Abstract
Acid phosphatases (APases) catalyze the hydrolysis of inorganic phosphate (Pi) from a broad range of Pi-monoesters with an acidic pH optimum. The liberated Pi is reassimilated into cellular metabolism via mitochondrial or chloroplastic ATP synthases of respiration or photosynthesis, respectively. Eukaryotic APases exist as a wide variety of tissue- and/or cellular compartment-specific isozymes that display marked differences in their physical and kinetic properties. Increases in intracellular (vacuolar) and secreted APase activities are useful biochemical markers of plant nutritional Pi deficiency. The protocols for protein extraction, APase activity determination and measurement of soluble protein concentration from plant tissues or cell suspension cultures are presented.
Materials and Reagents
Equipment
Procedure
This protocol applies to extraction of intracellular (vacuolar) APases from plant tissues and suspension cell cultures (e.g., Tran et al., 2010a; Veljanovski et al., 2006). Refer to Tran et al. (2010b) and Robinson et al. (2012) for information on the isolation and analysis of plant cell wall localized and secreted APase isoforms.
This is a ‘stopped-time’ APase assay based upon the hydrolysis of the synthetic substrate, para-nitrophenyl-phosphate (pNPP), to para-nitrophenol (pNP) and Pi (Figure 2). The pNP product forms a yellow color at alkaline pH (λmax = 410 nm; extinction coefficient = ε410 = 18.2/mM/cm; meaning a 1 mM solution of pNP should have an A410 of 18.2). The amount of yellow color formed is thus directly proportional to the amount of pNP produced and is therefore an indicator of the APase activity. This assay tends to be more popular in the APase literature. However, care needs to be taken to ensure that the amount of pNP being formed is proportional to the assay time and volume of clarified extract being assayed.
Figure 2. APase activity is often assayed spectrophotometrically at 410 nm by determining the amount of pNP produced following the hydrolysis of Pi from pNPP. Addition of NaOH after a specified assay time (e.g., 10 min) serves to stop the APase reaction while simultaneously converting the product p-nitrophenol into the yellow colored p-nitrophenolate (λmax = 410 nm).
Recipes
Acknowledgments
Research in our laboratory has been generously funded by research and equipment grants from The Natural Sciences and Engineering Research Council of Canada (NSERC) and Queen’s Research Chairs program to William Plaxton.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.