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Updated: Jul 11, 2026
07:38
Measuring In Vitro ATPase Activity for Enzymatic Characterization
Published on: August 23, 2016
An ATPase activity associated with the rotavirus phosphoprotein NSP5
Tamara Bar-Magen1, Eugenio Spencer, John T Patton
1Laboratorio de Virologia, Facultad de Quimica y Biologia, Universidad de Santiago, Alameda 3363, casilla 33 correo 40, Santiago, Chile.
Virology
|September 11, 2007
Summary
Rotavirus protein NSP5 exhibits Mg2+-dependent ATPase activity, potentially linked to its autokinase function. NSP2 may support NSP5 phosphorylation by providing ATP.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Rotavirus non-structural protein 5 (NSP5) interacts with NSP2 to form viroplasms, crucial for viral replication and packaging.
- NSP5 undergoes phosphorylation changes, transitioning between hypo- and hyper-phosphorylated states during the rotavirus replication cycle.
Purpose of the Study:
- To investigate the enzymatic activities of purified recombinant NSP5, specifically its ATPase and autokinase functions.
- To elucidate the relationship between NSP5 phosphorylation status and its enzymatic activities.
- To understand the role of NSP2 in modulating NSP5 phosphorylation.
Main Methods:
- Purification of recombinant NSP5.
- Measurement of Mg2+-dependent ATP-specific triphosphatase activity using ADP and Pi generation assays.
- Site-directed mutagenesis of NSP5 (Ser67 residue) to assess its role in phosphorylation and enzymatic activities.
- Analysis of NSP2's nucleoside diphosphate (NDP) kinase activity.
Main Results:
- Purified recombinant NSP5 demonstrated Mg2+-dependent ATP-specific triphosphatase activity (Vmax = 19.33 fmol/min/pmol).
- ATPase activity correlated with lower levels of NSP5 phosphorylation, suggesting a link to autokinase activity.
- Mutagenesis of Ser67, critical for cellular hyperphosphorylation, did not affect NSP5's ATPase or autokinase activities.
- NSP2's NDP kinase activity can generate ATP, potentially supporting NSP5 autokinase and cellular kinase activities.
Conclusions:
- NSP5 possesses intrinsic Mg2+-dependent ATPase and autokinase activities, independent of cellular casein kinase-like enzymes.
- NSP2's enzymatic function may sustain NSP5 phosphorylation by providing ATP, influencing rotavirus replication.
- The interplay between NSP5 phosphorylation, ATP hydrolysis, and NSP2 activity is critical for viroplasm formation and rotavirus propagation.
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