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Hydrolysis of ATP01:08

Hydrolysis of ATP

82.9K
The bonds of adenosine triphosphate (ATP) can be broken through the addition of water, releasing one or two phosphate groups in an exergonic process called hydrolysis. This reaction liberates the energy in the bonds for use in the cell—for instance, to synthesize proteins from amino acids.
If one phosphate group is removed, a molecule of ADP—adenosine diphosphate—remains, along with inorganic phosphate. ADP can be further hydrolyzed to AMP—adenosine...
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ATP Yield01:31

ATP Yield

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Cellular respiration produces 30 - 32 ATP per glucose molecule. Although most of the ATP results from oxidative phosphorylation and the electron transport chain (ETC), 4 ATP are gained beforehand (2 from glycolysis and 2 from the citric acid cycle).
The ETC is embedded in the inner mitochondrial membrane and is comprised of four main protein complexes and an ATP synthase. NADH and FADH2 pass electrons to these complexes, which pump protons into the intermembrane space. This distribution of...
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Related Experiment Video

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Measuring In Vitro ATPase Activity for Enzymatic Characterization
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Published on: August 23, 2016

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Measuring In Vitro ATPase Activity for Enzymatic Characterization.

Chelsea S Rule1, Marcella Patrick2, Maria Sandkvist2

  • 1Department of Microbiology and Immunology, University of Michigan; csrule@umich.edu.

Journal of Visualized Experiments : Jove
|September 2, 2016
PubMed
Summary

This study presents a basic colorimetric assay for measuring purified adenosine triphosphate-hydrolyzing enzymes (ATPases) in vitro. The adaptable protocol quantifies inorganic phosphate release, enabling functional characterization of ATPases like EpsE.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Adenosine triphosphate-hydrolyzing enzymes (ATPases) are crucial for cellular energy transduction.
  • These enzymes drive essential mechanical work, including protein transport, degradation, solute transport, and cell motility.
  • Functional characterization of purified ATPases is vital for understanding their roles in biological processes.

Purpose of the Study:

  • To describe a basic, adaptable in vitro assay for measuring purified ATPase activity.
  • To provide a framework for functional characterization of ATPases.
  • To detail a representative protocol based on the AAA+ ATPase EpsE from Vibrio cholerae.

Main Methods:

  • Enzymatic assay measuring ATP hydrolysis.
  • Quantification of inorganic phosphate release using a colorimetric method.
  • Adaptable protocol for kinetic or endpoint measurements.

Main Results:

  • Demonstrated a basic protocol for measuring in vitro ATPase activity.
  • The assay quantifies phosphate liberation, a direct product of ATP hydrolysis.
  • The protocol is adaptable for various ATPases with potential optimization required.

Conclusions:

  • The described colorimetric assay provides a simple and adjustable method for ATPase functional characterization.
  • This protocol serves as a foundational framework for researchers studying diverse ATPases.
  • The assay's adaptability allows for tailored application across different enzymatic systems.