Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy

Abstract

The nitrogen-15 chemical shift of the N1 (7)-nitrogen of ^(15)N-labeled histidine and the half-height line widths of proton-coupled resonances of the δ- and ω,ω-nitrogens of ^(15)N-labeled arginine and of the a-nitrogens of ^(15)N-labeled alanine and proline were measured in intact mycelia of Neurospora crassa to obtain estimates of intracellular pH. For intracellular ^(15)N-labeled histidine, the N1 (T)-nitrogen chemical shift was 200.2 ppm. In vitro measurements showed that the chemical shift was slightly affected by the presence of phosphate, with which the basic amino acids may be associated in vivo. These considerations indicate a pH of 5.7-6.0 for the environment of intracellular histidine. The half-height line widths of the δ- and ω,ω-nitrogens of [^(15)N]arginine were 15 and 26 Hz, respectively. In vitro studies showed that these line widths also are influenced by the presence of phosphate, and, after suitable allowance for this, the line widths indicate pH 6.1-6.5 for intracellular arginine. The half-height line widths for intracellular alanine and proline were 17 and 12 Hz, respectively, which are consistent with an intracellular pH of 7.1-7.2. Pools of histidine and arginine are found principally in the vacuole of Neurospora, most likely in association with polyphosphates. Proline and alanine are cytoplasmic. The results reported here are consistent with these localizations and indicate that the vacuolar pH is 6.1 ± 0.4 while the cytoplasmic pH is 7.15 ± 0.10. Comparisons of these estimates with those obtained by other techniques and their implications for vacuolar function are discussed.

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