Cleavage of phosphate from 5'-nucleotides: 5'NT/5PN activity

• Page authors: PRIMO
• Responsible curator: Hagen Buck-Wiese

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5'-nucleotidase activity (5'NT)
Approach: radiolabelled ATP substrate assay
Context: incubation, lab
Spatial scale: point sample
Temporal scale: hours to days
Units: mol ATP L-1 h-1
Community captured: heterotrophic bacteria
Co-measurements: cell abundance

Method Overview

5'-nucleotidase (5'NT, also called 5'-phosphonucleoside phosphohydrolase) cleaves phosphate from the 5' position of nucleotides such as ATP, ADP, and AMP. It represents a specific pathway for phosphorus regeneration from dissolved organic nucleotides, which are important components of dissolved organic phosphorus. Activity is measured by adding radiolabelled ³²P- or ³³P-labelled ATP at tracer concentrations to seawater samples incubated at in situ temperature. After incubation, the reaction is stopped and the phosphate liberated from ^32/33P-ATP (free Pi) is separated from nucleotide-bound phosphate by ion-exchange chromatography or activated charcoal adsorption. The radioactivity in the Pi fraction reflects 5'NT activity.

Scale of measurement

Sampling of incubations run for hours to days.

Data generated

5'NT activity (mol ATP equivalents hydrolyzed L^-1 h^-1) at saturating substrate, representing the community's capacity to regenerate phosphate from dissolved nucleotides.

Units & currency

Units are mol ATP L^-1 h^-1. The currency is phosphorus.

Sample size

Typical samples are < 1 L in volume.

Limitations

Rates represent V_max at saturating substrate; in situ rates depend on the ambient nucleotide concentration, which is typically in the low nM range and sub-saturating. The radiotracer method requires handling of ³²P, a high-energy beta emitter.

Example Applications & Protocols

Classic examples

• Ammerman & Azam (1985) Bacterial 5'-nucleotidase in aquatic ecosystems: a novel mechanism of phosphorus regeneration

Common calculations/conversions

• 5'NT activity (nmol Pi released L^-1 h^-1) = (cpm_{Pi fraction} / cpm_{total added}) × [ATP added] / incubation time.