Characterization of the 31P NMR spectrum of Manduca sexta and effects of antimetabolites

Abstract

High resolution 31P nuclear magnetic resonance spectroscopy (NMR) was successfully applied to 5th instar larvae of Manduca sexta. Conditions for in vivo analysis under non-saturating conditions are described. The 31P NMR spectrum of intact larvae was composed of six peaks. Their resonance frequencies are reported relative to orthophosphoric acid. Analysis of tissue extracts demonstrated the in vivo peaks to be composed of the β phosphorus resonance of nucleotide triphosphates (NTP) at −19.36 ppm; α phosphorus of NTP and nucleotide diphosphates (NDP) at −10.51 ppm; β and γ phosphorus of NDP and NTP, respectively, at −5.42 ppm; phosphoarginine (PA) at −3.45 ppm; inorganic phosphate (Pi) at +2.76 ppm and sugar phosphates at +3.34 ppm. The major sugar phosphate present in fat body extracts was trehalose-6-phosphate and this was the major phosphorus component of the spectrum of hemolymph. The spin-lattice relaxation times for each in vivo peak were determined. Titration of aqueous fat body and hemolymph extracts was carried out and the relationship between the chemical shift of Pi and pH determined. On this basis the pH of the hemolymph was estimated at approx. 6.7. The metabolic inhibitors, iodoacetate and dinitrophenol, had significant effects on the 31P NMR spectrum of intact larvae. Administration of iodoacetate caused a rapid increase in the levels of sugar phosphates together with decreases in NTP and PA. Dinitrophenol also caused declines in the relative levels of NTP and PA but sugar phosphates decreased as well. The experiments demonstrated the potential of in vivo NMR analysis for metabolic studies on high energy phosphate metabolites in M. sexta.