31P nuclear-magnetic-resonance studies on the developing embryos of Xenopus laevis

Abstract

The concentrations of nucleoside triphosphate, inorganic phosphate and the yolk proteins, phosvitin and lipovitellin, have been monitored in living embryos of Xenopus laevis by 31P nuclear magnetic resonance (NMR) spectroscopy. The nucleoside triphosphate levels remain relatively constant at about 3.5-4.5 nmol/embryo at least until the 'spontaneous movement' stage of development. By the swimming tadpole stage an inorganic phosphate resonance representing about 30 nmol/embryo becomes evident in the NMR spectrum. Computer manipulation also shows such a resonance, although smaller, to be present at a somewhat earlier developmental stage; these findings are confirmed biochemically. The major contribution to the NMR spectrum of oocytes, unfertilized eggs and early embryos is the yolk phosphoprotein resonance. On isolation of the yolk from the embryos it is possible to quantify the contribution to the NMR spectrum from the lipid-phosphate and protein-phosphate moieties of the yolk proteins. During development, as the yolk is used up, it is found that the protein-phosphate resonance disappears at a greater rate than the lipid-phosphate peak. The total phosphorus content of the embryo (approximately 200 nmol/embryo) is shown biochemically to remain constant during development; however, the total amount of phosphorus observed by NMR decreases by about 40% during development. From the resonance positions of their alpha, beta and gamma phosphate groups it is deduced that the nucleoside triphosphate molecules are liganded in vivo to a divalent cation which is not manganese, but could be either magnesium or calcium. From the position of the inorganic phosphate resonance it is deduced that the internal pH of embryos where this resonance is evident is 6.8 +/- 0.2.