Human renal cytoplasmic carbonic anhydrase Tissue levels and kinetic properties under near physiological conditions

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Abstract
The cytoplasmic form of human renal carbonic anhydrase, CA-C (carbonate dehydratase, EC 4.2.1.1), purified by affinity chromatography, was characterized kinetically at 37.degree. C in 25 mM N-methyl imidazole buffer, I [ionic strength] = 0.15, pH 7.1, using a pH-indicator stopped-flow technique. Under these conditions, the rate constants for the uncatalyzed hydration of CO2 and dehydration of H2CO3 were 0.12 .cntdot. s-1 and 0.60 .cntdot. s-1, respectively. The kinetic parameters for CA-C were: hydration reaction, Km = 11.8 mM, V/[E]0 = 10.6 .times. 105 .cntdot. s-1, dehydration reaction, Km = 70 mM, V/[E]0 = 5 .times. 105 .cntdot. s-1. In the hydration reaction CA-C was non-competitively inhibited by acetazolamide, Ki = 16 nM, sulfanilamide, Ki = 8 .mu.M and chlorothiazide, Ki = 1 .mu.M. The levels of immunoassayable CA-C in cortex, medulla and papilla of perfused donor kidneys were 1.3, 1.0 and 0.6 mg enzyme protein/g tissue protein, respectively, which corresponded with levels measured catalytically. The erythrocyte form, HCA-B, was detected immunochemically (.apprx. 0.1 mg/g protein) but is thought to be a contaminant. Calculations indicated that the uncatalyzed hydration of CO2 in the tubular cells can support 17 or 0.7% of the rate of urine acidification, dependent on whether the cellular alkaline pH-disequilibrium during acid secretion is 0.1 or 0.01 pH units, respectively. CA-C accelerates the hydration rate 6800-fold which enables the cell to sustain high rates of proton generation while maintaining near CO2-equilibrium and maximal buffering capacity. Even at an assumed pH -disequilibrium of only 0.01 pH-unit, CA-C is present in 50-fold excess of apparent physiological needs. When the enzyme is inhibited the rate of the uncatalyzed reaction increases which partly overcomes the effect of inhibition.