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Metabolism and Enzymology

Compartmentation of Nucleotides in Corn Root Tips Studied by ³¹P-NMR and HPLC ¹

Department of Biochemistry, University of California, Riverside, California 92521, United States Salinity Laboratory, 4500 Glenwood Drive, Riverside, California 92501

Corn (Zea mays L.) root tips were subjected to different conditions so that nucleotide levels varied over a wide range. Levels of nucleotides in corn root tips were measured using ³¹P nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography. Results indicate: (a) Similar amounts of NTP and sugar nucleotides were observed by in vivo NMR and in extracts. In contrast, a significant amount of NDP observed in root tip extracts was not detected by in vivo NMR. Thus, for a given sample, [NTP]/[NDP] ratios determined in vivo by ³¹P-NMR are always higher than ratios observed in extracts, deviating by 4-fold at the highest ratios. The NMR-invisible pool of NDP appeared quite metabolically inert, barely changing in size as total cell NDP changed. We conclude that NDP in corn root tips is compartmented with respect to NMR visibility, and that it is the NMR-visible pool which responds dynamically to metabolic state. The NMR-invisible NDP could either be immobilized (and so have broad, undetectable NMR signals), or be complexed with species that cause the chemical shift of NDP to change (so it does not contribute to the NMR signal of free NDP), or both. (b) ³¹P-NMR cannot distinguish between bases (A, U, C, and G) of nucleotides. HPLC analysis of root tip extracts showed that the relative amount of each base in the NTP and NDP pools was quite constant in the different samples. (c) In extracts, for each of the nonadenylate nucleotides, [NTP]/[NDP] was linearly proportional to [ATP]/[ADP], indicating near equilibrium in the nucleoside diphosphokinase (NDPK) reaction. However, the apparent equilibrium constants for the phosphorylation of GDP and UDP by ATP were significantly lower than 1, the true equilibrium constant for the NDPK reaction. Thus, for a given sample, [ATP]/[ADP] [CTP]/[CDP] > [UTP]/[UDP] > [GTP]/[GDP]. This result suggests that the different NDPs in corn root tips do not have equal access to NDPK.