Measurement of Chloroplastic NAD Kinase Activity and Whole Tissue NAD Kinase Assay.
Bio-protocol
confidence
Key findings
Methods paper describing novel assay for chloroplastic NAD kinase activity; no clinical or biological endpoints reported.
View source on PubMed (PMID 33654713) ↗
- Sample size
- Not reported
- Population
- Plant tissue (chloroplasts)
- Dosing
- Not applicable
- Duration
- Not reported
- Route
- Not applicable
- Blinding
- not_reported
- Controls
- none
- Drug class
- coenzyme
Full abstract
Nicotinamide adenine dinucleotide phosphate (NADP) synthesis requires nicotinamide adenine dinucleotide (NAD) kinase activity, substrate NAD and ATP. The NAD kinase responds to various environmental stimuli and its activity is regulated via various regulatory pathways, such as Ca2+-dependent and redox-dependent signals. Conventional in vitro NAD kinase assay has been useful to evaluate enzyme activity; however, recent reports revealed a dynamics of NADP pool (the sum of NADP+ and NADPH) under fluctuating light condition, indicating that the rate of NADP synthesis is not always determined by NAD kinase activity. Here, we developed a novel method for the estimation of chloroplastic NAD kinase activity by quantifying the changes in the NADP amounts in response to illumination. As our approach does not involve protein extraction, it saves time (compared to the in vitro assay), thereby allowing for a sequence of assays, and provides several clues in the investigation of regulatory mechanisms behind NADP synthesis under various environmental conditions.