Analysis of phosphorylated metabolites in crayfish extracts by two-dimensional 1H-31P NMR heteronuclear total correlation spectroscopy (heteroTOCSY).
In vivo and extract analyses by one-dimensional 31P NMR have been a key tool in investigating energy-related metabolism. Although many phosphorylated metabolites have been observed, many of them have yet to be identified. This reflects the difficulty in identifying them using 31P NMR alone. Two-dimensional 1H-31P correlation experiments have been shown to be useful for assigning phosphorylated metabolites. To obtain better sensitivity and structure information, 1H-detected 31P-1H heteronuclear total correlation spectroscopy (heteroTOCSY) was implemented and a complete chemical shift assignment for a number of phosphorylated standards was made. The time courses of 1D heteroTOCSY signal intensity versus spin-locking time were established for these standards to aid the optimization of the 2D experiment. This method was applied to crayfish extracts for the assignment of glucose 6-phosphate, alpha-glycerophosphate, ribose 5-phosphate, fructose 1,6-bisphosphate, phosphocholine, phosphoethanolamine, glucose 1-phosphate, glycerophosphoethanolamine, glycerophosphocholine, ATP, ADP, and AMP. An alkyl phosphate, a hexose 1-phosphate, and a UDP-hexose were also observed. These assignments allowed the identification of many changes in the 31P NMR spectra of crayfish extracts elicited by treatment with the organophosphate pesticide chlorpyrifos. The assignment of an in vivo 31P spectrum of a live crayfish was also made based on the extract assignment. This approach should be a powerful tool for examining stress-associated changes in the metabolism of phosphorylated compounds.
Gradwell
M J
MJ
MRC Biomedical NMR Centre, National Institute for Medical Research, London, The Ridgeway, NW7 1AA, United Kingdom.
Fan
T W
TW
Lane
A N
AN
D000818:Animals; D003400:Astacoidea / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D010179:Pancreas / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011522:Protons
D010761:Phosphorus Radioisotopes; D011522:Protons