An ATPase assay using scintillation proximity beads for high-throughput screening or kinetic analysis.
A new procedure for measuring ATPase activity in which gamma-(33)P-labeled inorganic orthophoshate is detected by addition of ammonium molybdate followed by selective adsorption of the resulting phosphomolybdate to scintillation proximity beads in the presence of cesium chloride is described. This method is shown to give accurate and reproducible results over a wide range of detection conditions and product concentrations. It requires no separation or filtration steps and is highly compatible with automated high-throughput screening. Rates of hydrolysis are easily and accurately determined over a wide range, and thus the method is useful for kinetic studies also. We show that this scintillation proximity assay is useful for the study of the E1 helicase of human papillomavirus, but it is a general procedure which could also be applied to any ATPase or other nucleotide triphosphate-hydrolyzing enzyme or any other enzyme which generates orthophosphate as a reaction product.
Jeffery
Jamie A
JA
Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard St., Laval, Quebec, H7S 2G5, Canada.
Sharom
Jeffrey R
JR
Fazekas
Monika
M
Rudd
Penny
P
Welchner
Ewald
E
Thauvette
Louise
L
White
Peter W
PW
D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000378:metabolism; D004265:DNA Helicases / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis; D006801:Humans; D007700:Kinetics; D009856:Oncogene Proteins, Viral / Q000032:analysis; D027383:Papillomaviridae / Q000201:enzymology; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins / Q000032:analysis; D012588:Scintillation Counting; D014764:Viral Proteins / Q000032:analysis
D004268:DNA-Binding Proteins; C084304:E1 protein, Human papillomavirus type 11; C118861:E1 protein, Human papillomavirus type 6; D009856:Oncogene Proteins, Viral; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D014764:Viral Proteins; D000251:Adenosine Triphosphatases; D004265:DNA Helicases