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

Abstract

The nitrogen-15 spin-lattice relaxation time, T1, 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 15N gamma of glutamine, 15N alpha of alanine, and 15N omega, omega ' of arginine in N. crassa were found, on the basis of their NOE values, to be predominantly the result of 15N-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 15N gamma of glutamine in the cytoplasm, the intracellular T1 (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 15N omega, omega ' of arginine, which is sequestered in vacuoles containing polyphosphates, the intracellular T1 (1.1 s) was only one-fourth of that in the medium (4.6 s). In model systems, the T1 of 15N omega, omega ' 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 omega, omega '-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.

15N‐NMR studies of nitrogen assimilation and amino acid biosynthesis in the ectomycorrhizal fungus Cenococcum graniforme

“…Chemical shifts are reported relative to "NH: at 0 ppm. Assignments of "N resonances were made by comparison with known spectra of amino acids and published data [2,3]. The assignments are additionally supported by protoncoupled "N-NMR spectra of the samples.…”

“…NMR spectroscopy is a useful technique for observing the flow of metabolites through pathways in vivo, as has been demonstrated by 13C-NMR studies of glycolysis and Krebs cycle in bacteria, fungi, and mammalian tissues [l]. The feasibility of observing intracellular ["Nlamino acids in intact cells of Brevibacterium lactofermenturn [2] and mycelia of Neurospora crassa [3] has recently been demonstrated by using highresolution "N-NMR. Despite these promising pioneering works, "N has not been used extensively as an NMR probe.…”

H+-ATPases from mitochondria, plasma membranes, and vacuoles of fungal cells

“…Measurements of the internal pH of the N. crassa vacuole, observed in vivo with NMR, showed it to be an acidic compartment, pH 6.1, while the surrounding cytosol was neutral, pH 7.1 [81]. Less is known about the electrical gradient, but, in a few instances, measurements with microelectrodes have shown a slightly positive value (25-40 mV) for the interior of the vacuole relative to the cytosol (C.L.…”

Analysis of mutational lesions of acetate metabolism in Neurospora crassa by 13C nuclear magnetic resonance

“…Direct isolation of vacuoles (27) has shown that less than 1% lies in this organelle, and only about 6% is present in other organelles. Similarly, in vivo experiments using "5N NMR have demonstrated that the bulk of the alanine lies in an environment different in both pH (20) and viscosity (15) from that of vacuolar amino acids such as arginine and histidine. However, by the time the alanine pool was restored to its initial level (Fig.…”

“…4), the '3C-enriched pool was 50% of the cellular alanine. That this degree of compartmentalization of the alanine pool preexisted on SM appears unlikely (15,20,27), suggesting that the sequestration increased as part of the adaptation process. Given that the unenriched alanine pool and gluconeogenesis were cytosolic and that there was evidence for a TCA cycle contribution to the latter in the '3C enrichments of C-3,3' and C-4,4' of trehalose (Fig.…”