title: ATPase Activity Measurements Using Radiolabeled ATP.
abstract: ATP provides the energy that is essential for all P-type ATPases to actively transport their substrates against an existing gradient. This ATP hydrolysis can be measured using different methods. Here, we describe a method that uses radiolabeled [γ-(32)P]ATP, which is hydrolyzed by P-type ATPases to ADP and (32)Pi. Activated charcoal is used to bind the excess of [γ-(32)P]ATP, which can be separated from the unbound (32)Pi by centrifugation. With this method, a wide range (0.1 μM-10 mM) of ATP can be used. In addition, we also describe in detail how ATP hydrolysis is translated into ATPase activity.
authors.affiliation: Radboud University Medical Center, Biochemistry 286, Nijmegen, The Netherlands.
authors.forename: Herman G P
authors.initials: HG
authors.lastname: Swarts
authors.affiliation: Pharmacology/Toxicology 149, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, 9101, 6500 HB, Nijmegen, The Netherlands. Jan.Koenderink@radboudumc.nl.
authors.forename: Jan B
authors.initials: JB
authors.lastname: Koenderink
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000096:biosynthesis* / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D010761:Phosphorus Radioisotopes / Q000737:chemistry*; D011188:Potassium / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D010761:Phosphorus Radioisotopes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium
keywords: ADP; ATP; ATPase activity; Adenosine triphosphate; Charcoal; H,K-ATPase; Na,K-ATPase; Pi