Jurkat T‐cells chronically growing at acidic pH 7.2 show increased CO 2 hydration activity

In our previous study (Vega et al. Faseb J. 32, S787.14) we reported that a clone of Jurkat T‐cell line that we have been chronically propagating at pH 7.2 (10% CO 2 ) showed a significantly different growth pattern than the control clone at pH 7.4 (5% CO 2 ). Since carbonic anhydrase (CA) is a key contributor to cellular pH regulation, here we tested if pH7.2 and pH7.4 clones have a different CA activity that was measured with the Wilbur‐Anderson assay; briefly, one unit is defined as 2*(T 0 ‐T)/T, where T 0 and T are the times in seconds in the absence and presence of enzyme, respectively, required for a saturated CO 2 solution (4 mL) to lower pH of 20 mM TRIS buffer (6 mL) from 8.3 to 6.3 at 0°C. In a control experiment, CA with the reported activity (≥ 3,000 units/mg dry weight) showed in duplicate experiments 3,048 (T 0 =50.5 sec, T=10.5 sec, 0.0025 mg, determined with Hanna HI10832) and 3,111 (T 0 =66 sec, T=13.5 sec, 0.0025 mg, determined with a timer). To avoid the osmotic shock to the cells, we added 0.85% NaCl to all the assay’s solutions and tested if the presence of 0.85% NaCl affects CA activity. In the presence of 0.85% NaCl of NaCl, the values were 1,869±95 (T 0 =72.3±0.9 sec, T=21.8±0.8 sec, n=5), both blank T 0 and enzyme T times being slower most likely because CO 2 content in the saturated aqueous solution was smaller due to the presence of another solute, 0.85% NaCl. For the experiment, cell suspensions were span twice in the TRIS (20 mM)+NaCl 0.85% buffer at 0°C to achieve 1 mL of 10 million cells/mL density that was added to 5 mL of the TRIS buffer (pH 8.3), then immediately 4 mL of 0.85% NaCl saturated with CO 2 was added, and time for pH to drop from 8.3 to 6.3 was measured; the final cell count being 10 million/mL in 10 mL of the reaction volume. For the pH7.4 clone, T= 82.3±1.1 sec (n=6) was slower than blank determination in 0.85% NaCl resulting in the apparent negative CA activity of −0.24 formally calculated as 2*(T 0 ‐T)/T; while for the pH7.2 clone, the T= 77.3±1.4 sec (n=6) resulted in −0.13 (p<0.05). If the increased CO 2 hydration in the pH7.2 clone is caused by the increase in CA molecules alone, we would expect that these cells would have an additional 0.11/(10*10 6 cells)/(3000 units/mg)/(1000 mg/g)/(30,000 g/mole) * 6.022*10 23 = 7.4*10 4 CA molecules per cell. We conclude that 1) isotonic NaCl significantly inhibits apparent CA activity in the context of Wilbur‐Anderson assay units, 2) both pH7.2 and 7.4 clones at 1 million/mL cell density inhibited CO 2 hydration compared to the blank CO 2 hydration in the Wilbur‐Anderson assay supplemented with 0.85% NaCl; 3) in relation to each other, pH7.2 clone has a higher CO 2 hydration activity than pH7.4 clone speculatively due to estimated additional 7.4*10 4 CA molecules. Differential CA activity and expression in the pH7.2 and 7.4 clones will be further compared using physiology and molecular biology tools.