Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity.
Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-L-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K(m) and V(max) values for the same substrate by Lineweaver-Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K(m) values at optimum pH and 20 degrees C were 0.625 mM, 0.541 mM, 0.785 mM and 0.862 mM with p-nitro phenyl acetate, for all CA localizations. The respective V(max) values at optimum pH and 20 degrees C were 0.875 micromol/L min, 0.186 micromol/L min, 0.214 micromol/L min and 0.253 micromol/L min with the same substrate. The K(i) and I50 values for the inhibitors sulphanilamide, KSCN, NaN3 and acetazolamide were determined for all the CA localizations.
Demir
Yaşar
Y
Department of Chemistry, Faculty of Education, Atatürk University, 25240 Erzurum, Turkey. demirn@yahoo.com
Nadaroğlu
Hayrunnisa
H
Demir
Nazan
N
D000086:Acetazolamide / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology*; D002256:Carbonic Anhydrases / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism; D019810:Sodium Azide / Q000494:pharmacology; D013270:Stomach / Q000201:enzymology*; D013861:Thiocyanates / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism
D000577:Amides; D002257:Carbonic Anhydrase Inhibitors; D009596:Nitrophenols; D013861:Thiocyanates; C008642:4-nitrophenyl acetate; D019810:Sodium Azide; D002256:Carbonic Anhydrases; D015032:Zinc; D000086:Acetazolamide; C009941:potassium thiocyanate