Renal membrane-bound carbonic anhydrase. Purification and properties.
Microsomes from perfused human donor kidneys were separated by differential centrifugation in sucrose, and thoroughly washed before solubilization by the nonionic detergent nonyl-beta-D-glucoside. The solubilized material was first applied onto an affinity chromatographic column of acetazolamide-oxirane-SepharoseR-CL-4B to remove contaminating cytoplasmic carbonic anhydrase isozymes CA I and CA II. It was then added onto an affinity column of p-aminomethylbenzene sulfonamide coupled to CM Bio-gel AR to purify the membrane-bound carbonic anhydrase activity. This resulted in a 50% pure enzyme. It was then concentrated and fractionated on an anion-exchange column, and desalted and purified to homogeneity (SDS-PAGE and isoelectric focusing) by gel filtration. The enzyme was now purified 411-fold from extractable membrane protein. Its molecular weight was 34.4 kDa from gel filtration and SDS-PAGE, and 36.7 kDa from amino acid analysis. The amino acid composition differed from that of the cytoplasmic isozymes CA I, II, and III. Antisera, produced in rabbits against the purified SDS-treated enzyme, reacted with native nondenatured membrane enzyme protein but only weakly with CA II. Kinetically the enzyme was similar to CA II with respect to hydrase and esterase activities and to inhibition by various sulfonamides. Considered together, the data suggest that the human kidney contains a membrane-bound carbonic anhydrase protein that differs from the cytoplasmic isozymes CA I, II, and III and the secretory form (CA VI) in the saliva.
Wistrand
P J
PJ
Department of Medical Pharmacology, University of Uppsala, Sweden.
Knuuttila
K G
KG
D002256:Carbonic Anhydrases / Q000302:isolation & purification*; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D007668:Kidney / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology
D002256:Carbonic Anhydrases