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

• Published in: Acta physiologica Scandinavica 1980-07, Vol.109 (3), p.239-248
• Main Author: WISTRAND, PER J.
• Format: Article
• Language: English
• Subjects: affinity chromatography; bicarbonate reabsorbtion; carbonic anhydrase; Carbonic Anhydrases - metabolism; Cytoplasm - enzymology; diuretics; Humans; Kidney; Kidney - enzymology; Kidney Cortex - enzymology; Kidney Medulla - enzymology; Kidney Tubules - physiology; Kinetics; radioimmunoassay; Radioimmunosorbent Test - methods; Spectrophotometry; stopped-flow technique; urine acidification
• ISSN: 0001-6772; 1365-201X
• DOI: 10.1111/j.1748-1716.1980.tb06593.x

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°C in 25 mM N‐methyl imidazole buffer, 1=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 s‐M1 and 0.60‐s‐1, respectively. The kinetic parameters for CA‐C were found to be: Hydration reaction, Km=11.8 mM, V/[E]0=10.6 × 105 · s‐1, dehydration reaction, Km=70 mM, V/[E]0=5×105· s‐1. In the hydration reaction CA‐C was non‐competitively inhibited by acetazolamide, Ki= 16 nM, sulfanilamide, Ki=8 μM, and chlorothiazide, Kil μM. 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 well with the levels measured catalytically. The erythrocyte form, HCA‐B, was also detected immunochemically (˜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.