Microquantitative determination of Pi-ATP and ADP-ATP exchange kinetics using thin-layer chromatography on silica gel

A new method for determination of 32 P i-ATP and [ 14C]ADP-ATP exchange rates is described. It is based upon separation of nucleotides and P i by thin-layer chromatography on commercial aluminum or plastic sheets precoated with silica gel. The method permits avoiding special procedures for stopping of the reaction and for preparation of aliquots for thin-layer chromatography separation. It also allows to separate all adenine nucleotides and P i in one chromatography procedure, and to work with a double label. The volume of the reaction mixture was 50–100 μl. Aliquots (2–6 μl) of the reaction mixture were taken at various moments without stopping the reaction and were layered immediately on a heated silica gel sheets. The nucleotides and P i were separated in a solvent system which consisted of dioxane, isopropanol, 25% ammonia, and water (4:2:3:4, v/v). The nucleotide spots were detected in ultraviolet light, cut out, and their radioactivity was measured with a liquid scintillation counter. The method for measurement of kinetics of exchange reactions catalyzed by reconstituted into liposomes H +-ATPase complexes from beef heart mitochondria and high plant chloroplasts, is described.