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JBC, Vol. 251, Issue 14, 4428-4435, Jul, 1976
D. N. Silverman, C. Tu and G. C. Wynns
The depletion of 18O from CO2, caused by the exchange of oxygen between CO2
and water during the hydration-dehydration cycle, is catalyzed by carbonic
anhydrase. This depletion process at chemical equilibrium in the presence
of erythrocytes is biphasic, exhibiting a very rapid depletion rate
immediately following the addition of cells to an isotonic solution
containing 18O-enriched CO2, followed by a much slower depletion rate. It
is hypothesized that these depletion characteristics are caused by the
diffusion of labeled CO2 into erythrocytes where depletion occurs rapidly
due to the large intracellular carbonic anhydrase content. Kinetic
equations which describe this hypothesis are solved and a rate constant is
obtained which represents the depletion of 18O in CO2 caused by the
presence of red cells. These are equilibrium experiments with no net uptake
or loss of CO2 in the cells. Consequently, depletion processes are not
limited in rate by bicarbonate-chloride exchange or proton distribution
across the membrane. The purpose of these measurements is to determine
whether the rate of 18O depletion in red cell suspensions is determined by
carbonic anhydrase activity in the cell or by the diffusion process by
which CO2 enters the cell. This goal is achieved by partially inhibiting
carbonic anhydrase with acetazolamide. The rate constant representing 18O
depletion caused by the presence of red cells is unchanged, even though up
to 90% of carbonic anhydrase is inhibited. From this rate constant the
permeability constant of the membrane of rat erythrocytes to CO2 at 25
degrees and pH 7.4 is determined to be (7.6 +/- 1.2) X 10(-3) cm s-1 in the
presence of 3.2 mM picrate, a passive anion diffusion inhibitor intended to
block HCO3 -flux across the membrane. Using no picrate and allowing
HCO3-flux to introduce an error in the measurements, the permeability
constant is (1.6 +/- 0.4) X 10(-2) cm s-1. The permeability constants
measured by this technique include the diffusion barrier to CO2 not only of
the red cell membrane but also of a portion of the intracellular medium.
Depletion of 18O from C18O2 in erythrocyte suspensions. The permeability of the erythrocyte membrane to CO2
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