Carbonic anhydrase IV (CAIV) is a membrane-associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8-A resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azoptm). The overall structure of murine CAIV is generally similar to that of human CAW, however, some local structural differences are found in the active site resulting from amino acid sequence differences in the "130's segment" and the residue-63 loop (these may affect the nearby catalytic proton shuttle, His-64). Similar to human CAN, the C-terminus of murine CAIV is surrounded by a substantial electropositive surface potential that may stabilize the interaction with the phospholipid membrane. Binding interactions observed for brinzolamide rationalize the generally weaker affinity of inhibitors used in glaucoma therapy toward CAIV compared with CAII.

A cDNA encoding the murine carbonic anhydrase IV (mCA IV) gene, modified to resemble a form of mature human carbonic anhydrase IV (Okuyama, T., Waheed, A., Kusumoto, W., Zhu, X. L., and Sly, W. S. (1995) Arch. Biochem. Biophys. 320, 315-322), was expressed in Escherichia coli. Inactive inclusion bodies were collected and refolded, and active enzyme was purified; the resulting mCA IV was used to characterize the catalysis of CO 2 hydration using stopped flow spectrophotometry and 18 O exchange between CO 2 and water. Unlike previously studied isozymes in this class of carbonic anhydrase, the pH profile for k cat for hydration of CO 2 catalyzed by mCA IV could not be described by a single ionization, suggesting multiple proton transfer pathways between the zinc-bound water molecule and solution. A role for His 64 in transferring protons between the zinc-bound water and solution was confirmed by the 100-fold lower activity of the mutant of mCA IV containing the replacement His 64 3 Ala. The remaining activity in this mutant at pH levels near 9 suggested a second proton shuttle mechanism. The maximal turnover number k cat for hydration of CO 2 catalyzed by mCA IV was 1

The mitochondrial enzyme, manganese superoxide dismutase (MnSOD) is an integral component of the cell's defense against superoxide-mediated cellular damage. We have isolated and characterized four cDNA clones and the structural gene for rat MnSOD. Northern analyses using MnSOD cDNA probes detected at least five mRNAs in all tissues and cell types examined. Southern and Northern analysis using a 3' non-coding sequence probe, common to all the cDNAs, showed hybridization only to genomic restriction fragments that correspond to our genomic clone and the five MnSOD mRNAs. These data demonstrate that all of the rat MnSOD transcripts are derived from a single functional gene. Primer extension data indicate that transcription initiation is clustered within a few bases. Northern analysis using intron probes demonstrates that all five transcripts are fully processed. Northern analysis using cDNA and genomic probes from sequences progressively 3' to the end of the coding sequence indicates that size heterogeneity in the MnSOD transcripts results from variations in the length of the 3' non-coding sequence. From this data and the location of potential polyadenylation signals near the expected sites of transcript termination, we conclude that the existence of multiple MnSOD mRNA species originate as the result of alternate polyadenylation.

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