Effect of the nitrogen source on glutamine and alanine biosynthesis in Neurospora crassa. An in vivo 15N nuclear magnetic resonance study

The influences of different nitrogen sources on the relative rates of biosynthesis of glutamine and alanine have been studied by 15N nuclear magnetic resonance spectroscopy of intact Neurospora crassa mycelia suspensions. The rate of glutamine synthesis was fastest after growth in media deficient in free ammonium ion, whereas it was slowest following growth in media containing both glutamic acid and glutamine. The reverse trend was observed for the biosynthesis of alanine. A competition between the two biosynthetic pathways for the same substrate, glutamic acid, was found to limit the rate of alanine synthesis when glutamine synthesis was rapid. The observed in vivo rates of these reactions are compared to the reported specific activities of the enzymes catalyzing the reactions, and implications of these results for nitrogen regulation of these pathways under various physiological conditions are discussed

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

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

Nitrogen-15 spin-lattice relaxation times of amino acids in Neurospora crassa as a probe of intracellular environment

The nitrogen- 15 spin-lattice relaxation time, T_1, and the nuclear Overhauser enhancement (NOE) have been measured for intracellular glutamine, alanine, and arginine in intact Neurospora crassa mycelia to probe their various intracellular environments. The relaxations of ^(15)N_y of glutamine, ^(15)Nɑ of alanine, and ^(15)N ω,ω' of arginine in N. crassa were found, on the basis of their NOE values, to be predominantly the result of ^(15)N-H dipolar relaxation. These relaxations are therefore related to the microviscosities of the various environments and associations of the respective molecules with other cellular components that act to increase the effective molecular sizes. For ^(l5)Ny of glutamine in the cytoplasm, the intracellular T_1 (4.1 s) was only slightly shorter than that in the culture medium (4.9 s). This indicates that the microviscosity of the cytoplasm surrounding the glutamine molecules is not much greater than 1.3 cP. By contrast, for ^(15)Nω,ω,of arginine, which is sequestered in vacuoles containing polyphosphates, the intracellular T_1 (1.1 s) was only one-fourth of that in the medium (4.6 s). In model systems, the T_1, of ^(15)Nω,ω' in a 1 M aqueous solution of arginine containing 0.2 M pentaphosphate was 0.95 s, whereas in an isoviscous (2.8 cP) solution without pentaphosphate, the T1 was 1.8 s. These results suggest either that the vacuolar viscosity is substantially above 2.8 cP or that the ω,ω’-nitrogens of vacuolar arginine are associated with a polyanion, possibly polyphosphate. The implications of these results for the properties of the vacuolar interior are discussed in relation to the mechanism of amino acid compartmentation