Carbonic anhydrase IV: purification of a secretory form of the recombinant human enzyme and identification of the positions and importance of its disulfide bonds.

Chinese hamster ovary cells were stably transfected with the cDNA for human carbonic anhydrase IV that was engineered to encode a secretory form of the normally glycosylphosphatidylinositol-anchored membrane protein. Overexpression was achieved by amplification of the cDNA and its dihydrofolate reductase-containing expression vector by growth in the presence of methotrexate. The 33-kDa secretory form of the enzyme was purified to homogeneity from cellular secretions by inhibitor affinity chromatography. Occasional CA IV preparations contained proteolytic fragments of 18 and 15 kDa held together by disulfide bonds. N-terminal sequencing identified the 18-kDa fragment as the N-terminus and the 15-kDa fragment as the C-terminal portion. The specific activity of the purified enzyme preparations (2587 +/- 149 U/mg protein) was comparable to that of enzyme purified from human tissues. In order to identify the cysteines involved in the two disulfide bonds, enzyme purified following metabolic labeling with [35S]cysteine was subjected to proteolytic cleavage and the N-terminal amino acid sequence determined on the labeled peptides isolated by HPLC. Results indicated that the disulfide bonds in the native enzyme link Cys6 to Cys18 and Cys28 to Cys211. Reduction of the enzyme or reduction followed by alkylation both destroy 70% of the enzyme activity and make the enzyme susceptible to inactivation by denaturants. Furthermore, the loss of activity of reduced enzyme on exposure to denaturants is not recovered on removal of denaturants. By contrast, disulfide-bonded enzyme is not only more resistant to inactivation by denaturants, but any loss of activity is reversed on removal of denaturants. Fluorescence anisotropy measurements provided further evidence that the disulfide-bonded enzyme retains structure in the presence of denaturants. Taken together, these results show that the disulfide bonds contribute significantly both to the retention of structure and of catalytic activity in the presence of denaturants, and to the ability to renature following removal of denaturants.