Identification of Cytosolic and Noncytosolic Carbonic Anhydrases in Brain

Abstract

Carbonic anhydrase is an enzyme found in many mammalian tissues, but it has not previously been determined whether it is present in the bovine brain. In this work, carbonic anhydrase was purified and characterized according to localizations: outer peripheral, cytosolic, inner peripheral and integral in four steps. Affinity chromatography was used for purification of the enzyme from the four different cell regions. The affinity column was prepared with Sepharose-4B-L-tyrosine-sulfanilamide. Purified enzymes obtained at each step activity were determined by hydratase activity and esterase activity methods. Optimum pH and optimum temperature values were defined for the purified enzymes. The behavior of carbonic anhydrase with specific inhibitors, sulfanilamide, KSCN and NaN3, was investigated. Molecular weights of enzymes were determined by gel filtration, and its purity controlled by SDS-PAGE electrophoresis. In addition, the enzyme's K-M and V-max values were determined with the Lineweaver-Burk method. The results obtained are discussed in comparison with other mammalian carbonic anhydrases.