ATPase activity is a measurement of the amount of NADH which is transformed to NAD+ over a specific period of time while Na+/K+ ATPase is inhibited by ouabain in one set of samples and uninhibited in another set. In clam gill tissue, ATPase activity is a reflection of the overall metabolic activity in the tissue, given it’s central role in mitochondrial function. We hope to use ATPase activity assays to understand whether OA has an effect on the energetics of clam gills.

The protocol is as follows:

Kinetic Assay

Gill is preserved by snap freezing in SEI buffer (Sucrose, Na2EDTA, Imidazole).

• When doing the assay, sample is thawed and handled quickly (<30 min. after thawing).
• A small piece of tissue is removed and place in SEI buffer with added Na Deoxycholic Acid
• Tissue is homogenated and centrifuged at 5500g, with the supernatant retained.
• Two mixtures are prepared of a salt solution and an assay solution
• The first solution contains:
  • Imidazole
  • NaCl
  • MgCl2
  • KCl
  • NADH
  • Phosphoenolpyruvate (PEP)
  • Lactic Dehydrogenase
  • Pyruvate Kinase
  • ATP
• The second solution contains the same components with the exceptions:
  • added ouadbain
  • minus KCl
• On a 96 well plate, homogenate supernatant is put in quadruplicate with two wells receiving each of the two solutions.
• Plate is read over ten minutes in all wells, with the difference in the final values between the two solutions used as the data for ATPase activity.

Protein

To normalize ATPase activity measurements, protein concentration is determined for the sample homogenate.

• samples are pipetted into a 96 well plate in duplicate, along with a set of serially diluted protein standards.
• into each well is added a mixture of:
  • Na2CO3
  • NaOH
  • CuSO4
  • Na-K tartrate
• Plate is agitated for 5 minutes
• Folin & Ciocalteu’s reagent is added to each well
• Plate is agitated for 1 minute, placed in water bath at 37 C for five minutes, and placed in the dark for 30 minutes.
• plate is read in plate reader