Microquantitative determination of Pi-ATP and ADP-ATP exchange kinetics using thin-layer chromatography on silica gel.
A new method for determination of 32Pi-ATP and [14C]ADP-ATP exchange rates is described. It is based upon separation of nucleotides and Pi 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 Pi in one chromatography procedure, and to work with a double label. The volume of the reaction mixture was 50-100 microliters. Aliquots (2-6 microliters) 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 Pi 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.
D000244:Adenosine Diphosphate / Q000302:isolation & purification*; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate / Q000302:isolation & purification*; D002855:Chromatography, Thin Layer; D004146:Dioxanes; D007700:Kinetics; D010710:Phosphates / Q000302:isolation & purification*; D058428:Silica Gel; D012822:Silicon Dioxide
D004146:Dioxanes; D010710:Phosphates; D058428:Silica Gel; D000244:Adenosine Diphosphate; D012822:Silicon Dioxide; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases