Comparison of pH-dependence of Carbonic Anhydrase Activity in vitro and in Living Cells

Abstract

Carbonic anhydrase (CA) plays an important role in acid-base homeostasis. It catalyses carbonic buffering, increases intracellular H+-mobility and facilitates the activity of pHi regulatory transporters. In vitro, CA activity is known to be inhibited by a fall of pH. This observation has been largely overlooked when considering mechanisms of pHi regulation. There is no information, however, on the pH-sensitivity of CA in intact cells. We have therefore investigated the pH-sensitivity of CA both in vitro, and in situ in living cells. In vitro CA activity was assessed from the time-course of pH change after addition of CO2-saturated water to a buffered solution (pH 8.0-6.5) containing purified bovine CA2, membrane fragments of HCT116 cells stably transfected with CA9, or cardiac homogenates from rat ventricle. In situ CA activity was assessed from the rate of CO2-induced fall of pHi in rat superfused ventricular myocytes, AM-loaded with the pH-fluorophore, carboxy-SNARF-1. The pH-sensitivity was estimated by fitting a kinetic algorithm to the pH time-course data. Results show that both in vitro and in situ CA-activity displays strong pH-dependence, activity declining with a fall of pH. Apparent pK and Hill coefficient (nH) values for in vitro pH-dependence were 7.00 and 1.10 respectively for purified CA2, 6.85 & 1.73 for CA9, and 7.15 and 0.80 for cardiac homogenates. The intracellular pKa and nH values for generic cardiac CA were 7.20 and 2.50. These results imply that the enzyme's contribution to both intracellular and extracellular pH-regulation may vary dynamically over the physiological pH range. Work funded by British Heart Foundation, Cancer Research UK, and Royal Society.