Kanamori K, Legerton T L, Weiss R L, Roberts J D
Biochemistry. 1982 Sep 28;21(20):4916-20. doi: 10.1021/bi00263a013.
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.
已对完整的粗糙脉孢菌菌丝体中的细胞内谷氨酰胺、丙氨酸和精氨酸的氮-15自旋晶格弛豫时间T1以及核Overhauser效应（NOE）进行了测量，以探究它们在细胞内的各种环境。基于它们的NOE值，发现粗糙脉孢菌中谷氨酰胺的15Nγ、丙氨酸的15Nα以及精氨酸的15Nω、ω′的弛豫主要是15N-H偶极弛豫的结果。因此，这些弛豫与各种环境的微粘度以及各个分子与其他细胞成分的缔合有关，这些缔合作用会增加有效分子大小。对于细胞质中谷氨酰胺的15Nγ，细胞内T1（4.1秒）仅略短于培养基中的T1（4.9秒）。这表明谷氨酰胺分子周围细胞质的微粘度不大于1.3厘泊。相比之下，对于隔离在含有多磷酸盐的液泡中的精氨酸的15Nω、ω′，细胞内T1（1.1秒）仅为培养基中T1（4.6秒）的四分之一。在模型系统中，含有0.2 M五磷酸盐的1 M精氨酸水溶液中15Nω、ω′的T1为0.95秒，而在没有五磷酸盐的等粘度（2.8厘泊）溶液中，T1为1.8秒。这些结果表明，要么液泡粘度大幅高于2.8厘泊，要么液泡精氨酸的ω、ω′-氮与一种聚阴离子（可能是多磷酸盐）缔合。结合氨基酸区室化机制讨论了这些结果对液泡内部性质的影响。
