SUMMARYFlounders Platichthys flesus were investigated with respect to isolation, purification, and cellular localization of carbonic anhydrase (CA) in the respiratory system. CA was purified from gills and erythrocytes and was shown to exclusively represent a soluble enzyme with an apparent molecular weight of 30 kD. Inhibition constants (K I ) towards acetazolamide (ACTZ) were 8.4·10 Ϫ 9 M for erythrocyte CA and 7.6·10 Ϫ 9 M for gill CA, indicating a high sensitivity to sulfonamides, as exhibited by human CA II. Specific CA activity did not differ significantly in seawater-and freshwater-acclimated fish. Antibodies were raised against purified gill and erythrocyte CA. Both antisera crossreacted and were used to localize CA in the gills of seawater and freshwater flounders at the light microscopic level. Independent of the salinity, a positive reaction of variable intensity was found in the following cell types: pavement cells (PVCs), forming the gill epithelial surface layer; mucous cells (MCs); pillar cells (PCs), bordering the vascular channels of the secondary lamellae; and chloride cells (CCs), mitochondria-rich cells located in the primary epithelium, the interlamellar regions, and at the bases of the secondary lamellae.

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Carbonic anhydrase, found at high levels in crustacean gill, is capable of catalyzing the formation of C 0 2 in the forward reaction or the production of HCOBp in the reverse reaction.Thus the enzyme may participate in both respiratory and ion regulatory functions. Its subcellular distribution in crab gill epithelial cells suggests that a large proportion of the enzyme is membranebound, where it would be available to pharmacological treatment with membrane-impermeable carbonic anhydrase inhibitors. These studies suggest that the enzyme plays a central role in COz excretion.Treatment with the permeable inhibitor acetazolamide affects osmoregulation and ion transport in addition to CO, excretion, suggesting that the cytosolic form of carbonic anhydrase may provide HC03 for C1 /HC03 exchange and H + for Na+/H + exchange across the gill epithelium. o 1993 Wi~ey-Liss,~nc.

Activities of carbonic anhydrase (CA) were determined in various organs of shore crabs, Carcinus maenas, acclimated to salinities of 10, 20, 30, 40, and 50%0. In addition, some properties of the branchial enzyme (effects of detergents, storage conditions, and specific inhibitors) were tested. Subcellular distribution of gill CA was analyzed by means of centrifugation procedures. In hemolymph no CA activity could be detected. In all other organs except gills activities were low and insensitive to salinity change. High activities of CA were found in the gills. Activities in posterior gills were-in comparison with anterior gills-more than twofold increased. Acclimation of crabs to reduced ambient salinities resulted in significant increases in CA only in posterior gills. In the gill enzyme specific activities and stability during storage could be increased by means of the detergent Triton X-100 used a t 1% (viv) in the homogenizing medium. Sulfonamide inhibitors reduced CA activities effectively. Inhibition constants Ki of the gill enzyme were 7.0 x lo-' M for acetazolamide and 3.1 x lo-' M for methazolamide. Measurements of CA activities in supernatants and pellets obtained by differential centrifugation and in fractions obtained by centrifugation of 7,500g supernatants in a sucrose density gradient indicated that the major portion of CA activity in the gill (ca. 75%) was bound to celluIar particulates including plasma membranes and ca. 25% occurred in a soluble form in the cytoplasm. Both the bound and the soluble forms of CA were increased in crabs acclimated to low salinity, suggesting that CA in crab gills, besides its known role in C02 excretion and acid-base balance, functions in osmoregulatory processes.

Abstract. Excretion of total CO2 and uptake of sodium and chloride ions across the branchial epithelium of the posterior gills of the shore crab Carcinus maenas, collected from Kiel Bay (Baltic Sea) in 1989, were measured using isolated perfused gill preparations. Total CO 2 effluxes depended on the HCO~-concentration of the internal perfusate in a saturable mode and were inhibited by internally and externally applied acetazolamide at 10-4 M. Potential differences between hemolymph space and medium did not change significantly during experimental treatments. Neither a bicarbonate gradient (6 mM) directed from the internal perfusate to external bath solution nor symmetrically applied 10-4 M acetazolamide significantly influenced the influxes of Na + and C1-. Results confirmed the role of carbonic anhydrase in CO 2 excretion but called into question the assumed functioning of the enzyme in branchial ion transport processes.