Bovine lens carbonic anhydrases: Purification and properties

Abstract

Carbonic anhydrase (EC 4.2.1.1) was isolated from bovine lenses by ion-exchange and affinity chromatography. The isolation yielded two soluble forms of the enzyme, here called lens CI and lens CII, which were homogenous with respect to sedimentation in the ultracentrifuge and SDS-gel electrophoresis. They had the same molecular weights (28 000) and amino acid composition within the limits of the methods. They also exhibited similar antigenic and kinetic properties (hydrase and esterase activities). However, they had different isoelectric points (lens CI = 5·9, lens CII = 5·6) and could be separated by DEAE-chromatography. Each constituted about half of the amount of carbonic anhydrase present in the lens. Lens-CI and lens-CII were similar, if not identical with the corresponding forms RBC-CI ∗ ∗ Abbreviations: Enzymes RBC-CI and RBC-CII are two electrophoretically different forms of carbonic anhydrase from bovine erythrocytes, recently claimed to have different amino acid compositions (Gulian, Limozin, Mallet, Di Costanzo and Charrel, 1977). The were previously called BCA-B and BCA-A, respectively. Anti-RBC-CI is the rabbit antiserum against RBC-CI. HCA-C and HCA-B are isoenzymes of carbonic anhydrase from human erythrocytes. SDS: sodium dodecyl sulfate. and RBC-CII, respectively, in the erythrocytes. The results therefore suggest that the lens and the erythrocytes contain the same high-activity types (with respect to CO 2) of soluble carbonic anhydrases. Small amounts of carbonic anhydrase activity (about 0·2% of total) was found in the thoroughly washed precipitate fraction of the centrifuged bovine lens homogenate. This fraction could be solubilized by Triton X-100 and was antigenically identical with erythrocyte soluble RBC-CI. Histochemically the enzyme activity was found to be distributed throughout the lens fibres, and the epithelium, but not in the capsule.