Before measurement of Na,K-ATPase activity, lens epithelium membrane preparation was incubated in kinase reaction buffer containing 1 mM EGTA, 10 mM Tris (pH 7.2), 20 mM MgCl2, 1 mM ATP, 0.2 mM sodium orthovanadate, 10 μg/mL pepstatin A, 10 μg/mL antipain, 10 μg/mL leupeptin, 1 mM PMSF, 5 mM DTT, and Src, Lyn, Lck, or Fyn tyrosine kinases (0.04–0.12 U/μg protein) or Fes tyrosine kinase (0.4 ng/μg protein) at 30°C. The tyrosine kinases were obtained from Upstate Biotechnology, Lake Placid, NY. Tyrosine kinase concentrations were selected to elicit a similar degree of tyrosine phosphorylation. Treated epithelium membrane preparation was then used either for Western blot analysis or Na,K-ATPase activity measurements. To prevent interference with the Na,K-ATPase activity assay, sodium orthovanadate and exogenous tyrosine kinases were removed by discarding the supernatant after subjecting the membrane preparation to centrifugation at 100,000g for 3 minutes. The membrane pellet was resuspended in centrifugation buffer (10 mM Tris [pH 7.2], 5 mM DTT, and 10% [vol/vol] glycerol) and centrifuged at 100,000g for 3 minutes. This step was repeated, and the final pellet was resuspended in ∼100 μL Na,K-ATPase buffer and assayed immediately for Na,K-ATPase activity.
Na,K-ATPase activity was determined according to a methodology described by Okafor et al.
6 Aliquots of protein kinase-treated and untreated epithelium membrane preparation (∼100 μg) were incubated in Na,K-ATPase buffer (100 mM NaCl, 5 mM KCl, 3 mM MgCl
2, 1 mM EGTA, 40 mM histidine [pH 7.4]). Ouabain, a specific inhibitor of Na,K-ATPase,
15 was added to half the samples at a final concentration of 1 mM. Samples were then preincubated with gentle agitation for 15 minutes at 37°C. ATP was added to a final concentration of 1 mM to initiate ATP hydrolysis. The ATP hydrolysis reaction was performed with gentle agitation for 45 minutes at 37°C. The reaction was then stopped by the addition of 15% ice-cold trichloroacetic acid. ATP hydrolysis was then quantified. The amount of inorganic phosphate released in each reaction sample was measured using a colorimetric method.
6 Less than 20% of the available ATP was hydrolyzed. Na,K-ATPase activity was calculated as the difference between ATP hydrolysis in the presence and absence of ouabain.
As described previously, separate studies were conducted to confirm Na,K-ATPase activity was not inhibited by residual vanadate.
7 Na,K-ATPase activity was 9.7 ± 0.4 nanomoles Pi/mg protein per minute (mean ± SE;
n = 5) in samples that had been subjected to vanadate treatment and then washed. This was not significantly different from the activity of 10.2 ± 0.6 nanomoles Pi/mg protein per minute measured in control samples.