se and to prolong perfusion, A. niger was immobilized in Ca2+-alginate bead s. Deteriorated spectra recorded under hypoxia could be completely restored in the presence of oxygen. With this system perfusion in the presence of c itrate could be maintained for at least 18 h at much higher rates (15 ml mi n(-1) compared with 4 ml min(-1) for free mycelium). During this period P-3 1 NMR spectra were highly invariable, indicating approximate steady-state i ntracellular conditions during long term measurements. Perfusion in the pre sence of glucose resulted in complete depletion of the vacuolar inorganic p hosphate pool within 45 min and yielded a higher pH gradient over the tonop last than when citrate was used (Delta pH = 1.6 and 1.4, respectively). (C) 2000 Elsevier Science B.V. All rights reserved.