Thin-Layer Chromatography and Real-Time Coupled Assays to Measure ATP Hydrolysis

Methods Mol Biol. 2019:1999:245-253. doi: 10.1007/978-1-4939-9500-4_15.

Abstract

Many chemical reactions in the cell are thermodynamically unfavorable. To overcome this barrier, the energy released from the hydrolysis of adenosine triphosphate (ATP) is coupled to these reactions via ATP hydrolyzing enzymes known as ATPases. These enzymes are ubiquitous in nature and frequently act as molecular motors in processes ranging from DNA replication to protein degradation. Assays that characterize ATPase activity in vitro are important tools to gain insight into their functions in vivo. Here, we describe a direct and flexible thin-layer chromatography method for detecting ATPase activity using radiolabeled ATP. Additionally, we describe a high-throughput coupled reaction assay pairing ATP hydrolysis with nicotinamide adenine dinucleotide (NADH) oxidation to monitor ATP hydrolysis in real time.

Keywords: ATPase; Coupled assay; NADH; Thin-layer chromatography (TLC).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Adenosine Triphosphate / isolation & purification*
  • Adenosine Triphosphate / metabolism
  • Chromatography, Thin Layer / methods
  • High-Throughput Screening Assays / methods*
  • Hydrolysis
  • NAD / metabolism
  • Oxidation-Reduction
  • Thermodynamics

Substances

  • NAD
  • Adenosine Triphosphate
  • Adenosine Triphosphatases