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, 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.