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

{{BreadcrumbsNutrients}}

* [[Page authors|Page authors]]: [[PRIMO]]
* [[Responsible curator|Responsible curator]]:  [[User:Hagi BucknWise|Hagen Buck-Wiese]]
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! 5'-nucleotidase activity (5'NT)
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| '''Approach:''' radiolabelled ATP substrate assay
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| '''Context:''' incubation, lab
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| '''Spatial scale:''' point sample
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| '''Temporal scale:''' hours to days
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| '''Units:''' mol ATP L<sup>-1</sup> h<sup>-1</sup>
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| '''Community captured:''' heterotrophic bacteria
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| '''Co-measurements:''' cell abundance
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== 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 <sup>32</sup>P- or <sup>33</sup>P-labelled ATP at tracer concentrations to seawater samples incubated at ''in situ'' temperature. After incubation, the reaction is stopped and the phosphate liberated from <sup>32/33</sup>P-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<ref name="Ammerman1985">Ammerman, J. W., & Azam, F. (1985). Bacterial 5'-nucleotidase in aquatic ecosystems: a novel mechanism of phosphorus regeneration. ''Science'', 227(4692), 1338–1340. https://doi.org/10.1126/science.227.4692.1338</ref>.

=== Scale of measurement ===

Sampling of incubations run for hours to days.

=== Data generated ===

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

=== Units & currency ===

Units are mol ATP L<sup>-1</sup> h<sup>-1</sup>. The currency is phosphorus.

=== Sample size ===

Typical samples are < 1 L in volume.

=== Repositories & databases ===

== Limitations ==

Rates represent V<sub>max</sub> 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 <sup>32</sup>P, 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'' <ref name="Ammerman1985" />

=== Recent applications ===
*

=== Common calculations/conversions ===
* 5'NT activity (nmol Pi released L<sup>-1</sup> h<sup>-1</sup>) = (cpm<sub>Pi fraction</sub> / cpm<sub>total added</sub>) × [ATP added] / incubation time.

== References ==

[[Category:Main Pages|Model types]]