Na, K-ATPase Assay for Crustacean and Fish Gills and Kidneys [as used in Holliday, C.W. (1985). Salinity-induced changes in gill Na, K-ATPase activity in the mud fiddler crab, Uca pugnax. J. Exp. Zool. 233: 199-208.]

(http://ww2.lafayette.edu/~hollidac/ATPase.html)

Solutions:

Homogenizing medium (HM): 0.250 M sucrose (85.6 g/l), 6.00 mM EDTA (1.75) g/l)

Albumin standard: 0.500 mg/ml bovine serum albumin (Sigma A-4503)

Phosphate standard: 0.600 mM Na2HPO4 (anhydrous, store desiccated), 0.0852 g/l)

Bonting's color reagent: in a 2000 ml graduated cylinder put:
a) 1000 ml DH2O;
b) 64.5 ml conc. sulfuric acid, mix well;
c) 20.0 g molybdic acid (ammonium molybdate, Sigma M-0878), allow to dissolve;
d) make to 2000 ml with DH2O and store in fridge.

Add 7.32 g FeSO4 (Fisher I-146)/150 ml of above to make stop solution; use within 4h.

Assay medium - "plus K":

167 mM NaCl (4.88 g/500 ml)
50 mM KCl (1.864 g/500 ml)
33.3 mM imidazole (Sigma I-0250; 1.135 g/500 ml)

Adjust to pH 7.20 w/HCl

Assay medium - "minus K":

217 mM Nacl (6.34 g/500 ml)
33.3 mM imidazole (1.135 g/500 ml)
1.67 mM ouabain (Sigma O-3125, 0.608 g/500 ml)

Adjust to pH 7.20 w/HCl

ATP/Mg "start" solution:

25 mM Na2ATP (Sigma A-5394, "vanadium-free"; 0.392 g/25 ml)
50 mM MgCl2.6H2O (Sigma M-9272; 0.254 g/25 ml)

Adjust to pH 7.20 by adding crystalline imidazole with stirring (pH is <3 without imidazole!) and store frozen in 3-5 ml aliquots in flint vials or plastic tubes. Once thawed and used in an assay, do not refreeze and/or reuse this solution.

Assay:

In this assay crude homogenates of gills or other ion transport tissues are prepared and the liberation of phosphate ion from ATP by the action of the Na, K-ATPase is measured in vitro. The assay has four parts: 1) preparation of the homogenates, 2) homogenate ATPase assay, 3) homogenate protein assay and 4) calculations. The results are expressed as micromoles phosphate liberated per mg protein per hour. It is also possible to use this assay with membrane vesicles prepared by centrifugation of crude homogenates (see Holliday, 1985 for details).

1. Homogenate preparation:

Excise gills and rinse in ice-cold homogenizing medium using a volume (in microliters) of HM equal to 100-200 times the mass (in mg) of the tissue. NOTE: it is particularly important to avoid contaminating the gills with digestive juices from the crab's stomach or hepatopancreas when excising gills - a small amount of this fluid will ruin the assay because it is chock-full of proteolytic enzymes. Homogenize in a ground glass homogenizer (I prefer Kontes "Duall") for 20-40 strokes (use the same number of strokes for each homogenate). Homogenize just enough to eliminate any pieces of gill lamellae which remain. Store homogenates on ice until assayed and assay as soon as possible to minimize loss of enzyme activity.

2. ATPase assay:

Na, K-ATP ase activity is measured as the difference between phosphate liberated in an assay medium with K+ present at optimal concentration and a medium without K+ (and with 1 mM ouabain, a specific inhibitor of the enzyme). "Other" or "residual" ATPase activity (Mg, Ca, HCO3 and other ATPases) may also be measured if a third assay tube, the homogenate blank is included. The homogenate is added after the reaction is stopped in the third tube and this allows calculation of phosphate present in the homogenate and/or released by non-enzymatic hydrolysis of ATP.

Make fresh "stop" solution as noted above and keep it on ice until needed for the assay. Do not use this solution more than 4 hours after the FeSO4 is added.

For each homogenate prepare two 10 x 75 mm flint glass test tubes (three if "other" ATPase is to be measured); the first is for the "+K" medium and the second is fo the "-K" medium. For each batch of test tubes (no more than 60 tubes total if 15-sec intervals are used between tubes - see below) place two 5ml tubes in the test tube rack; these are for the blank and standard solutions.

Pipette 200 microliters of the proper medium into each tube, alternating "+K" and "-K" tubes for each homogenate in the rack. This will leave you with a pair of tubes for each homogenate. Pipette 1.00 ml of DH2O into the 5ml blank tube and 1.00 ml of phosphate standard solution into the 5ml standard tube; these tubes get no homogenate or Mg/ATP solution during the assay. Pipette 66.7 microliters of the appropriate homogenate into each pair of tubes (one pair of tubes for each homogenate) and mix each tube on the Vortex Genie. Place the test tube rack with the tubes in the water bath at 30 deg C for 5 min for thermal equilibration.

At 15-second intervals (12, 10 or 8 sec when you are adept) start the reactions by adding 66.7 microliters of the Mg/ATP "start" solution, vortex the tube and return it to the rack in the water bath. After exactly 15 min begin adding 1.50 ml aliquots of the ice-cold "stop" solution to each tube in the same order and time interval (15, 12, 10 or, if you dared, 8 sec) that you added the "start" solution, vortexing each tube before putting it back in a different rack out of the water bath. If this is done properly, each tube will have incubated exactly 15 minutes. When you have "stopped" all of the reaction tubes, add 4.50 ml of the stop solution to each blank and standard tube (this allows for extra blank and standard solution if needed for blanking or extra standard readings), vortex them, too, and allow 20 min for color development. Read OD700 of each tube against the blank in a spectrophotometer.

3. Homogenate protein assay:

Set up duplicate 10 x 75 mm flint glass tubes for each homogenate and 5 ml tubes for the blank and standard solutions. Pipette 40 microliters of each homogenate into the appropriate pairs of tubes and pipette 80 microliters of DH2O into the blank tube and 80 microliters of the protein standard solution into the standard tube. Add 2.00 ml of diluted BIO-RAD protein assay concentrate to each homogenate tube and vortex; add 4.00 ml to the blank and standard tubes (again, this allows for extra blank and standard solution if needed) and allow color to develp for 20 minutes, but not more than one hour.

Measure OD595 against the blank for each tube and average the values for each pair of tubes.

4. Calculations:

Na, K-ATPase enzyme specific activity is calculated for each homogenate as follows:

OD595 protein std. / OD595 homogenate X

{[(+K OD700) - (-K OD700)] / PO4 std. OD700} X 23.96


The 23.96 is a constant derived as follows:

[0.600 micromoles PO4 / ml X 0.333 ml reaction volume X 60 min / h ] divided by

[15 min X 0.0667 ml homogenate X 0.500 mg protein per ml]


The units are micromoles PO4 per mg protein per hour.

Happy gill grinding!

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