Human renal cytoplasmic carbonic anhydrase. Tissue levels and kinetic properties under near physiological conditions.
Acta Physiol Scand
; 109(3): 239-48, 1980 Jul.
Article
en En
| MEDLINE
| ID: mdl-6778076
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 degrees C in 25 mM N-methyl imidazole buffer, I = 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-1 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 x 10(5) . s-1, dehydration reaction Km = 70 mM, V/[E]0 = 5 x 10(5) . s-1. In the hydration reaction CA-C was non-competitively inhibited by acetazolamide, Ki = 16 nM, sulfanilamide, Ki = 8 micrometer, and chlorothiazide, Ki = 1 micrometer. 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 (approximately 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.
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Banco de datos:
MEDLINE
Asunto principal:
Anhidrasas Carbónicas
/
Riñón
Límite:
Humans
Idioma:
En
Año:
1980
Tipo del documento:
Article