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Molecular Pharmacology, Vol 6, 430-440, Copyright © 1970 by the American Society for Pharmacology and Experimental Therapeutics

Kinetics of Carbonic Anhydrase in Whole Red Cells as Measured by Transfer of Carbon Dioxide and Ammonia

THOMAS H. MAREN 1 and CHRISTINE E. WILEY 1

1 Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida 32601

The activity and inhibition of carbonic anhydrase (EC 4.2.1.1.) were studied in intact red blood cells by measuring the hemolytic rates induced when the cells were suspended in a solution of NH4Cl and NaHCO3. The inward diffusion of CO2 and its subsequent hydration to HCO3- within the cell are matched by the inward diffusion of NH3 and formation of NH4+. The new ionic pair attracts water, leading to swelling and hemolysis of the red cells. The rate-limiting step in the over-all process is the conversion of CO2 to HCO3-. In the native cell, the calculated hemolysis time is about 0.1 sec; our observed hemolysis time is 20 sec. When the enzyme is totally inhibited by 100 µM ethoxzolamide, the hemolysis time is increased to 50 min. This corresponds to the time in which the uncatalyzed hydration of CO2 generates an increase in ionic strength of about 60% in the cell. This intact erythrocyte system differs from the usual carbonic anhydrase assay in solution, in the important point that the catalytic inydration rate greatly exceeds the noncatalytic. This reflects the very high (approximately 0.1 mM) enzyme concentration in the red cell, analogous to that in certain secretory sites. Under these conditions, we have determined the KI values of drugs at 99.99% inhibition, and have compared such values with the more conventional I50 data obtained from dilute enzyme in solution.

Note:
ACKNOWLEDGMENT We thank Dr. Betty Vogh for her careful and helpful reading of the manuscript.

Submitted on April 13, 1970




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