Method for real-time detection of inorganic pyrophosphatase activity.
A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.
Eriksson
J
J
Department of Biotechnology, Royal Institute of Technology, Teknikringen 34, SE-100 44 Stockholm, Sweden.
Karamohamed
S
S
Nyrén
P
P
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001517:Coleoptera / Q000201:enzymology; D011756:Diphosphates / Q000494:pharmacology; D008156:Luciferases / Q000037:antagonists & inhibitors; D008163:Luminescent Measurements; D010710:Phosphates / Q000032:analysis*; D011755:Pyrophosphatases / Q000032:analysis*; D012969:Sodium Fluoride / Q000494:pharmacology
D011756:Diphosphates; D010710:Phosphates; D000255:Adenosine Triphosphate; D012969:Sodium Fluoride; D008156:Luciferases; D011755:Pyrophosphatases