Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase II enhances CO2 fluxes across Xenopus oocyte plasma membranes

摘要

The a-carbonic an-hydrases (CAs) are zinc-containing enzymes that catalyze the inter-conversion of CO2 and HCOJ. Here, we focus on human CA II (CA II), a ubiquitous cytoplasmic enzyme. In the second paper in this series, we examine CA IV at the extracellular surface. After micro-injecting recombinant CA II in a Tris solution (or just Tris) into oocytes, we expose oocytes to 1.5% CO2/IO mM HCO^/pH 7.50 while using microelectrodes to monitor intracellular pH (pHi) and surface pH (pHs). CO2 influx causes the familiar sustained pHi fall as well as a transient pHs rise; CO2 efflux does the opposite. Both during CO2 addition and removal, CA II increases the magnitudes of the maximal rate of pHi change, (dpHi/dOmax, and the maximal change in pHs, ApHs- Preincubating oocytes with the inhibitor ethoxzolamide eliminates the effects of CA II. Compared with pHs, pHi begins to change only after a delay of ~9 s and its relaxation has a larger (i.e., slower) time constant (tpHj > TpHs)- Simultaneous measurements with two pHi electrodes, one superficial and one deep, suggest that impalement depth contributes to pHi delay and higher TpHi. Using higher CO2/HCO3" levels, i.e., 5%/33 mM HCO3" or 10%/66 mM HCO3", increases (dpHi/dOmax and ApHs, though not in proportion to the increase in [CO2]. A reaction-diffusion mathematical model (described in the third paper in this series) accounts for the above general features and supports the conclusion that cytosolic CA—consuming entering CO2 or replenishing exiting CO2—increases CO2 fluxes across the cell membrane.