title: Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity.
abstract: 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.
authors.affiliation: Department of Chemistry, Faculty of Education, Atatürk University, 25240 Erzurum, Turkey. demirn@yahoo.com
authors.forename: Yaşar
authors.initials: Y
authors.lastname: Demir
authors.forename: Hayrunnisa
authors.initials: H
authors.lastname: Nadaroğlu
authors.forename: Nazan
authors.initials: N
authors.lastname: Demir
mesh_terms: 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
chemical_list: 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