Bioluminescent assay of ATPase activity in embryonic material using firefly luciferase.
The continuous bioluminescent assay of ATP has been adapted to the study of Mg2+-dependent ATPases, including the (Na+; K+) pump, in amphibian tissues. A discrete bioluminescent assay procedure for ATPase has also been developed. Components of the firefly luciferase assay reagent modify the observed ATPase activity but this can be circumvented by performing discrete instead of continuous measurements of enzyme activity. In assays with commercial ATPase preparations the continuous bioluminescent assay procedure gave ATPase activities 2.2-fold lower than obtained with the discrete procedure. In Xenopus oocyte or egg homogenates, in contrast, the total ATPase activity measured is stimulated eight times by the luciferase reagent, mainly through an unexplained activation of a Mg2+-independent ATPase. In other tissues, such as Xenopus brain homogenates, both the continuous and discrete monitoring procedures are equally suitable for the determination of ATPase activity.
Hanocq-Quertier
J
J
Département de Biologie Moléculaire, Université libre de Bruxelles, Rhode-Saint-Genèse, Belgium.
Baltus
E
E
Schram
E
E
D000818:Animals; D001755:Blastocyst / Q000201:enzymology*; D001921:Brain / Q000201:enzymology; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D001517:Coleoptera / Q000201:enzymology; D005260:Female; D005306:Fertilization; D007700:Kinetics; D008156:Luciferases; D008163:Luminescent Measurements; D008297:Male; D009865:Oocytes / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014982:Xenopus laevis
D008156:Luciferases; D017301:Ca(2+) Mg(2+)-ATPase; D000254:Sodium-Potassium-Exchanging ATPase