Determination of Specific Radioactivities of Mononucleotides and Glycolytic Intermediates from Mouse Liver after Labelling <i>in vivo</i> with [³²P]Orthophosphate

Abstract

Experimental procedures and conditions are described for the determination of specific radioactivities of 16 different P ‐positions in 12 phosphorylated liver compounds after labelling in vivo with [ 32 P]orthophosphate. These data are necessary for calculations of flux rates within the mononucleotides and the glycolytic pathway. Purification procedures for the following compounds are given: ATP, ADP, GTP, UTP, UDPG, glucose 6‐phosphate, fructose 6‐phosphate, fructose 1,6‐biphosphate, dihydroxyacetone‐phosphate, glycerol 3‐phosphate, phosphoglyceric acid and phosphoenolpyruvate. The purification of each compound consists of 3–5 chromatographic steps, in most cases including a specific enzymic conversion. A preparation procedure is described for mouse liver which shows only a negligible artificial isotope exchange between the β‐ and γ‐ P ‐positions of ATP. This was proved by addition of labelled ATP to non‐labelled livers. Five and ten min after intravenous injection of carrier‐free [ 32 P]orthophosphate the specific radioactivities of the compounds examined show identical values, thereby excluding compartmentations with isotope kinetic relevance.